1
Calcium pumps and interacting BON1 protein modulate calcium signature stomatal 1
closure and plant immunity 2
Dong-Lei Yang12sect Zhenying Shi23sect Yongmei Bao12sect Jiapei Yan2sect Ziyuan Yang1 Huiyun 3
Yu2 Yun Li1 Mingyue Gou24 Shu Wang2 Baohong Zou12 Dachao Xu1 Zhiqi Ma1 Jitae Kim2 4
Jian Hua21 5
1State Key Laboratory of Crop Genetics and Germplasm Enhancement Nanjing Agricultural 6
University Nanjing 210095 China 7
2 School of Integrative Plant Science Plant Biology Section Cornell University Ithaca NY 8
14853 USA 9
3present address Key Laboratory of Insect Developmental and Evolutionary Biology Institute of 10
Plant Physiology and Ecology Shanghai Institutes for Biological Sciences Chinese Academy of 11
Sciences Shanghai 200032 China 12
4present address Biology Department Brookhaven National Laboratory 50 Bell Avenue Upton 13
NY 11973 USA 14
sectThese authors contribute equally to this work 15
Correspondence should be addressed JH (email jh299cornelledu) or D-LY (email 16
dlyangnjaueducn) 17
18
Author Contributions 19
JH and DLY conceived the project DLY ZS YB JY and JH designed the experiments analyzed 20
the data and made the figures DLY ZS YB JY ZY HY YL MG SW BZ DX ZM and JK 21
conducted experiments JH and DLY wrote the paper 22
23
Plant Physiology Preview Published on July 12 2017 as DOI101104pp1700495
Copyright 2017 by the American Society of Plant Biologists
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One-sentence summary 24
Calcium pumps ACA10 and ACA8 and their interacting protein BON1 regulate calcium 25
signatures and impact stomatal movement and plant immunity in Arabidopsis 26
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Abstract 27
Calcium signaling is essential for environmental responses including immune responses 28
Here we provide evidence that the evolutionarily conserved protein BONZAI1 (BON1) functions 29
together with autoinhibited calcium ATPase 10 (ACA10) and ACA8 to regulate calcium signals 30
in Arabidopsis BON1 is a plasma membrane localized protein that negatively regulates the 31
expression of immune receptor genes and positively regulates stomatal closure We found that 32
BON1 interacts with the autoinhibitory domains of ACA10 and ACA8 and the aca10 loss of 33
function (LOF) mutants have an autoimmune phenotype similar to that of the bon1 LOF mutants 34
Genetic evidences indicate that BON1 positively regulates the activities of ACA10 and ACA8 35
Consistent with this idea the steady level of calcium concentration is increased in both aca10 36
and bon1 mutants Most strikingly cytosolic calcium oscillation imposed by external calcium 37
treatment was altered in aca10 aca8 and bon1 mutants in guard cells In addition calcium and 38
pathogen induced stomatal closure was compromised in the aca10 and bon1 mutants Taken 39
together this study indicates that ACA108 and BON1 physically interact on plasma membrane 40
and function in the generation of cytosol calcium signatures that are critical for stomatal 41
movement and impact plant immunity 42
43
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Introduction 44
Calcium ion (Ca2+) is an important cellular second messenger for diverse developmental 45
processes and environmental responses in both plants and animals (Lewis 2001 Dodd et al 46
2010 Kudla et al 2010) The extreme low calcium concentration in cytosol (100-200 nM) 47
creates a unique environment where calcium concentration can be regulated dynamically (Bush 48
1995) Increases of calcium transient in cytosol are activated in various environmental and 49
developmental processes including root growth stomatal movement pollen growth abiotic 50
stress responses and plant-microbe interaction Calcium spiking with unique magnitude 51
frequency shape and duration in response to environmental and endogenous cues is referred as 52
ldquocalcium signaturerdquo and is thought to encode stimulus-specific information It is shown that in 53
the guard cell where calcium is a key signal for stomatal movement control (Murata et al 2015) 54
calcium oscillation is indeed essential for stomatal closure (Allen et al 2000) 55
The stimulus-specific calcium signature is thought to be generated by coordinated action of 56
various Ca2+ influx and efflux systems including channels pumps and exchangers located at 57
different cellular membranes However the molecular identities of calcium specific influx 58
channels remain controversial (Steinhorst and Kudla 2013) Plasma membrane (PM) localized 59
cyclic nucleotide-gated channels (CNGCs) glutamate receptor like (GLR) and TWO-PORE 60
CHANNEL (TPC) are considered as main channels that release Ca2+ in plant (Dodd et al 2010 61
Kudla et al 2010) Extrusion of Ca2+ from cytosol to external cell and intercellular store 62
compartment is believed to be achieved by Ca2+-ATPases (Ca2+ pumps) and Ca2+H+ antiporters 63
(CAXs) driven by ATP and proton motive force respectively (Geisler et al 2000 Sze et al 64
2000 Shigaki and Hirschi 2006) Ca2+ pumps fall into two groups type IIA or ECA for ER type 65
Ca2+-ATPases and type IIB or ACA for autoinhibited Ca2+-ATPase (Geisler et al 2000) ACAs 66
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have an N-terminal calmodulin-binding autoinhibitory domain that inhibits the ATPase activity 67
in the C-terminus and they are localized to diverse membrane compartments (Sze et al 2000 68
Boursiac and Harper 2007) Calcium pumps in vacuole ER and nucleus were found to be 69
important for calcium signal generation in response to environmental stimuli The loss of 70
function (LOF) of PCA1 a vacuole localized ACA resulted in sustained rather than transient 71
elevated Ca2+ in cytosol under salt treatment in the moss Physcomitrella patens (Qudeimat et al 72
2008) In tobacco knock-down of NbCA1 an ER localized ACA greatly increased the 73
amplitude and duration of calcium spikes induced by cryptogein (Zhu et al 2010) The LOF of 74
MCA a nucleus localized EAC resulted in greatly reduced nuclear calcium spiking in response 75
to Nod factor in Medicago truncatula (Capoen et al 2011) The involvement of PM localized 76
calcium pumps in calcium signal is not known yet in plants 77
Calcium signaling has recently been genetically connected with plant immunity Distinct 78
calcium signatures are rapidly induced upon pathogen invasion (Lecourieux et al 2006 Ma and 79
Berkowitz 2007) Disruption of putative calcium channels such as PM-localized CNGCs and 80
GLR either enhance or compromise plant immune responses (Clough et al 2000 Ford and 81
Roberts 2014) The PM-localized calcium pumps ACA8 and ACA10 are found to be associated 82
with immune receptor FLS2 and their LOF mutants were susceptible to bacterial pathogens (Frei 83
dit Frey et al 2012) Multiple calcium binding proteins are also involved in plant immunity 84
regulation For instance a calmodulin (CaM)-binding protein MLO negatively regulates 85
resistance to powdery mildew in barley (Kim et al 2002) and a Ca2+- and CaM-binding 86
transcription factor AtSR1CAMTA3 is a negative regulator of salicylic acid (SA)-dependent 87
defense responses in Arabidopsis (Du et al 2009) A CaM gene in Nicotiana benthamiana (N 88
benthamiana) is required for immune responses triggered by silencing of the ER calcium pump 89
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CA1 (Zhu et al 2010) and overexpression of a pepper CaM gene induces programmed cell 90
death (PCD) and enhances disease resistance (Choi et al 2009) A chloroplast calcium regulated 91
protein CAS also plays an important role in plant immunity (Nomura et al 2012) In addition 92
calcium-dependent protein kinases (CDPK or CPK) are critical regulators of plant immune 93
responses both to pathogen-associated molecular patterns (PAMP) and effectors (Boudsocq and 94
Sheen 2013) Four CDPKs (CPK45611) are found to be critical for transcriptional 95
reprogramming and ROS production in responses to PAMPs (Boudsocq et al 2010) 96
CPK1245611 are shown to be involved in downstream events including transcriptional 97
reprogramming and reactive oxygen species (ROS) production after activation of plant immune 98
receptor NLR (NOD1 like Receptors) genes in response to pathogen effectors (Gao et al 2013) 99
Recently CPK28 is shown to phosphorylate BIK1 a substrate of multiple PAMP receptors and 100
therefore attenuating PAMP signaling (Monaghan et al 2014) 101
One intriguing component involved in calcium signaling and plant immunity is the 102
Arabidopsis BON1 gene BON1 is a member of an evolutionarily conserved copine family found 103
in protozoa plants nematodes and mammals (Creutz et al 1998) The copine proteins have two 104
calcium-dependent phospholipid-binding C2 domains at their amino (N)-terminus and a putative 105
protein-protein interaction VWA (von Willebrand A) or A domain at their carboxyl (C)-terminus 106
(Rizo and Sudhof 1998 Whittaker and Hynes 2002) The BON1 protein resides on the PM 107
mainly through myristoylation of its second residue glycine (Hua et al 2001 Li et al 2010) 108
Mutating residue glycine (G) 2 to alanine (A) abolishes PM localization of BON1 and results in 109
the loss of BON1 activity (Li et al 2010) BON1 has conserved aspartate residues important for 110
calcium binding in the two C2 domains Mutating all these Asp residues in either C2A or C2B 111
domains abolishes BON1 activity in rescuing the bon1-1 defects indicating that calcium binding 112
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is required for BON1 function (Li et al 2010) 113
BON1 is a negative regulator of plant immune responses and a positive regulator of stomatal 114
closure response In the LOF mutant bon1-1 in Col-0 background a plant immune receptor NLR 115
gene SNC1 (suppressor of NPR1 constitutive 1) is increased at transcript level through initial 116
upregulation followed by SA-mediated amplification (Yang and Hua 2004 Li et al 2007) The 117
bon1-1 mutant thus has constitutive defense (autoimmune) responses and is consequently dwarf 118
However these defects observed is dependent on a Col-0 specific NB-LRR gene SNC1 and the 119
LOF bon1-2 allele in the Wassilewakija (Ws) accession does not exhibit constitutive defense 120
activation because there is no functional SNC1 in Ws (Yang and Hua 2004) In addition to the 121
function in negatively regulating SNC1 BON1 is recently shown to promote stomatal closure in 122
response to stimuli including abscisic acid (ABA) and bacterial pathogen (Gou et al 2015) This 123
function is independent of SNC1 or NB-LRR signaling and the bon1 mutant is defective in 124
stomatal closure when autoimmunity is suppressed by the SNC1 LOF mutation snc1-11 (Gou et 125
al 2015) Therefore BON1 has a more general role in regulating signaling events in addition to 126
NLR gene expression 127
It is intriguing that an evolutionarily conserved calcium binding copine protein can regulate 128
stomatal closure and impact NLR gene expression Here we identified calcium pumps ACA10 129
and ACA8 as interacting proteins of BON1 both physically and genetically The LOF mutants of 130
these genes have altered calcium signature and calcium homeostasis and are defective in 131
stomatal closure and plant immunity Thus we have uncovered a critical role for PM-localized 132
calcium pumps ACA108 in calcium signature generation as well as their regulation by an 133
evolutionarily conserved BON1 protein in Arabidopsis 134
135
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136
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Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
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to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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17
1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
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2
One-sentence summary 24
Calcium pumps ACA10 and ACA8 and their interacting protein BON1 regulate calcium 25
signatures and impact stomatal movement and plant immunity in Arabidopsis 26
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3
Abstract 27
Calcium signaling is essential for environmental responses including immune responses 28
Here we provide evidence that the evolutionarily conserved protein BONZAI1 (BON1) functions 29
together with autoinhibited calcium ATPase 10 (ACA10) and ACA8 to regulate calcium signals 30
in Arabidopsis BON1 is a plasma membrane localized protein that negatively regulates the 31
expression of immune receptor genes and positively regulates stomatal closure We found that 32
BON1 interacts with the autoinhibitory domains of ACA10 and ACA8 and the aca10 loss of 33
function (LOF) mutants have an autoimmune phenotype similar to that of the bon1 LOF mutants 34
Genetic evidences indicate that BON1 positively regulates the activities of ACA10 and ACA8 35
Consistent with this idea the steady level of calcium concentration is increased in both aca10 36
and bon1 mutants Most strikingly cytosolic calcium oscillation imposed by external calcium 37
treatment was altered in aca10 aca8 and bon1 mutants in guard cells In addition calcium and 38
pathogen induced stomatal closure was compromised in the aca10 and bon1 mutants Taken 39
together this study indicates that ACA108 and BON1 physically interact on plasma membrane 40
and function in the generation of cytosol calcium signatures that are critical for stomatal 41
movement and impact plant immunity 42
43
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4
Introduction 44
Calcium ion (Ca2+) is an important cellular second messenger for diverse developmental 45
processes and environmental responses in both plants and animals (Lewis 2001 Dodd et al 46
2010 Kudla et al 2010) The extreme low calcium concentration in cytosol (100-200 nM) 47
creates a unique environment where calcium concentration can be regulated dynamically (Bush 48
1995) Increases of calcium transient in cytosol are activated in various environmental and 49
developmental processes including root growth stomatal movement pollen growth abiotic 50
stress responses and plant-microbe interaction Calcium spiking with unique magnitude 51
frequency shape and duration in response to environmental and endogenous cues is referred as 52
ldquocalcium signaturerdquo and is thought to encode stimulus-specific information It is shown that in 53
the guard cell where calcium is a key signal for stomatal movement control (Murata et al 2015) 54
calcium oscillation is indeed essential for stomatal closure (Allen et al 2000) 55
The stimulus-specific calcium signature is thought to be generated by coordinated action of 56
various Ca2+ influx and efflux systems including channels pumps and exchangers located at 57
different cellular membranes However the molecular identities of calcium specific influx 58
channels remain controversial (Steinhorst and Kudla 2013) Plasma membrane (PM) localized 59
cyclic nucleotide-gated channels (CNGCs) glutamate receptor like (GLR) and TWO-PORE 60
CHANNEL (TPC) are considered as main channels that release Ca2+ in plant (Dodd et al 2010 61
Kudla et al 2010) Extrusion of Ca2+ from cytosol to external cell and intercellular store 62
compartment is believed to be achieved by Ca2+-ATPases (Ca2+ pumps) and Ca2+H+ antiporters 63
(CAXs) driven by ATP and proton motive force respectively (Geisler et al 2000 Sze et al 64
2000 Shigaki and Hirschi 2006) Ca2+ pumps fall into two groups type IIA or ECA for ER type 65
Ca2+-ATPases and type IIB or ACA for autoinhibited Ca2+-ATPase (Geisler et al 2000) ACAs 66
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5
have an N-terminal calmodulin-binding autoinhibitory domain that inhibits the ATPase activity 67
in the C-terminus and they are localized to diverse membrane compartments (Sze et al 2000 68
Boursiac and Harper 2007) Calcium pumps in vacuole ER and nucleus were found to be 69
important for calcium signal generation in response to environmental stimuli The loss of 70
function (LOF) of PCA1 a vacuole localized ACA resulted in sustained rather than transient 71
elevated Ca2+ in cytosol under salt treatment in the moss Physcomitrella patens (Qudeimat et al 72
2008) In tobacco knock-down of NbCA1 an ER localized ACA greatly increased the 73
amplitude and duration of calcium spikes induced by cryptogein (Zhu et al 2010) The LOF of 74
MCA a nucleus localized EAC resulted in greatly reduced nuclear calcium spiking in response 75
to Nod factor in Medicago truncatula (Capoen et al 2011) The involvement of PM localized 76
calcium pumps in calcium signal is not known yet in plants 77
Calcium signaling has recently been genetically connected with plant immunity Distinct 78
calcium signatures are rapidly induced upon pathogen invasion (Lecourieux et al 2006 Ma and 79
Berkowitz 2007) Disruption of putative calcium channels such as PM-localized CNGCs and 80
GLR either enhance or compromise plant immune responses (Clough et al 2000 Ford and 81
Roberts 2014) The PM-localized calcium pumps ACA8 and ACA10 are found to be associated 82
with immune receptor FLS2 and their LOF mutants were susceptible to bacterial pathogens (Frei 83
dit Frey et al 2012) Multiple calcium binding proteins are also involved in plant immunity 84
regulation For instance a calmodulin (CaM)-binding protein MLO negatively regulates 85
resistance to powdery mildew in barley (Kim et al 2002) and a Ca2+- and CaM-binding 86
transcription factor AtSR1CAMTA3 is a negative regulator of salicylic acid (SA)-dependent 87
defense responses in Arabidopsis (Du et al 2009) A CaM gene in Nicotiana benthamiana (N 88
benthamiana) is required for immune responses triggered by silencing of the ER calcium pump 89
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6
CA1 (Zhu et al 2010) and overexpression of a pepper CaM gene induces programmed cell 90
death (PCD) and enhances disease resistance (Choi et al 2009) A chloroplast calcium regulated 91
protein CAS also plays an important role in plant immunity (Nomura et al 2012) In addition 92
calcium-dependent protein kinases (CDPK or CPK) are critical regulators of plant immune 93
responses both to pathogen-associated molecular patterns (PAMP) and effectors (Boudsocq and 94
Sheen 2013) Four CDPKs (CPK45611) are found to be critical for transcriptional 95
reprogramming and ROS production in responses to PAMPs (Boudsocq et al 2010) 96
CPK1245611 are shown to be involved in downstream events including transcriptional 97
reprogramming and reactive oxygen species (ROS) production after activation of plant immune 98
receptor NLR (NOD1 like Receptors) genes in response to pathogen effectors (Gao et al 2013) 99
Recently CPK28 is shown to phosphorylate BIK1 a substrate of multiple PAMP receptors and 100
therefore attenuating PAMP signaling (Monaghan et al 2014) 101
One intriguing component involved in calcium signaling and plant immunity is the 102
Arabidopsis BON1 gene BON1 is a member of an evolutionarily conserved copine family found 103
in protozoa plants nematodes and mammals (Creutz et al 1998) The copine proteins have two 104
calcium-dependent phospholipid-binding C2 domains at their amino (N)-terminus and a putative 105
protein-protein interaction VWA (von Willebrand A) or A domain at their carboxyl (C)-terminus 106
(Rizo and Sudhof 1998 Whittaker and Hynes 2002) The BON1 protein resides on the PM 107
mainly through myristoylation of its second residue glycine (Hua et al 2001 Li et al 2010) 108
Mutating residue glycine (G) 2 to alanine (A) abolishes PM localization of BON1 and results in 109
the loss of BON1 activity (Li et al 2010) BON1 has conserved aspartate residues important for 110
calcium binding in the two C2 domains Mutating all these Asp residues in either C2A or C2B 111
domains abolishes BON1 activity in rescuing the bon1-1 defects indicating that calcium binding 112
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7
is required for BON1 function (Li et al 2010) 113
BON1 is a negative regulator of plant immune responses and a positive regulator of stomatal 114
closure response In the LOF mutant bon1-1 in Col-0 background a plant immune receptor NLR 115
gene SNC1 (suppressor of NPR1 constitutive 1) is increased at transcript level through initial 116
upregulation followed by SA-mediated amplification (Yang and Hua 2004 Li et al 2007) The 117
bon1-1 mutant thus has constitutive defense (autoimmune) responses and is consequently dwarf 118
However these defects observed is dependent on a Col-0 specific NB-LRR gene SNC1 and the 119
LOF bon1-2 allele in the Wassilewakija (Ws) accession does not exhibit constitutive defense 120
activation because there is no functional SNC1 in Ws (Yang and Hua 2004) In addition to the 121
function in negatively regulating SNC1 BON1 is recently shown to promote stomatal closure in 122
response to stimuli including abscisic acid (ABA) and bacterial pathogen (Gou et al 2015) This 123
function is independent of SNC1 or NB-LRR signaling and the bon1 mutant is defective in 124
stomatal closure when autoimmunity is suppressed by the SNC1 LOF mutation snc1-11 (Gou et 125
al 2015) Therefore BON1 has a more general role in regulating signaling events in addition to 126
NLR gene expression 127
It is intriguing that an evolutionarily conserved calcium binding copine protein can regulate 128
stomatal closure and impact NLR gene expression Here we identified calcium pumps ACA10 129
and ACA8 as interacting proteins of BON1 both physically and genetically The LOF mutants of 130
these genes have altered calcium signature and calcium homeostasis and are defective in 131
stomatal closure and plant immunity Thus we have uncovered a critical role for PM-localized 132
calcium pumps ACA108 in calcium signature generation as well as their regulation by an 133
evolutionarily conserved BON1 protein in Arabidopsis 134
135
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8
136
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9
Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
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10
to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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11
were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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13
due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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3
Abstract 27
Calcium signaling is essential for environmental responses including immune responses 28
Here we provide evidence that the evolutionarily conserved protein BONZAI1 (BON1) functions 29
together with autoinhibited calcium ATPase 10 (ACA10) and ACA8 to regulate calcium signals 30
in Arabidopsis BON1 is a plasma membrane localized protein that negatively regulates the 31
expression of immune receptor genes and positively regulates stomatal closure We found that 32
BON1 interacts with the autoinhibitory domains of ACA10 and ACA8 and the aca10 loss of 33
function (LOF) mutants have an autoimmune phenotype similar to that of the bon1 LOF mutants 34
Genetic evidences indicate that BON1 positively regulates the activities of ACA10 and ACA8 35
Consistent with this idea the steady level of calcium concentration is increased in both aca10 36
and bon1 mutants Most strikingly cytosolic calcium oscillation imposed by external calcium 37
treatment was altered in aca10 aca8 and bon1 mutants in guard cells In addition calcium and 38
pathogen induced stomatal closure was compromised in the aca10 and bon1 mutants Taken 39
together this study indicates that ACA108 and BON1 physically interact on plasma membrane 40
and function in the generation of cytosol calcium signatures that are critical for stomatal 41
movement and impact plant immunity 42
43
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4
Introduction 44
Calcium ion (Ca2+) is an important cellular second messenger for diverse developmental 45
processes and environmental responses in both plants and animals (Lewis 2001 Dodd et al 46
2010 Kudla et al 2010) The extreme low calcium concentration in cytosol (100-200 nM) 47
creates a unique environment where calcium concentration can be regulated dynamically (Bush 48
1995) Increases of calcium transient in cytosol are activated in various environmental and 49
developmental processes including root growth stomatal movement pollen growth abiotic 50
stress responses and plant-microbe interaction Calcium spiking with unique magnitude 51
frequency shape and duration in response to environmental and endogenous cues is referred as 52
ldquocalcium signaturerdquo and is thought to encode stimulus-specific information It is shown that in 53
the guard cell where calcium is a key signal for stomatal movement control (Murata et al 2015) 54
calcium oscillation is indeed essential for stomatal closure (Allen et al 2000) 55
The stimulus-specific calcium signature is thought to be generated by coordinated action of 56
various Ca2+ influx and efflux systems including channels pumps and exchangers located at 57
different cellular membranes However the molecular identities of calcium specific influx 58
channels remain controversial (Steinhorst and Kudla 2013) Plasma membrane (PM) localized 59
cyclic nucleotide-gated channels (CNGCs) glutamate receptor like (GLR) and TWO-PORE 60
CHANNEL (TPC) are considered as main channels that release Ca2+ in plant (Dodd et al 2010 61
Kudla et al 2010) Extrusion of Ca2+ from cytosol to external cell and intercellular store 62
compartment is believed to be achieved by Ca2+-ATPases (Ca2+ pumps) and Ca2+H+ antiporters 63
(CAXs) driven by ATP and proton motive force respectively (Geisler et al 2000 Sze et al 64
2000 Shigaki and Hirschi 2006) Ca2+ pumps fall into two groups type IIA or ECA for ER type 65
Ca2+-ATPases and type IIB or ACA for autoinhibited Ca2+-ATPase (Geisler et al 2000) ACAs 66
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5
have an N-terminal calmodulin-binding autoinhibitory domain that inhibits the ATPase activity 67
in the C-terminus and they are localized to diverse membrane compartments (Sze et al 2000 68
Boursiac and Harper 2007) Calcium pumps in vacuole ER and nucleus were found to be 69
important for calcium signal generation in response to environmental stimuli The loss of 70
function (LOF) of PCA1 a vacuole localized ACA resulted in sustained rather than transient 71
elevated Ca2+ in cytosol under salt treatment in the moss Physcomitrella patens (Qudeimat et al 72
2008) In tobacco knock-down of NbCA1 an ER localized ACA greatly increased the 73
amplitude and duration of calcium spikes induced by cryptogein (Zhu et al 2010) The LOF of 74
MCA a nucleus localized EAC resulted in greatly reduced nuclear calcium spiking in response 75
to Nod factor in Medicago truncatula (Capoen et al 2011) The involvement of PM localized 76
calcium pumps in calcium signal is not known yet in plants 77
Calcium signaling has recently been genetically connected with plant immunity Distinct 78
calcium signatures are rapidly induced upon pathogen invasion (Lecourieux et al 2006 Ma and 79
Berkowitz 2007) Disruption of putative calcium channels such as PM-localized CNGCs and 80
GLR either enhance or compromise plant immune responses (Clough et al 2000 Ford and 81
Roberts 2014) The PM-localized calcium pumps ACA8 and ACA10 are found to be associated 82
with immune receptor FLS2 and their LOF mutants were susceptible to bacterial pathogens (Frei 83
dit Frey et al 2012) Multiple calcium binding proteins are also involved in plant immunity 84
regulation For instance a calmodulin (CaM)-binding protein MLO negatively regulates 85
resistance to powdery mildew in barley (Kim et al 2002) and a Ca2+- and CaM-binding 86
transcription factor AtSR1CAMTA3 is a negative regulator of salicylic acid (SA)-dependent 87
defense responses in Arabidopsis (Du et al 2009) A CaM gene in Nicotiana benthamiana (N 88
benthamiana) is required for immune responses triggered by silencing of the ER calcium pump 89
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6
CA1 (Zhu et al 2010) and overexpression of a pepper CaM gene induces programmed cell 90
death (PCD) and enhances disease resistance (Choi et al 2009) A chloroplast calcium regulated 91
protein CAS also plays an important role in plant immunity (Nomura et al 2012) In addition 92
calcium-dependent protein kinases (CDPK or CPK) are critical regulators of plant immune 93
responses both to pathogen-associated molecular patterns (PAMP) and effectors (Boudsocq and 94
Sheen 2013) Four CDPKs (CPK45611) are found to be critical for transcriptional 95
reprogramming and ROS production in responses to PAMPs (Boudsocq et al 2010) 96
CPK1245611 are shown to be involved in downstream events including transcriptional 97
reprogramming and reactive oxygen species (ROS) production after activation of plant immune 98
receptor NLR (NOD1 like Receptors) genes in response to pathogen effectors (Gao et al 2013) 99
Recently CPK28 is shown to phosphorylate BIK1 a substrate of multiple PAMP receptors and 100
therefore attenuating PAMP signaling (Monaghan et al 2014) 101
One intriguing component involved in calcium signaling and plant immunity is the 102
Arabidopsis BON1 gene BON1 is a member of an evolutionarily conserved copine family found 103
in protozoa plants nematodes and mammals (Creutz et al 1998) The copine proteins have two 104
calcium-dependent phospholipid-binding C2 domains at their amino (N)-terminus and a putative 105
protein-protein interaction VWA (von Willebrand A) or A domain at their carboxyl (C)-terminus 106
(Rizo and Sudhof 1998 Whittaker and Hynes 2002) The BON1 protein resides on the PM 107
mainly through myristoylation of its second residue glycine (Hua et al 2001 Li et al 2010) 108
Mutating residue glycine (G) 2 to alanine (A) abolishes PM localization of BON1 and results in 109
the loss of BON1 activity (Li et al 2010) BON1 has conserved aspartate residues important for 110
calcium binding in the two C2 domains Mutating all these Asp residues in either C2A or C2B 111
domains abolishes BON1 activity in rescuing the bon1-1 defects indicating that calcium binding 112
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7
is required for BON1 function (Li et al 2010) 113
BON1 is a negative regulator of plant immune responses and a positive regulator of stomatal 114
closure response In the LOF mutant bon1-1 in Col-0 background a plant immune receptor NLR 115
gene SNC1 (suppressor of NPR1 constitutive 1) is increased at transcript level through initial 116
upregulation followed by SA-mediated amplification (Yang and Hua 2004 Li et al 2007) The 117
bon1-1 mutant thus has constitutive defense (autoimmune) responses and is consequently dwarf 118
However these defects observed is dependent on a Col-0 specific NB-LRR gene SNC1 and the 119
LOF bon1-2 allele in the Wassilewakija (Ws) accession does not exhibit constitutive defense 120
activation because there is no functional SNC1 in Ws (Yang and Hua 2004) In addition to the 121
function in negatively regulating SNC1 BON1 is recently shown to promote stomatal closure in 122
response to stimuli including abscisic acid (ABA) and bacterial pathogen (Gou et al 2015) This 123
function is independent of SNC1 or NB-LRR signaling and the bon1 mutant is defective in 124
stomatal closure when autoimmunity is suppressed by the SNC1 LOF mutation snc1-11 (Gou et 125
al 2015) Therefore BON1 has a more general role in regulating signaling events in addition to 126
NLR gene expression 127
It is intriguing that an evolutionarily conserved calcium binding copine protein can regulate 128
stomatal closure and impact NLR gene expression Here we identified calcium pumps ACA10 129
and ACA8 as interacting proteins of BON1 both physically and genetically The LOF mutants of 130
these genes have altered calcium signature and calcium homeostasis and are defective in 131
stomatal closure and plant immunity Thus we have uncovered a critical role for PM-localized 132
calcium pumps ACA108 in calcium signature generation as well as their regulation by an 133
evolutionarily conserved BON1 protein in Arabidopsis 134
135
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8
136
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9
Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
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10
to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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11
were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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12
ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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13
due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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14
genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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17
1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
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Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574
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Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
4
Introduction 44
Calcium ion (Ca2+) is an important cellular second messenger for diverse developmental 45
processes and environmental responses in both plants and animals (Lewis 2001 Dodd et al 46
2010 Kudla et al 2010) The extreme low calcium concentration in cytosol (100-200 nM) 47
creates a unique environment where calcium concentration can be regulated dynamically (Bush 48
1995) Increases of calcium transient in cytosol are activated in various environmental and 49
developmental processes including root growth stomatal movement pollen growth abiotic 50
stress responses and plant-microbe interaction Calcium spiking with unique magnitude 51
frequency shape and duration in response to environmental and endogenous cues is referred as 52
ldquocalcium signaturerdquo and is thought to encode stimulus-specific information It is shown that in 53
the guard cell where calcium is a key signal for stomatal movement control (Murata et al 2015) 54
calcium oscillation is indeed essential for stomatal closure (Allen et al 2000) 55
The stimulus-specific calcium signature is thought to be generated by coordinated action of 56
various Ca2+ influx and efflux systems including channels pumps and exchangers located at 57
different cellular membranes However the molecular identities of calcium specific influx 58
channels remain controversial (Steinhorst and Kudla 2013) Plasma membrane (PM) localized 59
cyclic nucleotide-gated channels (CNGCs) glutamate receptor like (GLR) and TWO-PORE 60
CHANNEL (TPC) are considered as main channels that release Ca2+ in plant (Dodd et al 2010 61
Kudla et al 2010) Extrusion of Ca2+ from cytosol to external cell and intercellular store 62
compartment is believed to be achieved by Ca2+-ATPases (Ca2+ pumps) and Ca2+H+ antiporters 63
(CAXs) driven by ATP and proton motive force respectively (Geisler et al 2000 Sze et al 64
2000 Shigaki and Hirschi 2006) Ca2+ pumps fall into two groups type IIA or ECA for ER type 65
Ca2+-ATPases and type IIB or ACA for autoinhibited Ca2+-ATPase (Geisler et al 2000) ACAs 66
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
5
have an N-terminal calmodulin-binding autoinhibitory domain that inhibits the ATPase activity 67
in the C-terminus and they are localized to diverse membrane compartments (Sze et al 2000 68
Boursiac and Harper 2007) Calcium pumps in vacuole ER and nucleus were found to be 69
important for calcium signal generation in response to environmental stimuli The loss of 70
function (LOF) of PCA1 a vacuole localized ACA resulted in sustained rather than transient 71
elevated Ca2+ in cytosol under salt treatment in the moss Physcomitrella patens (Qudeimat et al 72
2008) In tobacco knock-down of NbCA1 an ER localized ACA greatly increased the 73
amplitude and duration of calcium spikes induced by cryptogein (Zhu et al 2010) The LOF of 74
MCA a nucleus localized EAC resulted in greatly reduced nuclear calcium spiking in response 75
to Nod factor in Medicago truncatula (Capoen et al 2011) The involvement of PM localized 76
calcium pumps in calcium signal is not known yet in plants 77
Calcium signaling has recently been genetically connected with plant immunity Distinct 78
calcium signatures are rapidly induced upon pathogen invasion (Lecourieux et al 2006 Ma and 79
Berkowitz 2007) Disruption of putative calcium channels such as PM-localized CNGCs and 80
GLR either enhance or compromise plant immune responses (Clough et al 2000 Ford and 81
Roberts 2014) The PM-localized calcium pumps ACA8 and ACA10 are found to be associated 82
with immune receptor FLS2 and their LOF mutants were susceptible to bacterial pathogens (Frei 83
dit Frey et al 2012) Multiple calcium binding proteins are also involved in plant immunity 84
regulation For instance a calmodulin (CaM)-binding protein MLO negatively regulates 85
resistance to powdery mildew in barley (Kim et al 2002) and a Ca2+- and CaM-binding 86
transcription factor AtSR1CAMTA3 is a negative regulator of salicylic acid (SA)-dependent 87
defense responses in Arabidopsis (Du et al 2009) A CaM gene in Nicotiana benthamiana (N 88
benthamiana) is required for immune responses triggered by silencing of the ER calcium pump 89
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
CA1 (Zhu et al 2010) and overexpression of a pepper CaM gene induces programmed cell 90
death (PCD) and enhances disease resistance (Choi et al 2009) A chloroplast calcium regulated 91
protein CAS also plays an important role in plant immunity (Nomura et al 2012) In addition 92
calcium-dependent protein kinases (CDPK or CPK) are critical regulators of plant immune 93
responses both to pathogen-associated molecular patterns (PAMP) and effectors (Boudsocq and 94
Sheen 2013) Four CDPKs (CPK45611) are found to be critical for transcriptional 95
reprogramming and ROS production in responses to PAMPs (Boudsocq et al 2010) 96
CPK1245611 are shown to be involved in downstream events including transcriptional 97
reprogramming and reactive oxygen species (ROS) production after activation of plant immune 98
receptor NLR (NOD1 like Receptors) genes in response to pathogen effectors (Gao et al 2013) 99
Recently CPK28 is shown to phosphorylate BIK1 a substrate of multiple PAMP receptors and 100
therefore attenuating PAMP signaling (Monaghan et al 2014) 101
One intriguing component involved in calcium signaling and plant immunity is the 102
Arabidopsis BON1 gene BON1 is a member of an evolutionarily conserved copine family found 103
in protozoa plants nematodes and mammals (Creutz et al 1998) The copine proteins have two 104
calcium-dependent phospholipid-binding C2 domains at their amino (N)-terminus and a putative 105
protein-protein interaction VWA (von Willebrand A) or A domain at their carboxyl (C)-terminus 106
(Rizo and Sudhof 1998 Whittaker and Hynes 2002) The BON1 protein resides on the PM 107
mainly through myristoylation of its second residue glycine (Hua et al 2001 Li et al 2010) 108
Mutating residue glycine (G) 2 to alanine (A) abolishes PM localization of BON1 and results in 109
the loss of BON1 activity (Li et al 2010) BON1 has conserved aspartate residues important for 110
calcium binding in the two C2 domains Mutating all these Asp residues in either C2A or C2B 111
domains abolishes BON1 activity in rescuing the bon1-1 defects indicating that calcium binding 112
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is required for BON1 function (Li et al 2010) 113
BON1 is a negative regulator of plant immune responses and a positive regulator of stomatal 114
closure response In the LOF mutant bon1-1 in Col-0 background a plant immune receptor NLR 115
gene SNC1 (suppressor of NPR1 constitutive 1) is increased at transcript level through initial 116
upregulation followed by SA-mediated amplification (Yang and Hua 2004 Li et al 2007) The 117
bon1-1 mutant thus has constitutive defense (autoimmune) responses and is consequently dwarf 118
However these defects observed is dependent on a Col-0 specific NB-LRR gene SNC1 and the 119
LOF bon1-2 allele in the Wassilewakija (Ws) accession does not exhibit constitutive defense 120
activation because there is no functional SNC1 in Ws (Yang and Hua 2004) In addition to the 121
function in negatively regulating SNC1 BON1 is recently shown to promote stomatal closure in 122
response to stimuli including abscisic acid (ABA) and bacterial pathogen (Gou et al 2015) This 123
function is independent of SNC1 or NB-LRR signaling and the bon1 mutant is defective in 124
stomatal closure when autoimmunity is suppressed by the SNC1 LOF mutation snc1-11 (Gou et 125
al 2015) Therefore BON1 has a more general role in regulating signaling events in addition to 126
NLR gene expression 127
It is intriguing that an evolutionarily conserved calcium binding copine protein can regulate 128
stomatal closure and impact NLR gene expression Here we identified calcium pumps ACA10 129
and ACA8 as interacting proteins of BON1 both physically and genetically The LOF mutants of 130
these genes have altered calcium signature and calcium homeostasis and are defective in 131
stomatal closure and plant immunity Thus we have uncovered a critical role for PM-localized 132
calcium pumps ACA108 in calcium signature generation as well as their regulation by an 133
evolutionarily conserved BON1 protein in Arabidopsis 134
135
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136
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Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
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to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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5
have an N-terminal calmodulin-binding autoinhibitory domain that inhibits the ATPase activity 67
in the C-terminus and they are localized to diverse membrane compartments (Sze et al 2000 68
Boursiac and Harper 2007) Calcium pumps in vacuole ER and nucleus were found to be 69
important for calcium signal generation in response to environmental stimuli The loss of 70
function (LOF) of PCA1 a vacuole localized ACA resulted in sustained rather than transient 71
elevated Ca2+ in cytosol under salt treatment in the moss Physcomitrella patens (Qudeimat et al 72
2008) In tobacco knock-down of NbCA1 an ER localized ACA greatly increased the 73
amplitude and duration of calcium spikes induced by cryptogein (Zhu et al 2010) The LOF of 74
MCA a nucleus localized EAC resulted in greatly reduced nuclear calcium spiking in response 75
to Nod factor in Medicago truncatula (Capoen et al 2011) The involvement of PM localized 76
calcium pumps in calcium signal is not known yet in plants 77
Calcium signaling has recently been genetically connected with plant immunity Distinct 78
calcium signatures are rapidly induced upon pathogen invasion (Lecourieux et al 2006 Ma and 79
Berkowitz 2007) Disruption of putative calcium channels such as PM-localized CNGCs and 80
GLR either enhance or compromise plant immune responses (Clough et al 2000 Ford and 81
Roberts 2014) The PM-localized calcium pumps ACA8 and ACA10 are found to be associated 82
with immune receptor FLS2 and their LOF mutants were susceptible to bacterial pathogens (Frei 83
dit Frey et al 2012) Multiple calcium binding proteins are also involved in plant immunity 84
regulation For instance a calmodulin (CaM)-binding protein MLO negatively regulates 85
resistance to powdery mildew in barley (Kim et al 2002) and a Ca2+- and CaM-binding 86
transcription factor AtSR1CAMTA3 is a negative regulator of salicylic acid (SA)-dependent 87
defense responses in Arabidopsis (Du et al 2009) A CaM gene in Nicotiana benthamiana (N 88
benthamiana) is required for immune responses triggered by silencing of the ER calcium pump 89
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6
CA1 (Zhu et al 2010) and overexpression of a pepper CaM gene induces programmed cell 90
death (PCD) and enhances disease resistance (Choi et al 2009) A chloroplast calcium regulated 91
protein CAS also plays an important role in plant immunity (Nomura et al 2012) In addition 92
calcium-dependent protein kinases (CDPK or CPK) are critical regulators of plant immune 93
responses both to pathogen-associated molecular patterns (PAMP) and effectors (Boudsocq and 94
Sheen 2013) Four CDPKs (CPK45611) are found to be critical for transcriptional 95
reprogramming and ROS production in responses to PAMPs (Boudsocq et al 2010) 96
CPK1245611 are shown to be involved in downstream events including transcriptional 97
reprogramming and reactive oxygen species (ROS) production after activation of plant immune 98
receptor NLR (NOD1 like Receptors) genes in response to pathogen effectors (Gao et al 2013) 99
Recently CPK28 is shown to phosphorylate BIK1 a substrate of multiple PAMP receptors and 100
therefore attenuating PAMP signaling (Monaghan et al 2014) 101
One intriguing component involved in calcium signaling and plant immunity is the 102
Arabidopsis BON1 gene BON1 is a member of an evolutionarily conserved copine family found 103
in protozoa plants nematodes and mammals (Creutz et al 1998) The copine proteins have two 104
calcium-dependent phospholipid-binding C2 domains at their amino (N)-terminus and a putative 105
protein-protein interaction VWA (von Willebrand A) or A domain at their carboxyl (C)-terminus 106
(Rizo and Sudhof 1998 Whittaker and Hynes 2002) The BON1 protein resides on the PM 107
mainly through myristoylation of its second residue glycine (Hua et al 2001 Li et al 2010) 108
Mutating residue glycine (G) 2 to alanine (A) abolishes PM localization of BON1 and results in 109
the loss of BON1 activity (Li et al 2010) BON1 has conserved aspartate residues important for 110
calcium binding in the two C2 domains Mutating all these Asp residues in either C2A or C2B 111
domains abolishes BON1 activity in rescuing the bon1-1 defects indicating that calcium binding 112
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is required for BON1 function (Li et al 2010) 113
BON1 is a negative regulator of plant immune responses and a positive regulator of stomatal 114
closure response In the LOF mutant bon1-1 in Col-0 background a plant immune receptor NLR 115
gene SNC1 (suppressor of NPR1 constitutive 1) is increased at transcript level through initial 116
upregulation followed by SA-mediated amplification (Yang and Hua 2004 Li et al 2007) The 117
bon1-1 mutant thus has constitutive defense (autoimmune) responses and is consequently dwarf 118
However these defects observed is dependent on a Col-0 specific NB-LRR gene SNC1 and the 119
LOF bon1-2 allele in the Wassilewakija (Ws) accession does not exhibit constitutive defense 120
activation because there is no functional SNC1 in Ws (Yang and Hua 2004) In addition to the 121
function in negatively regulating SNC1 BON1 is recently shown to promote stomatal closure in 122
response to stimuli including abscisic acid (ABA) and bacterial pathogen (Gou et al 2015) This 123
function is independent of SNC1 or NB-LRR signaling and the bon1 mutant is defective in 124
stomatal closure when autoimmunity is suppressed by the SNC1 LOF mutation snc1-11 (Gou et 125
al 2015) Therefore BON1 has a more general role in regulating signaling events in addition to 126
NLR gene expression 127
It is intriguing that an evolutionarily conserved calcium binding copine protein can regulate 128
stomatal closure and impact NLR gene expression Here we identified calcium pumps ACA10 129
and ACA8 as interacting proteins of BON1 both physically and genetically The LOF mutants of 130
these genes have altered calcium signature and calcium homeostasis and are defective in 131
stomatal closure and plant immunity Thus we have uncovered a critical role for PM-localized 132
calcium pumps ACA108 in calcium signature generation as well as their regulation by an 133
evolutionarily conserved BON1 protein in Arabidopsis 134
135
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136
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Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
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to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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11
were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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12
ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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13
due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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17
1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
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42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
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43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
6
CA1 (Zhu et al 2010) and overexpression of a pepper CaM gene induces programmed cell 90
death (PCD) and enhances disease resistance (Choi et al 2009) A chloroplast calcium regulated 91
protein CAS also plays an important role in plant immunity (Nomura et al 2012) In addition 92
calcium-dependent protein kinases (CDPK or CPK) are critical regulators of plant immune 93
responses both to pathogen-associated molecular patterns (PAMP) and effectors (Boudsocq and 94
Sheen 2013) Four CDPKs (CPK45611) are found to be critical for transcriptional 95
reprogramming and ROS production in responses to PAMPs (Boudsocq et al 2010) 96
CPK1245611 are shown to be involved in downstream events including transcriptional 97
reprogramming and reactive oxygen species (ROS) production after activation of plant immune 98
receptor NLR (NOD1 like Receptors) genes in response to pathogen effectors (Gao et al 2013) 99
Recently CPK28 is shown to phosphorylate BIK1 a substrate of multiple PAMP receptors and 100
therefore attenuating PAMP signaling (Monaghan et al 2014) 101
One intriguing component involved in calcium signaling and plant immunity is the 102
Arabidopsis BON1 gene BON1 is a member of an evolutionarily conserved copine family found 103
in protozoa plants nematodes and mammals (Creutz et al 1998) The copine proteins have two 104
calcium-dependent phospholipid-binding C2 domains at their amino (N)-terminus and a putative 105
protein-protein interaction VWA (von Willebrand A) or A domain at their carboxyl (C)-terminus 106
(Rizo and Sudhof 1998 Whittaker and Hynes 2002) The BON1 protein resides on the PM 107
mainly through myristoylation of its second residue glycine (Hua et al 2001 Li et al 2010) 108
Mutating residue glycine (G) 2 to alanine (A) abolishes PM localization of BON1 and results in 109
the loss of BON1 activity (Li et al 2010) BON1 has conserved aspartate residues important for 110
calcium binding in the two C2 domains Mutating all these Asp residues in either C2A or C2B 111
domains abolishes BON1 activity in rescuing the bon1-1 defects indicating that calcium binding 112
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
7
is required for BON1 function (Li et al 2010) 113
BON1 is a negative regulator of plant immune responses and a positive regulator of stomatal 114
closure response In the LOF mutant bon1-1 in Col-0 background a plant immune receptor NLR 115
gene SNC1 (suppressor of NPR1 constitutive 1) is increased at transcript level through initial 116
upregulation followed by SA-mediated amplification (Yang and Hua 2004 Li et al 2007) The 117
bon1-1 mutant thus has constitutive defense (autoimmune) responses and is consequently dwarf 118
However these defects observed is dependent on a Col-0 specific NB-LRR gene SNC1 and the 119
LOF bon1-2 allele in the Wassilewakija (Ws) accession does not exhibit constitutive defense 120
activation because there is no functional SNC1 in Ws (Yang and Hua 2004) In addition to the 121
function in negatively regulating SNC1 BON1 is recently shown to promote stomatal closure in 122
response to stimuli including abscisic acid (ABA) and bacterial pathogen (Gou et al 2015) This 123
function is independent of SNC1 or NB-LRR signaling and the bon1 mutant is defective in 124
stomatal closure when autoimmunity is suppressed by the SNC1 LOF mutation snc1-11 (Gou et 125
al 2015) Therefore BON1 has a more general role in regulating signaling events in addition to 126
NLR gene expression 127
It is intriguing that an evolutionarily conserved calcium binding copine protein can regulate 128
stomatal closure and impact NLR gene expression Here we identified calcium pumps ACA10 129
and ACA8 as interacting proteins of BON1 both physically and genetically The LOF mutants of 130
these genes have altered calcium signature and calcium homeostasis and are defective in 131
stomatal closure and plant immunity Thus we have uncovered a critical role for PM-localized 132
calcium pumps ACA108 in calcium signature generation as well as their regulation by an 133
evolutionarily conserved BON1 protein in Arabidopsis 134
135
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8
136
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
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10
to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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11
were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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13
due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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7
is required for BON1 function (Li et al 2010) 113
BON1 is a negative regulator of plant immune responses and a positive regulator of stomatal 114
closure response In the LOF mutant bon1-1 in Col-0 background a plant immune receptor NLR 115
gene SNC1 (suppressor of NPR1 constitutive 1) is increased at transcript level through initial 116
upregulation followed by SA-mediated amplification (Yang and Hua 2004 Li et al 2007) The 117
bon1-1 mutant thus has constitutive defense (autoimmune) responses and is consequently dwarf 118
However these defects observed is dependent on a Col-0 specific NB-LRR gene SNC1 and the 119
LOF bon1-2 allele in the Wassilewakija (Ws) accession does not exhibit constitutive defense 120
activation because there is no functional SNC1 in Ws (Yang and Hua 2004) In addition to the 121
function in negatively regulating SNC1 BON1 is recently shown to promote stomatal closure in 122
response to stimuli including abscisic acid (ABA) and bacterial pathogen (Gou et al 2015) This 123
function is independent of SNC1 or NB-LRR signaling and the bon1 mutant is defective in 124
stomatal closure when autoimmunity is suppressed by the SNC1 LOF mutation snc1-11 (Gou et 125
al 2015) Therefore BON1 has a more general role in regulating signaling events in addition to 126
NLR gene expression 127
It is intriguing that an evolutionarily conserved calcium binding copine protein can regulate 128
stomatal closure and impact NLR gene expression Here we identified calcium pumps ACA10 129
and ACA8 as interacting proteins of BON1 both physically and genetically The LOF mutants of 130
these genes have altered calcium signature and calcium homeostasis and are defective in 131
stomatal closure and plant immunity Thus we have uncovered a critical role for PM-localized 132
calcium pumps ACA108 in calcium signature generation as well as their regulation by an 133
evolutionarily conserved BON1 protein in Arabidopsis 134
135
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136
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Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
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to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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17
1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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136
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Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
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to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
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43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
9
Results 137
BON1 and the calcium ATPase ACA10 interact physically 138
To further understand how BON1 functions in immunity we searched for BON1 co-139
expressed genes as they might function together with BON1 Among the top 300 genes with high 140
co-expression values in the ATTED-II database (Obayashi et al 2009) 33 genes were annotated 141
as calcium binding proteins including calcium channels pumps calmodulin and calcium 142
dependent kinases (Supplementary Table 1) Five of them encode calcium efflux pumps 143
including ACA1 ACA2ACA10 ACA11 and ACA1 Previously ACA1 was identified as a 144
putative BON1 interacting protein by BON1 immunoprecipitation followed by Mass 145
Spectrometry (Gou et al 2015) suggesting that BON1 could physically interact with ACA 146
proteins Because ACA1 2 and 11 are localized to plastid envelope ER and vacuole 147
respectively (Huang et al 1993 Harper et al 1998 Lee et al 2007) we chose the PM-148
localized ACA10 for further analysis because BON1 is PM localized This is also due to the fact 149
that the loss of function (LOF) mutant of ACA10 in the Nossen-0 (No-0) accession had a 150
phenotype reminiscent to that of bon1 (see below section) 151
We first analyzed potential physical interaction between BON1 and ACA10 We were not 152
able to clone the full length of ACA10 cDNA without mutations in E coli which was also 153
reported for the full-length cDNAs of ACA9 the close homologue of ACA10 (Schiott et al 154
2004) We therefore used a cDNAgenomic DNA chimera (where the genomic fragment is used 155
after the 22nd exon) for ACA10 for expression analysis When expressed in N benthamiana the 156
ACA10-GFP fusion protein showed signals only on the PM (Supplementary Fig 1) confirming 157
that ACA10 is indeed a PM-localized protein (Bonza et al 2000 Schiott et al 2004) 158
Bimolecular Fluorescence Complementation (BiFC) (Bracha-Drori et al 2004) assays were used 159
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
10
to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
11
were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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12
ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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13
due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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14
genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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17
1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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10
to determine the interaction of BON1 and ACA10 in N benthamiana Strong fluorescence 160
signals were observed when both the ACA10 fusion with N-terminal of YFP and the BON1 161
fusion with C-terminal of YFP were expressed (Fig 1A supplemental Fig 2) and the signals 162
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11
were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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13
due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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11
were on the PM where both BON1 and ACA10 are localized (Supplemental Fig 1A) The 163
positive BiFC signal of ACA10 with BON1 is not a non-specific interaction of BON1 with any 164
PM protein as another PM protein OPT3 (oligopeptide transporter 3) did not give a positive 165
signal when co-expressed with BON1 (Fig 1A and supplemental Fig 2) 166
We subsequently used BiFC assay to test which segment of ACA10 (Supplemental Fig 1B) 167
interacts with BON1 and found that BON1 interacts with the N-terminal segment I that includes 168
the autoinhibited domain of the ACA10 (Fig 1B) Four other segments (II to IV) were also 169
analyzed but no positive signals were observed (Fig 1B) Further test will determine their 170
expression levels in order to determine whether or not these segments interact with BON1 in 171
addition to segment I The interaction between BON1 and the segment I of ACA10 was analyzed 172
further by splitndashLUC (luciferase) assay (Fig 1C) Co-expression of the fusion of BON1 and the 173
N-terminal LUC (BON1-NLUC) with the fusion of C-terminal LUC and segment I of ACA10 174
(CLUC-ACA10I) in N benthamiana gave a strong luminescence signal from the LUC activity 175
while the controls without co-expressing the two proteins showed no luminescence (Fig 1C) 176
This interaction was further verified by the yeast two hybrid (Y2H) assay where the C-terminal A 177
domain of BON1 (BON1-A) was found to interact with the segment I of ACA10 (Fig 1D) The 178
full-length BON1 did not exhibit interaction with segment I (Fig 1D) likely because the N-179
terminal part of BON1 targets to the PM and inhibits the fusion protein to go to the nucleus to 180
activate gene expression Because the segment I of ACA10 contains the putative autoinhibitory 181
motif that confers auto-inhibition of many ACA proteins (Bonza et al 2000 Curran et al 2000 182
Geisler et al 2000) BON1 may regulate ACA10 activity by binding to this motif or its nearby 183
sequences 184
185
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ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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12
ACA10 mutant in the Nossen-0 (No-0) background has an autoimmune phenotype 186
We found that ACA10 like BON1 is a negative regulator of plant immunity An aca10 187
mutant in No-0 accession named cif1-1 was reported to have a compact inflorescence (George 188
et al 2008) This aca10 mutant allele will be referred as aca10-cif1 to be consistent with other 189
mutant names We suspected that the growth phenotype of aca10-cif1 is at least partially due to 190
constitutive activation of immune responses induced by plant immune receptor NB-LRR genes 191
based on the following observations Under long day growth condition the young leaves of 192
aca10-cif1 display water soaked phenotype that is similar to the autoimmune mutant bon1-1 193
(Fig 2A Yang and Hua 2014) In addition the compact inflorescence phenotype of aca10-cif1 is 194
reminiscent of that of bon1 bon2 bon3 pad4 mutant (Yang et al 2006) Phenotype of water-195
soaked leaf was suppressed by a higher growth temperature of 28degC (Fig 2A) reminiscent of the 196
suppression of NB-LRR mediated plant defense responses by an elevated temperature (Wang and 197
Hua 2009) Furthermore the aca10-cif1 phenotype is only present in No-0 but not in Col-0 or 198
Ws which was due to an accession difference at the CIF2 locus (George et al 2008) The CIF2 199
gene is not yet cloned and it is likely to be a NB-LRR gene as the accession specificity is often 200
the property of such genes (Noel et al 1999 Clark et al 2007) 201
We therefore assayed the growth of virulent pathogen Pseudomonas syringe pv tomato (Pst) 202
DC3000 in aca10-cif1 The bacterial growth was reduced by 10 times in the aca10-cif1 mutant 203
compared to that in the wild-type No-0 plants at 22degC but not 28degC (Fig 2B) This enhanced 204
resistance likely results from a constitutive upregulation of defense responses at 22degC The 205
defense marker gene PR1 which is induced by pathogen in the wild type is upregulated in 206
aca10-cif1 in the absence of pathogen inoculation and this upregulation of PR1 expression was 207
observed at 22degC but not at 28degC (Fig 2C) To further test the hypothesis that growth defect is 208
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13
due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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17
1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
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40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
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43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllen GJ Chu SP Schumacher K Shimazaki CT Vafeados D Kemper A Hawke SD Tallman G Tsien RY Harper JF Chory JSchroeder JI (2000) Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutantScience 289 2338-2342
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Balagueacute C Lin B Alcon C Flottes G Malmstroumlm S Koumlhler C Neuhaus G Pelletier G Gaymard F Roby D (2003) HLM1 anessential signaling component in the hypersensitive response is a member of the cyclic nucleotide-gated channel ion channelfamily Plant Cell 15 365-379
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Bonza MC Morandini P Luoni L Geisler M Palmgren MG De Michelis MI (2000) At-ACA8 encodes a plasma membrane-localizedcalcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus Plant Physiol 123 1495-1506
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13
due to activation of NB-LRR mediated defense we introduced in aca10-cif1 mutant the LOF 209
mutation in the PAD4 gene that plays an important role in such a defense (Wiermer et al 2005) 210
The pad4-1 mutant in Col-0 background was crossed to aca10-cif1 in No-0 and F2 plants were 211
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14
genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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17
1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
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14
genotyped at the ACA10 CIF2 and PAD4 loci All plants with genotypes of aca10-cif1 cif2-n 212
(No-0 allele of CIF2) exhibited water-soaked leaf phenotype while all aca10-cif1 cif2-n pad4 213
plants exhibited a wild-type phenotype (Fig 2D) The suppression of cif1 mutant phenotype by 214
pad4 is further confirmed by analyzing progenies from F2 plants with aca10-cif1 cif2-n pad4+ 215
genotype In addition to the growth phenotype pad4 mutation also reduced PR1 expression in 216
aca10-cif1 (Fig 2E) Taken together ACA10 is a negative regulator of disease resistance in a 217
PAD4-dependent manner in the No-0 accession and constitutive immune responses result in 218
growth retardation in aca10-cif1 mutant 219
220
ACA10 and ACA8 are negative regulators of plant immune responses 221
Earlier study reported that ACA8 the closest homolog of ACA10 interacts with a PAMP 222
receptor FLS2 and is a positive regulator of plant immune response (Frei dit Frey et al 2012) It 223
was also reported that using surface inoculation method in which bacteria enter the plants 224
through stomata the aca8 aca10 and aca10aca8 mutants in the Col-0 background were more 225
susceptible to virulent pathogen Pst DC3000 compared to the wild type Col-0 (Frei dit Frey et 226
al 2012) However our characterization of the aca10-cif1 mutant as well as the genetic and 227
physical interaction of ACA10 and BON1 indicate that ACA10 is a negative regulator of immune 228
responses To investigate this discrepancy we analyzed the disease phenotype of aca10 and aca8 229
mutants in the Col-0 background The single LOF mutants of aca10-2 or aca8-2 in Col-0 did not 230
exhibit obvious growth defects in early developmental stage but the double mutant had water-231
soaked leaves and exhibited dwarfism (Fig 3A B) When analyzed for resistance to the virulent 232
pathogen Pst DC3000 the aca10-2 and the aca10-2aca8-2 mutants consistently exhibited 233
enhanced resistance compared to the wild-type Col-0 (Fig 3C D E) Using syringe infiltration 234
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
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15
and dipping methods aca10-2 but not aca8-2 had reduced pathogen growth while aca10 aca8 235
mutant had a further reduction of pathogen growth compared to the wild-type Col-0 (Fig 3C D) 236
We subsequently used the same spray method as described in the prior study (Frei dit Frey et al 237
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
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40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
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43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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16
2012) for inoculation and found a smaller but significant reduction of pathogen growth in the 238
aca10-2 and aca10-2aca8-2 mutants (Fig 3E) We could not explain the discrepancy between 239
our result and the prior result by different inoculation methods or plant growth conditions 240
although we cannot exclude the possibility that a difference in an undefined plant growth 241
condition or pathogen preparation caused a difference in disease resistance We conclude that 242
ACA10 and ACA8 are negative regulators of defense responses to bacterial pathogen Pst DC3000 243
from our analysis of their mutants This is supported by the upregulation of a defense response 244
marker gene PR1 in the aca10 and aca10aca8 mutants compared to the wild-type Col-0 (Fig 245
3F) 246
247
Mutants of ACA10 and BON1 have synergistic interactions 248
The physical interaction of BON1 and ACA10 proteins as well as a similar phenotype in 249
immunity indicates that they might function together To further test this hypothesis we 250
constructed double mutant of aca10 and bon1 in the Ws background as neither of the LOF 251
mutants aca10-1 nor bon1-2 in the Ws background exhibited visible developmental defects (Fig 252
4A and Supplemental Fig 3A) The double mutant had smaller curled and water soaked leaves 253
and produced a more compact inflorescence compared to the wild type or the single mutants at 254
22 but not 28degC (Fig 4A and Supplemental Fig 3A-D) Pathogen growth assay demonstrated 255
that the disease resistance was increased in the aca10-1bon1-2 double mutant compared to wild-256
type and single mutant under 22 but not 28degC (Fig 4b) The aca10-1bon1-2 double mutant had a 257
significant increase of PR1 expression compared to wild-type and the single mutants at 22 but 258
not 28degC (Fig 4C) All the defects in the double mutant were suppressed by either pad4 or eds1 259
The two triple mutants aca10-1 bon1-2 eds1-1 and aca10-1 bon1-2 pad4-5 in contrast to aca10-260
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
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43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Balagueacute C Lin B Alcon C Flottes G Malmstroumlm S Koumlhler C Neuhaus G Pelletier G Gaymard F Roby D (2003) HLM1 anessential signaling component in the hypersensitive response is a member of the cyclic nucleotide-gated channel ion channelfamily Plant Cell 15 365-379
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Bonza MC Morandini P Luoni L Geisler M Palmgren MG De Michelis MI (2000) At-ACA8 encodes a plasma membrane-localizedcalcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus Plant Physiol 123 1495-1506
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Boudsocq M Sheen J (2013) CDPKs in immune and stress signaling Trends Plant Sci 18 30-40Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Willmann MR McCormack M Lee H Shan L He P Bush J Cheng SH Sheen J (2010) Differential innate immunesignalling via Ca2+ sensor protein kinases Nature 464 418-422
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1 bon1-2 had a wild-type leaf and inflorescence morphology (Fig 4D and Supplemental Fig 261
3E) These triple mutants also had the wild-type level of disease resistance and PR1 upregulation 262
(Fig 4E F) Thus the aca10-1 and bon1-2 has synergistic effect on immune responses in the Ws 263
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background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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18
background We hypothesize that NLR genes other than SNC1 are activated in the bon1-2 aca10-264
1 mutant in the Ws background similar to SNC1 activation in the bon1-1 mutant in the Col-0 265
background 266
267
Overexpression of BON1 suppressed the aca10-cif1 but not the aca10aca8 defects 268
The synergistic genetic interaction between BON1 and ACA10 single mutants is not 269
apparently consistent with the hypothesis that BON1 and ACA10 function in the same protein 270
complex andor regulate each other in a signaling pathway However this can be explained if 271
members of the BON1 or the ACA10 family each have similar functions In this case over-272
expression of one family member of the upstream component might be able to compensate for 273
the loss of one but not all family members of the downstream component To test this hypothesis 274
we overexpressed BON1 in aca10-cif1 mutant More than 20 transgenic lines were generated 275
and majority of the lines showed a rescued phenotype with wild-type leaf and inflorescent 276
morphology (Fig 5A and Supplemental Fig 4A) In addition bacterial growth was increased and 277
the PR1 expression was reduced in BON1-OEaca10-cif1 compared to aca10-cif1 (Fig 5B C) 278
In contrast when we overexpressed BON1 in the double mutant aca10aca8 none of the over 279
10 transgenic lines of BON1-OEaca10aca8 exhibited any difference from the aca10aca8 mutant 280
(Fig 5D) To exclude the possibility that the non-rescue was due to lower expression of BON1 in 281
aca10aca8 than in aca10-cif we analyzed the BON1 protein level by protein blot BON1 protein 282
level was in general lower in BON1-OEaca10aca8 than in BON1-OEaca10-cif1 however one 283
BON1-OEaca10-cif1 line exhibiting a rescued phenotype had a lower expression than several 284
BON1-OEaca10aca8 lines with a non-rescued phenotype (Supplemental Fig 4B) To confirm 285
the non-rescue phenotype in aca10aca8 we crossed a high BON1-expression line in aca10-cif1 286
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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19
(in No-0) with aca10aca8 (in Col-0) and analyzed the F2 progenies The aca10aca8 plants (now 287
in mixed No-0 and Col-0 background) exhibited a similar growth defect irrespective of the 288
presence of the BON1-OE transgene The non-rescuing of aca10aca8 defects by BON1-OE was 289
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
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Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
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DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
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20
further confirmed in the F3 progenies of several lines of BON1-OEaca10aca8 in mixed 290
background (Fig 5E) Therefore BON1-OE rescues the defects of aca10 single mutant but not 291
the aca10 aca8 double mutant which suggests that BON1 functions upstream of both ACA10 292
and ACA8 293
294
ACA8 and BON1 have physical interaction 295
We subsequently tested the hypothesis that BON1 physically interacts with ACA8 as well 296
as ACA10 In the BiFC assay in N benthamiana co-expression of the full length ACA8 and 297
BON1 exhibited a strong fluorescence signal while co-expression of BON1 and a control PM 298
protein did not (Fig 6A supplemental Fig 2) The interaction between ACA8 and BON1 was 299
verified by a positive signal when the N-terminal segment I of ACA8 and BON1 were co-300
expressed in the split-LUC assay (Fig 6B) Therefore both ACA8 and ACA10 interact with 301
BON1 and the BON1-interacting domains in ACA8 and ACA10 are localized in the same region 302
of the protein 303
304
ACA10 ACA8 and BON1 affect calcium homeostasis and calcium signals 305
Because ACA10 and ACA8 are calcium pumps we hypothesize that the loss of the pump 306
function or its regulation will alter calcium homeostasis To test this hypothesis we monitored 307
calcium homeostasis and signature in plant cells using a FRET reporter Yellow Cameleon (YC) 308
A plant version of this calcium sensor YC36 under the control of the constitutive 35S promoter 309
(Yang et al 2008) was introduced into aca10-2 aca8-2 and bon1-1 mutants Steady levels of 310
calcium were measured in guard cells where YC36 has a strong expression Calcium 311
concentration was measured higher in the aca10 mutants compared to the wild type in both the 312
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
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22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
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40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
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43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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21
Ws and No-0 backgrounds (Fig 7A) which is consistent with earlier report based on another 313
calcium reporter (Frei dit Frey et al 2012) Interestingly the bon1 mutants also exhibited an 314
increase of calcium at steady status in both Ws and Col-0 backgrounds (Fig 7A) 315
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
22
Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
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40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
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43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Furthermore cytosolic calcium signals generated in response to imposed calcium were 316
altered in the aca10 and bon1 mutants In wild-type Col-0 an external application of 10 mM 317
calcium rapidly induced cytosolic calcium oscillation in guard cells (Fig 7B) as reported earlier 318
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(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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23
(Allen et al 2000 Hubbard et al 2012) In aca10-2 aca8-2 and bon1-1 LOF mutants only one 319
calcium spike was observed and no more calcium peaks followed (Fig 7B) Moreover the 320
decreasing of calcium level from the first spike in the three mutants was drastically delayed 321
compared to the wild-type Col-0 (Fig 7B) Therefore the initial influx of calcium ions happened 322
normally but the calcium oscillation pattern was lost This observation indicates an essential role 323
of BON1 ACA10 and ACA8 in cytosolic calcium oscillation and supports the hypothesis that 324
BON1 functions closely with ACA10 and ACA8 in regulating calcium signature 325
326
ACA10 ACA8 and BON1 regulate stomatal movement 327
Calcium signaling is critical in controlling stomatal movement (Kim et al 2010) The altered 328
calcium signature in bon1 aca10 and aca8 guard cells suggests that these mutants might have 329
defects in stomata closure in response to environmental stimuli Indeed the bon1 mutant does 330
not close stomata in response to calcium ABA or pathogen and this defect is not a consequence 331
of autoimmunity as it is independent of SNC1 or PAD4 (Gou et al 2015) (Fig 8A) None of the 332
aca10 aca8 and aca10aca8 mutants in No-0 Ws or Col-0 closed their stomata in response to 10 333
mM calcium either (Fig 8A) Furthermore ACA10 and ACA8 similar to BON1 functions in 334
pathogen induced stomata closure control as well When applied with a coronatine-deficient 335
(CORndash) Pst DC3000 strain which does not induce stomata reopening (Melotto et al 2006) none 336
of the aca10 and aca8 single or double mutants closed their stomata in contrast to the wild type 337
(Fig 8B) We further examined the stomatal movement over a time course in response to Pst 338
DC3000 CORndash as well as Pst DC3000 which causes reclose of stomata in the wild type Col-0 As 339
expected the wild type Col-0 opened stomata at 15 hours and 3 hours in response to both Pst 340
DC3000 CORndash and Pst DC3000 and closeed its stomata at 45 hours when applied with Pst 341
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24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
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25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
24
DC3000 but not Pst DC3000 CORndash (Fig 8C) In contrast the aca8 aca10 mutant kept its stomata 342
closed at 15 3 and 45 hours when applied with either of the pathogen strains except for a slight 343
opening at 3 hours when applied with Pst DC3000 (Fig 8C) This further supports that the 344
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
25
aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
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aca10 aca8 double mutant is insensitive or less sensitive to pathogens in stomatal closure 345
compared to the wild type Therefore the BON1 and ACA108 regulate stomata closure and 346
plant immune responses in a similar fashion and they might do so through modulating calcium 347
signatures 348
349
350
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Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
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31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
26
Discussion 351
Calcium signaling is universal and complex Despite rapid progress in deciphering calcium 352
signaling and network in various developmental and environmental responses it remains largely 353
unknown in many processes what calcium transporters are used to generate calcium signals and 354
how these transporters are regulated Here we identified Ca2+-ATPases ACA10 and ACA8 and 355
calcium-binding protein BON1 as important regulators of calcium signature (Fig 9) They are 356
essential to generate calcium oscillation in response to externally applied calcium and without 357
their functions only one single calcium spike is produced instead of multiple repeated spikes 358
This could be due to a failure to produce additional spikes or more likely to terminate the first 359
spike due to the lack of ACA108 function Prior studies also found an effect of ACAs on 360
calcium signatures For instances mutations of tonoplast-localized PCA in moss and ER-361
localized NbCA1 in tobacco caused elevated calcium concentration and increased the amplitude 362
and duration of calcium spikes respectively (Qudeimat et al 2008 Zhu et al 2010) Ca2+-363
ATPases are high affinity but low-capacity transporters whereas Ca2+-exchangers are low affinity 364
high-capacity transporters Based on their biochemistry characteristics it is hypothesized that 365
Ca2+-ATPase is the primarily component that terminates Ca2+ signaling whereas Ca2+-exchanger 366
is the primarily factor that removes Ca2+ following cytosolic Ca2+ elevation (Sze et al 2000) 367
The finding here that the loss of ACA108 function compromises cytosolic calcium oscillation 368
not only establishes a critical role of PM-localized ACAs in calcium signals but also indicates a 369
broader role of ACAs in calcium signature generation than previously thought 370
Environmental signals such as PAMPs trigger opening of calcium permeable channels for 371
calcium influx leading to the cytosolic calcium spike and sometimes multiple spikes forming an 372
oscillation (Thor and Peiter 2014) Our study indicates that calcium pumps ACA10 and ACA8 373
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
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28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
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39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
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40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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27
are needed for the oscillation but not the first spike It is likely that calcium level in cytosol are 374
reset after the first induction before subsequent spikes can be generated and calcium pumps are 375
needed for this resetting BON1 whose activity is promoted by calcium binding (Li et al 2010) 376
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
28
potentially modulates calcium oscillation through its binding to and activating ACA10 and 377
ACA8 ACA10 and ACA8 both have an auto-inhibitory domain that overlaps with the 378
calmodulin binding motif and calmodulin binding is thought to activate ACAs (Tidow et al 379
2012) The binding of BON1 to this auto-inhibitory domain of ACA108 may alter the protein 380
conformation of ACA108 or facilitate calmodulin binding of ACA108 leading to the release of 381
auto-inhibition of ACA108 (Fig 9) Because BON1 and calmodulin are both calcium binding 382
proteins the ACA108 pump activity could be controlled by calcium itself thus forming a 383
feedback regulation Future analysis of the interaction among ACA108 BON1 and calmodulin 384
will reveal further the regulatory mechanisms of calcium efflux conferred by ACA108 385
Calcium oscillation is generated in response to a number of stimuli in plants including NOD 386
factor in a variety of legume species (Granqvist et al 2015) In legume calcium oscillation 387
occurs in nucleus in response to NOD factor and an ER- or nuclear envelope- localized calcium 388
ATPase was found to be essential to generate nuclear calcium oscillation (Capoen et al 2011) 389
The finding of a role of PM-localized calcium ATPases in calcium oscillation in cytosol indicate 390
that calcium ATPases have general roles in calcium signature generation in various cellular 391
compartments Further study should determine whether or not ACA108 and BON1 affect 392
calcium signatures in response to other stimuli as well The role of BON1 in calcium signature 393
generation also has implications for the copine protein family where BON1 is a member Some 394
copines in animals were identified as genetic modifiers of channel or receptor mutants (Church 395
and Lambie 2003 Gottschalk et al 2005) and they have also been implicated in receptor 396
mediated signaling from cell culture analysis (Tomsig et al 2004 Heinrich et al 2010) It 397
would be interesting to test if copines have a regulatory role of calcium signals involved in those 398
processes 399
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
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33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
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35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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29
This study also finds opposing functions of ACA10 and ACA8 in two layers of plant 400
immunity similarly to BON1 On one hand the aca10 and aca8 mutants are insensitive to 401
calcium and pathogen in stomatal closure response similarly to the bon1 mutants This feature 402
presumably makes these mutant plants more susceptible to pathogen at the invasion stage An 403
earlier report did find a more susceptible phenotype for the aca8 and aca10 mutants (Frei dit 404
Frey et al 2012) However we found in this study that the aca10 and aca10 aca8 mutants have 405
enhanced resistance when bacterial propagation in plants is assayed by either infiltration or 406
surface inoculation methods This discrepancy might have resulted from differences in 407
unidentified plant or pathogen growth conditions because resistance phenotype was observed 408
even when we replicated the experiment reported in Frei dit Frey et al 2012 Nevertheless the 409
enhanced resistance we observed is temperature- and PAD4-dependent indicating of activation 410
of plant immune receptor NLR genes reminiscent of the activation of NLR genes in the bon1 411
mutant Apparently this layer of regulation over-rides the stomatal layer of immunity regulation 412
as both bon1 and aca10 mutants have enhanced resistance measured by pathogen growth 413
How do BON1 and ACA108 regulate two layers of plant immunity Conceivably their 414
positive role in stomatal closure control is directly tied with their roles in calcium signature 415
generating and this might represent a more direct role of these proteins Their roles in controlling 416
pathogen growth likely results from their negative regulation of immune receptor gene 417
expression which could be associated with calcium homeostasis Altered steady state of calcium 418
might mimic signals from sustained pathogen invasion and therefore upregulate or activate NLR 419
genes Indeed disruption of calcium channels and pumps have been shown to affect plant 420
immunity (Clough et al 2000 Zhu et al 2010) For instances two vacuole-localized ACA 421
members ACA4 and ACA11 repress cell death and innate immunity in Arabidopsis (Boursiac et 422
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
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32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202
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DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
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30
al 2010) Although these molecules are located in different parts of the cell including plasma 423
membrane ER or vacuole they could all regulate calcium levels in cytosol and perhaps nucleus 424
as well 425
Although both bon1 and aca10aca8 have autoimmunity their growth phenotypes are not 426
identical This might be due to specific functions these proteins have in addition to their 427
overlapping roles It could also be due to overlapping functions among the BON1 family 428
members or the ACA10 family members BON1 has two other homologs BON2 and BON3 in 429
Arabidopsis while ACA10 has ACA8 and ACA9 as close homologs as well as ACA12 and 430
ACA13 as homologs It is likely that BON1 family members interact with ACA10 family 431
members each pair with a different regulatory strength Therefore a partial loss of the family 432
activity could result in different statuses of calcium homeostasis This variation in calcium steady 433
level might be associated with activation of different NLR genes Identifying such NLR genes 434
activated in various mutants of calcium channels and pumps will lead to a further understanding 435
of the mode of action of calcium signatures as well as their roles in plant immunity 436
437
Materials and Methods 438
Arabidopsis Mutants and Plant Growth 439
The seeds of aca10-cif1 and aca10-1 were kindly provided by R Sharrock (George et al 440
2008) The aca10-2 (GK-044H01) and aca8-2 (GK-688H09) lines were obtained from the 441
Arabidopsis Stock Centre (httparabidopsisinfo) For growth phenotyping the Arabidopsis 442
plants were grown under constant light condition with 100 mmol mndash2 sndash1 and relative humidity at 443
50 to 70 For pathogen growth assays plants were grown under 12 or 16 hour light 444
condition N benthamiana plants were grown in the greenhouse at 24degC for 4 to 6 weeks before 445
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
31
use for transient expression studies 446
447
Protein Subcellular Localization Assay 448
An ACA10 genomiccDNA hybrid was constructed and cloned into the Gateway entry 449
vector pCR8 TOPO TA vector (Invitrogen) For localization of ACA10 protein the ACA10 gene 450
was transferred from the entry vector to the Hpt-psatn1-ACA10 (GW) vector and transformed 451
into the Agrobacterium tumefaciens (A tumefaciens) strain GV3101 for infiltration into the 452
abaxial surface side of 4- to 6-week-old N benthamiana plant leaves as previously described 453
(Gou et al 2015) Fluorescence of the epidermal cell layer of the lower leaf surface was 454
examined at 2 to 4 days post inoculation (DPI) Images were captured by a Leica TCS SP2 455
Confocal Microscope with excitation wavelength at 488 and 496 nm and emission wavelength 456
between 520 and 535 nm for GFP signals 457
458
BiFC Assay 459
The full-length complementary DNA (cDNA) fragments (without stop codon) of BON1 460
ACA8 OPT3 (Zhai et al 2014) and ACA10 (genomiccDNA hybrid) were amplified using 461
primers in Supplemental Table S2 and cloned into the Gateway entry vectors pCR8 TOPO TA or 462
pENTRD TOPO (Invitrogen) For BiFC experiments BON1 was cloned into pSPYCE-35S GW 463
(Schutze et al 2009) using LR clonase (Invitrogen) to generate BON1cYFP constructs while 464
ACA8 ACA10 and OPT3 were cloned similarly into pSPYNE-35S GW to generate 465
corresponding nYFP constructs respectively A previously described protocol (Schutze et al 466
2009) was followed to observe BiFC signals with minor modification The constructs were 467
transformed into the A tumefaciens strain GV3101 Overnight cell cultures were collected and 468
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
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Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
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Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
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Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472
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Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094
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Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349
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Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387
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Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071
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Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
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Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6
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Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
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Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
32
resuspended in 1 mL of AS medium (10 mM MES-KOH pH56 10 mM MgCl2 and 150 microM 469
acetosyringone) to optical density at 600 nm (OD600) to 08 The working suspensions were 470
prepared by mixing appropriate clones containing the BiFC constructs and the gene-silencing 471
inhibitor pBA-HcPro plasmid (Menke et al 2005) at 111 ratio and let them stand for 2 to 4 472
hours The A tumefaciens suspensions were then co-infiltrated into the abaxial surface of 4-473
week-old N benthamiana plant leaves Fluorescence of the epidermal cell layer of the lower leaf 474
surface was examined at 3 to 4 DPI Images were captured by a Leica SP5 confocal microscope 475
with excitation wavelength at 514 nm and emission wavelength between 500 and 550 nm for 476
YFP signals 477
478
Split-Luc Assay 479
The full length cDNA of BON1 was amplified using the oligos listed in Supplemental Table 480
2 The PCR fragment of BON1 was ligated into the pCAMBIA-NLuc vector (Chen et al 2008) 481
digested by BamH1 and SalI using the ClonExpressTM MultiS One Step Cloning Kit (Vazyme) to 482
generate BON1-NLuc The N-terminal parts of ACA8 and ACA10 were each amplified using the 483
oligos listed in Supplemental Table 2 The PCR fragments of ACA8 and ACA10 were ligated 484
into the pCAMBIA-CLuc vector (Chen et al 2008) digested by Kpn1 and SalI using a similar 485
strategy as above to generate ACA8I-CLuc and ACA10I-CLuc These constructs were 486
transformed into A tumefaciens strain GV3101 487
A tumefaciens GV3101 strains containing recombinant constructs were grown in liquid 488
Luria-Bertani medium with rifampicin and kanamycin for 2 days pelleted and resuspended in 489
Murashige and Skoog with 10 mM MES medium containing 200 μM acetosyringone to a final 490
concentration of OD600=06 After 2 hoursrsquo induction the bacterial suspensions were infiltrated 491
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
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36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
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38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
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Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
33
into young expanded leaves of N benthamiana plants with a needleless syringe After infiltration 492
the plants were covered with dark bag at 23degC for 48 hours The plants were then kept under 493
light for 16 hours sprayed with 1 mM luciferin in 001 Triton X100 solution and kept in dark 494
for 5 min to quench the fluorescence A deep cooling CCD imaging apparatus (DU934P-BV 495
Andor) was used to capture the fluorescence image The camera was cooled to -80degC and all 496
images were taken after 3 min exposure 497
498
Pathogen Growth Assay 499
Pst DC3000 grown on plates with Kingrsquos B medium were washed collected and diluted 500
with 10 mM MgCl2 and 002 Silwet L-77 Syringe infiltration was used for inoculation unless 501
stated otherwise where dipping or spray methods were used For syringe infiltration bacteria 502
were diluted to OD600 of 0002 and syringe-infiltrated on leaves of 3 to 4 week old plants Six 503
inoculated leaf were collected as one sample weighed ground in 1 ml of 10 mM MgCl2 and 504
shaken at room temperature for 1 hour Dipping inoculation was performed as previously 505
described (Gou et al 2015) and bacteria were diluted to OD600=005 for dipping seedlings of 2 506
weeks old Three whole seedlings were collected as one sample Spray inoculation was 507
performed as described previously (Frei dit Frey et al 2012) and the bacteria were diluted to 508
OD600=02 for spray 509
To determine bacterial growth (for all inoculation methods) serial dilutions of the ground 510
tissue solution were spotted on KB medium and the number of cfu (colony forming unit) per 511
fresh weight was calculated Three to four samples were analyzed for each genotype and time 512
point 513
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
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34
514
Yeast Two Hybrid Assay 515
The yeast (Saccharomyces cerevisiae) two-hybrid constructs were made in the pDEST-516
GBKT7 and pDEST-GADT7 Gateway vectors (Rossignol et al 2007) The DNA fragment of 517
the N-terminal segment I of ACA10 was cloned into the pCR8 TOPO TA vector and transferred 518
to the pDEST-GBKT7 vector to obtain the BDACA10-I construct The ADBON1 ADBON1-519
A and BD-BAP1 constructs were previously described (Hua et al 2001) The yeast two-hybrid 520
assay was performed as previously described (Hua et al 2001 Li et al 2010) 521
522
Northern Blotting and Real-time RT-PCR 523
Total RNA was extracted from 3-week old seedling using Trizol reagent (Invitrogen) as 524
instructed RNA of 20 μg was resolved in a 12 gel containing formaldehyde RNA blots were 525
hybridized with gene specific and 32P labeled single strand DNA probe 526
For Real-time PCR SuperScript II Reverse Transcriptase (Invitrogen) was used to 527
synthesize cDNA from isolated RNA Real time RT-PCR was performed using PR1 primers 528
listed in Supplemental Table S2 The ACTIN2 gene was used as internal controls Advanced 529
Universal SYBR Green Supermix (Bio-Rad) was used for Real time RT-PCR 530
531
Western Blotting 532
The vector of 35SBON1-HA used to overexpression BON1 was reported previously (Gou 533
et al 2015) Arabidopsis leaves grown in soil under constant light for 3 weeks were used for 534
protein extraction and western blotting with anti-HA antibody (Sigma Aldrich Catalog H3663) 535
following a previously described method (Gou et al 2015) 536
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllen GJ Chu SP Schumacher K Shimazaki CT Vafeados D Kemper A Hawke SD Tallman G Tsien RY Harper JF Chory JSchroeder JI (2000) Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutantScience 289 2338-2342
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Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574
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Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342
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Clough SJ Fengler KA Yu IC Lippok B Smith RK Jr Bent AF (2000) The Arabidopsis dnd1 defense no death gene encodes amutated cyclic nucleotide-gated ion channel Proc Natl Acad Sci U S A 97 9323-9328
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Du L Ali GS Simons KA Hou J Yang T Reddy AS Poovaiah BW (2009) Ca2+calmodulin regulates salicylic-acid-mediated plantimmunity Nature 457 1154-1158
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Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37
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Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127
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George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728
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Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
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Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
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Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
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Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
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Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
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Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
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DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
35
537
Stomatal Closure Assay 538
Stomatal aperture assay was performed as described (Nomura et al 2012) Young rosette 539
leaves from 17- to 25-day-old Arabidopsis plants were detached floated on the stomatal opening 540
buffer containing 10 mM Tris-Mes (pH 615) 5 mM KCl and 50 μM CaCl2 and incubated for 541
25 hours under white light (200-250μmol m-2 s-1) at 22degC Each leaf was then clung onto a cover 542
slip with a medical adhesive (Hollister Libertyville USA) and their mesophyll cells were 543
removed by a razor blade The epidermal strips were then transferred into the stomatal closing 544
buffer containing 10 mM Tris-Mes (pH 615) 5mM KCl and 10mM CaCl2 and incubated for 545
another 2 hours under white light Then the epidermal strips were observed under an inverted 546
microscopy (Model D1 Carl Zeiss) before and after the closing buffer treatment The stomatal 547
aperture was calculated as the ratio of the inner widthouter length of each pair of stomata For 548
each sample more than 50 guard cells were calculated and the experiments were repeated for 4 549
times 550
551
Calcium Level Measurement 552
For calcium oscillation experiment the epidermal strips after the light treatment was 553
acquired as described above Cover slips were then placed in a perfusion chamber which was fit 554
to an inverted microscopy (Model D1 Carl Zeiss Germany) equipped with an emission filter 555
wheel (Lambda XL Novato USA) and a CCD camera (AndorTM Technology Belfast Northern 556
Ireland) Imaging calcium in the guard cells was conducted by monitoring the ratio 557
(535nm442nm) of YC36 using the MetaFluor fluorescence ratio imaging software The 558
epidermal strips were firstly measured for approximately 100 seconds and then the opening 559
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
36
buffer was changed into the closing buffer by an injector when the same epidermal strips were 560
measured for 700 seconds The interval of image acquisition was 3 seconds For each genotype 561
more than 30 guard cells were measured and the experiments were repeated for 3 times 562
Measurement of steady level of Ca2+ concentration using the YC36 system was performed 563
with ZESS LSM710 confocal laser scanning microscope following the protocol previously 564
described (Krebs et al 2012) 565
566
Acknowledgement 567
We thank Y Wang for the assistance in microscopy Y Tan for technical assistance on 568
YC36 assay R Sharrock for the seeds of cif1-1 and aca10-2 and J Schroeder for the seeds of 569
35SYC36 transgenic plant This work was supported by grants from National Science 570
Foundation of USA (IOSndash0919914 and IOS-1353738) to J Hua the National Key Research and 571
Development Program of China (2016YFD0100600) to D-L Yang Natural Science Foundation 572
of Jiangsu (BK20150659) to D-L Yang National Science Foundation of China (31330061) to B 573
Zou and Jiangsu Collaborative Innovation Center for Modern Crop Production to J Hua and D-574
L Yang We thank China Scholarship Council for fellowships to B Zou H Yu and Y Bao and 575
Shanghai Institute of Plant Physiology and Ecology for a fellowship to Z Shi 576
577
Figure legends 578
Figure 1 Protein-protein interaction between ACA10 and BON1 579
A BiFC assay of ACA10 and BON1 interaction ACA10 fused with N-terminal part of YFP 580
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
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Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342
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Frei dit Frey N Mbengue M Kwaaitaal M Nitsch L Altenbach D Haweker H Lozano-Duran R Njo MF Beeckman T Huettel BBorst JW Panstruga R Robatzek S (2012) Plasma membrane calcium ATPases are important components of receptor-mediatedsignaling in plant immune responses and development Plant Physiol 159 798-809
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37
(ACA10-NE) and BON1 fused with C-terminal part of YFP (BON1-CE) were transiently 581
expressed in Nicotiana benthamiana (N benthamiana) by Agrobacterium mediated 582
transformation A control plasma membrane protein OPT3 co-expressed with BON1 is shown at 583
the bottom panel Bar=50microm 584
B BiFC assay of interaction between BON1 and each of the five segments of ACA10 I II III 585
IV and V (supplemental Fig 1B) Bar=50microm 586
C Split-LUC assay of interaction of BON1 and the first segment of ACA10 Fusion of segment I 587
of ACA10 with C-terminus LUC (ACA10I-Cluc) was co-expressed with fusion of BON1 with 588
N-terminus LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of 589
ACA10I-Cluc with Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc 590
(lower right) were used as controls 591
D Yeast-two-hybrid assay between the first segment of ACA10 and the A domain of BON1 592
BAP1 was used as a positive control Shown is growth of serial dilutions of yeasts on synthetic 593
medium without leucine or tryptophan (SC-LT) or synthetic medium without leucine tryptophan 594
adenine or histidine (SC-LTAH) AD Activation domain of GAL4 BD DNA-binding domain 595
of GAL4 596
597
Figure 2 The loss of ACA10 in Nossen-0 (No-0) background leads to autoimmune responses 598
A Growth phenotype of aca10-cif1 and wild-type No-0 plants at 22ordmC and 28ordmC 599
B Pst DC3000 bacterial growth in aca10-cif1 and No-0 plants under 22ordmC and 28ordmC ldquordquo 600
indicates significant difference by studentrsquos t-test (P-valuelt0001) 601
C PR1 expression level in aca10-cif1 and No-0 plants at 22ordmC and 28ordmC analyzed by Northern 602
blotting 603
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Boursiac Y Lee SM Romanowsky S Blank R Sladek C Chung WS Harper JF (2010) Disruption of the vacuolar calcium-ATPasesin Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway Plant Physiol 154 1158-1171
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Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
38
D Growth phenotype of No-0 aca10-cif1 and aca10-cif1 pad4 plants grown at 22ordmC 604
E PR1 expression level in No-0 aca10-cif1 and aca10-cif1pad4 plants analyzed by real-time 605
RT-PCR assay Actin2 is used as a control gene ldquordquo indicates significant difference by studentrsquos 606
t-test (P-valuelt0001) 607
608
Figure 3 The growth and defense phenotypes of aca10 and aca8 mutant in Col-0 accession 609
background 610
A The growth phenotypes of Col-0 aca10-2 aca8-2 and aca10-2 aca8-2 at seedling stage 611
B The growth phenotypes of Col-0 aca8-2 aca10-2 and aca10-2 aca8-2 at seed setting stage 612
C-D-E Growth of virulent pathogen Pst DC3000 in the above genotypes Different inoculation 613
methods were used syringe infiltration (C) dipping (D) and spray (E) Different letters indicate 614
statistical difference (P-value lt0001 by Bonferonni test) among genotypes 615
F PR1 expression level in Col-0 aca10 aca8 and aca10aca8 plants analyzed by real-time RT-616
PCR assay Actin2 is used as a control gene 617
618
Figure 4 Genetic interaction between BON1 and ACA10 in Wassilewakija (Ws) background 619
A Growth phenotypes of wild type aca10-1 bon1-2 and aca10-1 bon1-2 in Ws background at 620
22ordmC and 28ordmC 621
B Pst DC3000 bacterial growth in above genotypes assayed by the syringae infiltration method 622
C PR1 expression level in above genotypes grown at 22degC and 28degC by RNA blotting Same 623
amount of total RNAs were loaded on gels and the blots were hybridized at the same time 624
D Growth phenotypes of aca10-1 bon1-2 mutants and their eds1-1 and pad4-5 combination 625
mutants grown at 22degC 626
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Balagueacute C Lin B Alcon C Flottes G Malmstroumlm S Koumlhler C Neuhaus G Pelletier G Gaymard F Roby D (2003) HLM1 anessential signaling component in the hypersensitive response is a member of the cyclic nucleotide-gated channel ion channelfamily Plant Cell 15 365-379
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Bonza MC Morandini P Luoni L Geisler M Palmgren MG De Michelis MI (2000) At-ACA8 encodes a plasma membrane-localizedcalcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus Plant Physiol 123 1495-1506
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Boudsocq M Sheen J (2013) CDPKs in immune and stress signaling Trends Plant Sci 18 30-40Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Willmann MR McCormack M Lee H Shan L He P Bush J Cheng SH Sheen J (2010) Differential innate immunesignalling via Ca2+ sensor protein kinases Nature 464 418-422
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Boursiac Y Harper JF (2007) The origin and function of calmodulin regulated Ca2+ pjmps in plants J Bioenerg Biomembr 39 409-414
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Boursiac Y Lee SM Romanowsky S Blank R Sladek C Chung WS Harper JF (2010) Disruption of the vacuolar calcium-ATPasesin Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway Plant Physiol 154 1158-1171
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Bracha-Drori K Shichrur K Katz A Oliva M Angelovici R Yalovsky S Ohad N (2004) Detection of protein-protein interactions inplants using bimolecular fluorescence complementation Plant Journal 40 419-427
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Capoen W Sun J Wysham D Otegui MS Venkateshwaran M Hirsch S Miwa H Downie JA Morris RJ Ane JM Oldroyd GE (2011)Nuclear membranes control symbiotic calcium signaling of legumes Proc Natl Acad Sci U S A 108 14348-14353
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Chen H Zou Y Shang Y Lin H Wang Y Cai R Tang X Zhou JM (2008) Firefly luciferase complementation imaging assay forprotein-protein interactions in plants Plant Physiol 146 368-376
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Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
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Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574
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Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342
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Clough SJ Fengler KA Yu IC Lippok B Smith RK Jr Bent AF (2000) The Arabidopsis dnd1 defense no death gene encodes amutated cyclic nucleotide-gated ion channel Proc Natl Acad Sci U S A 97 9323-9328
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Creutz CE Tomsig JL Snyder SL Gautier MC Skouri F Beisson J Cohen J (1998) The copines a novel class of C2 domain-containing calcium-dependent phospholipid-binding proteins conserved from Paramecium to humans J Biol Chem 273 1393-1402
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Curran AC Hwang I Corbin J Martinez S Rayle D Sze H Harper JF (2000) Autoinhibition of a calmodulin-dependent calciumpump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain J Biol Chem 27530301-30308
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Dodd AN Kudla J Sanders D (2010) The language of calcium signaling Annu Rev Plant Biol 61 593-620Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Du L Ali GS Simons KA Hou J Yang T Reddy AS Poovaiah BW (2009) Ca2+calmodulin regulates salicylic-acid-mediated plantimmunity Nature 457 1154-1158
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Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37
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Frei dit Frey N Mbengue M Kwaaitaal M Nitsch L Altenbach D Haweker H Lozano-Duran R Njo MF Beeckman T Huettel BBorst JW Panstruga R Robatzek S (2012) Plasma membrane calcium ATPases are important components of receptor-mediatedsignaling in plant immune responses and development Plant Physiol 159 798-809
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Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127
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Geisler M Axelsen KB Harper JF Palmgren MG (2000) Molecular aspects of higher plant P-type Ca2+-ATPases Biochim BiophysActa 1465 52-78
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George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728
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Gottschalk A Almedom RB Schedletzky T Anderson SD Yates JR 3rd Schafer WR (2005) Identification and characterization ofnovel nicotinic receptor-associated proteins in Caenorhabditis elegans Embo J 24 2566-2578
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Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
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Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106
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Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
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Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
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Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
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Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
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Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591
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Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192
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Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Lewis RS (2001) Calcium signaling mechanisms in T lymphocytes Annu Rev Immunol 19 497-521Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
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Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456
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Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
39
E Growth of Pst DC3000 in genotypes as in D assayed by the syringae infiltration method 627
F PR1 expression level in genotypes as in D assayed by real-time RT-PCR 628
Genotypes ab=aca10-1bon1-2 abeds1-1=aca10-1bon1-2eds1-1 abpad4-5=aca10-1bon1-629
2pad4-5 630
Different letters in (B) and (D) indicate statistical difference (P-value lt0001 by Bonferonni test) 631
of various genotypes 632
633
Figure 5 Overexpression of BON1 rescued defects of the aca10-cif1 mutant but not the 634
aca10aca8 mutants 635
A Growth phenotype of wild-type No-0 aca10-cif1 and BON1 overexpression in aca10-cif1 at 636
22degC 637
B Growth of Pst DC3000 in genotypes above assayed by the dipping inoculation method 638
C PR1 expression level in above genotypes by real-time RT-PCR assay Actin2 is used as a 639
control gene 640
D Growth phenotype of aca10-2 aca8-2 (in Col-0) and BON1 overexpression in aca10 aca8 at 641
22degC 642
E Growth phenotype of BON1 overexpression in aca10 aca8 double mutant in a mixed Col-0 643
and No-0 background Shown as the second and the last plant respectively are the two parental 644
lines used for the cross the BON1-OE line in aca10-cif1 and the aca10-2 aca8-2 in Col-0 The 645
two plants in the middle are F3s from the same F2 parent from the cross with one carrying the 646
BON1-OE transgene 647
Different letters in (B) and (C) indicate statistical difference (P-value lt0001 by Bonferonni test) 648
of various genotypes 649
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllen GJ Chu SP Schumacher K Shimazaki CT Vafeados D Kemper A Hawke SD Tallman G Tsien RY Harper JF Chory JSchroeder JI (2000) Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutantScience 289 2338-2342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Balagueacute C Lin B Alcon C Flottes G Malmstroumlm S Koumlhler C Neuhaus G Pelletier G Gaymard F Roby D (2003) HLM1 anessential signaling component in the hypersensitive response is a member of the cyclic nucleotide-gated channel ion channelfamily Plant Cell 15 365-379
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bonza MC Morandini P Luoni L Geisler M Palmgren MG De Michelis MI (2000) At-ACA8 encodes a plasma membrane-localizedcalcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus Plant Physiol 123 1495-1506
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Sheen J (2013) CDPKs in immune and stress signaling Trends Plant Sci 18 30-40Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Willmann MR McCormack M Lee H Shan L He P Bush J Cheng SH Sheen J (2010) Differential innate immunesignalling via Ca2+ sensor protein kinases Nature 464 418-422
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boursiac Y Harper JF (2007) The origin and function of calmodulin regulated Ca2+ pjmps in plants J Bioenerg Biomembr 39 409-414
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boursiac Y Lee SM Romanowsky S Blank R Sladek C Chung WS Harper JF (2010) Disruption of the vacuolar calcium-ATPasesin Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway Plant Physiol 154 1158-1171
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bracha-Drori K Shichrur K Katz A Oliva M Angelovici R Yalovsky S Ohad N (2004) Detection of protein-protein interactions inplants using bimolecular fluorescence complementation Plant Journal 40 419-427
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bush DS (1995) Calcium Regulation in Plant-Cells and Its Role in Signaling Annual Review of Plant Physiology and Plant MolecularBiology 46 95-122
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Capoen W Sun J Wysham D Otegui MS Venkateshwaran M Hirsch S Miwa H Downie JA Morris RJ Ane JM Oldroyd GE (2011)Nuclear membranes control symbiotic calcium signaling of legumes Proc Natl Acad Sci U S A 108 14348-14353
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen H Zou Y Shang Y Lin H Wang Y Cai R Tang X Zhou JM (2008) Firefly luciferase complementation imaging assay forprotein-protein interactions in plants Plant Physiol 146 368-376
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574
Pubmed Author and TitleCrossRef Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342
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Clough SJ Fengler KA Yu IC Lippok B Smith RK Jr Bent AF (2000) The Arabidopsis dnd1 defense no death gene encodes amutated cyclic nucleotide-gated ion channel Proc Natl Acad Sci U S A 97 9323-9328
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Creutz CE Tomsig JL Snyder SL Gautier MC Skouri F Beisson J Cohen J (1998) The copines a novel class of C2 domain-containing calcium-dependent phospholipid-binding proteins conserved from Paramecium to humans J Biol Chem 273 1393-1402
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Curran AC Hwang I Corbin J Martinez S Rayle D Sze H Harper JF (2000) Autoinhibition of a calmodulin-dependent calciumpump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain J Biol Chem 27530301-30308
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Dodd AN Kudla J Sanders D (2010) The language of calcium signaling Annu Rev Plant Biol 61 593-620Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Du L Ali GS Simons KA Hou J Yang T Reddy AS Poovaiah BW (2009) Ca2+calmodulin regulates salicylic-acid-mediated plantimmunity Nature 457 1154-1158
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Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37
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Frei dit Frey N Mbengue M Kwaaitaal M Nitsch L Altenbach D Haweker H Lozano-Duran R Njo MF Beeckman T Huettel BBorst JW Panstruga R Robatzek S (2012) Plasma membrane calcium ATPases are important components of receptor-mediatedsignaling in plant immune responses and development Plant Physiol 159 798-809
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Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127
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Geisler M Axelsen KB Harper JF Palmgren MG (2000) Molecular aspects of higher plant P-type Ca2+-ATPases Biochim BiophysActa 1465 52-78
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George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728
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Gottschalk A Almedom RB Schedletzky T Anderson SD Yates JR 3rd Schafer WR (2005) Identification and characterization ofnovel nicotinic receptor-associated proteins in Caenorhabditis elegans Embo J 24 2566-2578
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Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
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Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106
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Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
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Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
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Huang LQ Berkelman T Franklin AE Hoffman NE (1993) Characterization of a Gene Encoding a Ca2+-Atpase-Like Protein in thePlastid Envelope Proceedings of the National Academy of Sciences of the United States of America 90 10066-10070
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Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
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Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
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Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591
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Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192
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Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lee SM Kim HS Han HJ Moon BC Kim CY Harper JF Chung WS (2007) Identification of a calmodulin-regulated autoinhibitedCa2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis FEBS Lett 581 3943-3949
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Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
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Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456
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Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615
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Murata Y Mori IC Munemasa S (2015) Diverse stomatal signaling and the signal integration mechanism Annu Rev Plant Biol 66369-392
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Nomura H Komori T Uemura S Kanda Y Shimotani K Nakai K Furuichi T Takebayashi K Sugimoto T Sano S Suwastika INFukusaki E Yoshioka H Nakahira Y Shiina T (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis NatCommun 3 926
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Qudeimat E Faltusz AM Wheeler G Lang D Holtorf H Brownlee C Reski R Frank W (2008) A PIIB-type Ca2+-ATPase is essentialfor stress adaptation in Physcomitrella patens Proc Natl Acad Sci U S A 105 19555-19560
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Rossignol P Collier S Bush M Shaw P Doonan JH (2007) Arabidopsis POT1A interacts with TERT-V(I8) an N-terminal splicingvariant of telomerase J Cell Sci 120 3678-3687
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Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
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Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
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Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
40
650
Figure 6 Physical interaction of ACA8 and BON1 651
A BiFC assay of ACA8 and BON1 ACA8 fused with N-terminal part of YFP (ACA8-NE) and 652
BON1 fused with C-terminal part of YFP (BON1-CE) were transiently expressed in N 653
benthamiana by Agrobacterium-mediated transformation The plasma membrane protein OPT3 654
was used as a negative control Graphs show YFP-mediated fluorescence derived from the 655
protein-protein interaction chlorophyll autofluorescence (chlorophyll) and superimposed 656
images of chlorophyll auto-fluorescence and YFP (Merge) Bar=100 microm 657
B Split-LUC assay of BON1 with N-terminal segment I of ACA8 Fusion of segment I of ACA8 658
with C-terminus LUC (ACA8I-Cluc) was co-expressed with fusion of BON1 with N-terminus 659
LUC (BON1-Nluc) in N benthamiana (upper left panel) Co-expressions of ACA8I-Cluc with 660
Nluc (upper right) Cluc with BON1-Nluc (lower left) and Cluc with Nluc (lower right) were 661
used as controls 662
663
Figure 7 Calcium homeostasis and calcium oscillation are altered in the bon1 and aca10 664
mutants 665
A Steady state calcium levels in guard cells of No-0 aca10-cif1 Ws bon1-2 aca10-1 bon1-2 666
aca10-1 Col-0 and bon1-1 assayed by the YC36 reporter Shown are the average and standard 667
deviation of ratios of FRETCFP from at least 30 guard cells (from 5 plants 2 leavesplant and 3 668
guard cellsleaf) of each genotype ldquordquo and ldquordquo indicate significant difference between the wild 669
type and the mutant at P-valuelt0001 and P-valuelt005 respectively by Bonferroni test 670
B Calcium signals induced by exogenous application of calcium in Col-0 bon1-1 aca10-2 and 671
aca8-2 plants Shown is the representative image of calcium signals in guard cells of each 672
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllen GJ Chu SP Schumacher K Shimazaki CT Vafeados D Kemper A Hawke SD Tallman G Tsien RY Harper JF Chory JSchroeder JI (2000) Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutantScience 289 2338-2342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Balagueacute C Lin B Alcon C Flottes G Malmstroumlm S Koumlhler C Neuhaus G Pelletier G Gaymard F Roby D (2003) HLM1 anessential signaling component in the hypersensitive response is a member of the cyclic nucleotide-gated channel ion channelfamily Plant Cell 15 365-379
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bonza MC Morandini P Luoni L Geisler M Palmgren MG De Michelis MI (2000) At-ACA8 encodes a plasma membrane-localizedcalcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus Plant Physiol 123 1495-1506
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Sheen J (2013) CDPKs in immune and stress signaling Trends Plant Sci 18 30-40Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Willmann MR McCormack M Lee H Shan L He P Bush J Cheng SH Sheen J (2010) Differential innate immunesignalling via Ca2+ sensor protein kinases Nature 464 418-422
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boursiac Y Harper JF (2007) The origin and function of calmodulin regulated Ca2+ pjmps in plants J Bioenerg Biomembr 39 409-414
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boursiac Y Lee SM Romanowsky S Blank R Sladek C Chung WS Harper JF (2010) Disruption of the vacuolar calcium-ATPasesin Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway Plant Physiol 154 1158-1171
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bracha-Drori K Shichrur K Katz A Oliva M Angelovici R Yalovsky S Ohad N (2004) Detection of protein-protein interactions inplants using bimolecular fluorescence complementation Plant Journal 40 419-427
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bush DS (1995) Calcium Regulation in Plant-Cells and Its Role in Signaling Annual Review of Plant Physiology and Plant MolecularBiology 46 95-122
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Capoen W Sun J Wysham D Otegui MS Venkateshwaran M Hirsch S Miwa H Downie JA Morris RJ Ane JM Oldroyd GE (2011)Nuclear membranes control symbiotic calcium signaling of legumes Proc Natl Acad Sci U S A 108 14348-14353
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen H Zou Y Shang Y Lin H Wang Y Cai R Tang X Zhou JM (2008) Firefly luciferase complementation imaging assay forprotein-protein interactions in plants Plant Physiol 146 368-376
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574
Pubmed Author and TitleCrossRef Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Google Scholar Author Only Title Only Author and Title
Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Clough SJ Fengler KA Yu IC Lippok B Smith RK Jr Bent AF (2000) The Arabidopsis dnd1 defense no death gene encodes amutated cyclic nucleotide-gated ion channel Proc Natl Acad Sci U S A 97 9323-9328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Creutz CE Tomsig JL Snyder SL Gautier MC Skouri F Beisson J Cohen J (1998) The copines a novel class of C2 domain-containing calcium-dependent phospholipid-binding proteins conserved from Paramecium to humans J Biol Chem 273 1393-1402
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Curran AC Hwang I Corbin J Martinez S Rayle D Sze H Harper JF (2000) Autoinhibition of a calmodulin-dependent calciumpump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain J Biol Chem 27530301-30308
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dodd AN Kudla J Sanders D (2010) The language of calcium signaling Annu Rev Plant Biol 61 593-620Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Du L Ali GS Simons KA Hou J Yang T Reddy AS Poovaiah BW (2009) Ca2+calmodulin regulates salicylic-acid-mediated plantimmunity Nature 457 1154-1158
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Frei dit Frey N Mbengue M Kwaaitaal M Nitsch L Altenbach D Haweker H Lozano-Duran R Njo MF Beeckman T Huettel BBorst JW Panstruga R Robatzek S (2012) Plasma membrane calcium ATPases are important components of receptor-mediatedsignaling in plant immune responses and development Plant Physiol 159 798-809
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geisler M Axelsen KB Harper JF Palmgren MG (2000) Molecular aspects of higher plant P-type Ca2+-ATPases Biochim BiophysActa 1465 52-78
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gottschalk A Almedom RB Schedletzky T Anderson SD Yates JR 3rd Schafer WR (2005) Identification and characterization ofnovel nicotinic receptor-associated proteins in Caenorhabditis elegans Embo J 24 2566-2578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang LQ Berkelman T Franklin AE Hoffman NE (1993) Characterization of a Gene Encoding a Ca2+-Atpase-Like Protein in thePlastid Envelope Proceedings of the National Academy of Sciences of the United States of America 90 10066-10070
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lee SM Kim HS Han HJ Moon BC Kim CY Harper JF Chung WS (2007) Identification of a calmodulin-regulated autoinhibitedCa2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis FEBS Lett 581 3943-3949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lewis RS (2001) Calcium signaling mechanisms in T lymphocytes Annu Rev Immunol 19 497-521Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456
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Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980
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DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
41
genotype measured as FRETCFP ratio using the YC36 reporter The two numbers underneath 673
each genotype indicate the number of cells with the representative calcium pattern versus the 674
number of total cells analyzed 675
676
677
Figure 8 Stomatal movement is compromised in the bon1 aca10 and aca8 mutants 678
A Stomatal closure induced by exogenous application of CaCl2 in wild type aca10 aca8 and 679
bon1 in No-0 Ws andor Col-0 backgrounds 680
B Stomatal closure induced by exogenous application of Pst DC3000 COR- in wild type aca10 681
and bon1 in the Col-0 background 682
C Time course of stomatal movement by application of Pst DC3000 COR- and Pst DC3000 in 683
the wild type and the aca8aca10 plants 684
ldquordquoand ldquordquo indicate significant difference before and after treatment by studentrsquos t-test at P-685
valuelt0001 and P-valuelt005 respectively 686
687
Figure 9 Working model for the role of ACA108 and BON1 in calcium signature and immunity 688
responses 689
Extra-celluar calcium was released into cytosol by plasma membrane localized calcium channels 690
when the host cells use receptors to detect pathogen features The transient increase of Ca2+ 691
concentration activates BON1 and ACA108 complex which pumps calcium from cytosol to 692
extracellular space This Ca2+ exclusion is necessary to activate the following Ca2+ increase in 693
cytosol Ca2+ oscillation in cytosol is generated coordinately by Ca2+ channels that released 694
calcium from extracellular medium and subcellular compartments into cytosol and by Ca2+ 695
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
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Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574
Pubmed Author and TitleCrossRef Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Google Scholar Author Only Title Only Author and Title
Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Clough SJ Fengler KA Yu IC Lippok B Smith RK Jr Bent AF (2000) The Arabidopsis dnd1 defense no death gene encodes amutated cyclic nucleotide-gated ion channel Proc Natl Acad Sci U S A 97 9323-9328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Creutz CE Tomsig JL Snyder SL Gautier MC Skouri F Beisson J Cohen J (1998) The copines a novel class of C2 domain-containing calcium-dependent phospholipid-binding proteins conserved from Paramecium to humans J Biol Chem 273 1393-1402
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Curran AC Hwang I Corbin J Martinez S Rayle D Sze H Harper JF (2000) Autoinhibition of a calmodulin-dependent calciumpump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain J Biol Chem 27530301-30308
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dodd AN Kudla J Sanders D (2010) The language of calcium signaling Annu Rev Plant Biol 61 593-620Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Du L Ali GS Simons KA Hou J Yang T Reddy AS Poovaiah BW (2009) Ca2+calmodulin regulates salicylic-acid-mediated plantimmunity Nature 457 1154-1158
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Frei dit Frey N Mbengue M Kwaaitaal M Nitsch L Altenbach D Haweker H Lozano-Duran R Njo MF Beeckman T Huettel BBorst JW Panstruga R Robatzek S (2012) Plasma membrane calcium ATPases are important components of receptor-mediatedsignaling in plant immune responses and development Plant Physiol 159 798-809
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geisler M Axelsen KB Harper JF Palmgren MG (2000) Molecular aspects of higher plant P-type Ca2+-ATPases Biochim BiophysActa 1465 52-78
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gottschalk A Almedom RB Schedletzky T Anderson SD Yates JR 3rd Schafer WR (2005) Identification and characterization ofnovel nicotinic receptor-associated proteins in Caenorhabditis elegans Embo J 24 2566-2578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang LQ Berkelman T Franklin AE Hoffman NE (1993) Characterization of a Gene Encoding a Ca2+-Atpase-Like Protein in thePlastid Envelope Proceedings of the National Academy of Sciences of the United States of America 90 10066-10070
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lee SM Kim HS Han HJ Moon BC Kim CY Harper JF Chung WS (2007) Identification of a calmodulin-regulated autoinhibitedCa2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis FEBS Lett 581 3943-3949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lewis RS (2001) Calcium signaling mechanisms in T lymphocytes Annu Rev Immunol 19 497-521Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Menke FL Kang HG Chen Z Park JM Kumar D Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by theMAP kinase SIPK and mediates HR-like cell death in tobacco Mol Plant Microbe Interact 18 1027-1034
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Murata Y Mori IC Munemasa S (2015) Diverse stomatal signaling and the signal integration mechanism Annu Rev Plant Biol 66369-392
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Noel L Moores TL van Der Biezen EA Parniske M Daniels MJ Parker JE Jones JD (1999) Pronounced intraspecific haplotypedivergence at the RPP5 complex disease resistance locus of Arabidopsis Plant Cell 11 2099-2112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nomura H Komori T Uemura S Kanda Y Shimotani K Nakai K Furuichi T Takebayashi K Sugimoto T Sano S Suwastika INFukusaki E Yoshioka H Nakahira Y Shiina T (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis NatCommun 3 926
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Hayashi S Saeki M Ohta H Kinoshita K (2009) ATTED-II provides coexpressed gene networks for ArabidopsisNucleic Acids Res 37 D987-991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qudeimat E Faltusz AM Wheeler G Lang D Holtorf H Brownlee C Reski R Frank W (2008) A PIIB-type Ca2+-ATPase is essentialfor stress adaptation in Physcomitrella patens Proc Natl Acad Sci U S A 105 19555-19560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rizo J Sudhof TC (1998) C2-domains structure and function of a universal Ca2+-binding domain J Biol Chem 273 15879-15882Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rossignol P Collier S Bush M Shaw P Doonan JH (2007) Arabidopsis POT1A interacts with TERT-V(I8) an N-terminal splicingvariant of telomerase J Cell Sci 120 3678-3687
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schiott M Romanowsky SM Baekgaard L Jakobsen MK Palmgren MG Harper JF (2004) A plant plasma membrane Ca2+ pump isrequired for normal pollen tube growth and fertilization Proc Natl Acad Sci U S A 101 9502-9507
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
42
pumps that export Ca2+ from the cytosol The information within Ca2+ spiking was decoded by 696
Ca2+ binding proteins and transmitted to control stomatal movement and the expression of 697
defense response genes 698
699
700
Supplementary tables 701
Supplementary Table 1 List of BON1 co-expressed genes that code for calcium signaling 702
molecules 703
Supplementary Table 2 Primers used in this study 704
705
Supplementary Figures 706
Supplemental Figure 1 Subcellular localization and structure of the ACA10 protein 707
Supplemental Figure 2 Physical interaction of ACA810 with BON1 assayed by BiFC 708
Supplemental Figure 3 The growth defect of aca10-1 bon1-2 is dependent on temperature 709
EDS1 and PAD4 710
Supplemental Figure 4 Overexpression of BON1 in the aca10 mutants 711
712
713
714
715
716
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllen GJ Chu SP Schumacher K Shimazaki CT Vafeados D Kemper A Hawke SD Tallman G Tsien RY Harper JF Chory JSchroeder JI (2000) Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutantScience 289 2338-2342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Balagueacute C Lin B Alcon C Flottes G Malmstroumlm S Koumlhler C Neuhaus G Pelletier G Gaymard F Roby D (2003) HLM1 anessential signaling component in the hypersensitive response is a member of the cyclic nucleotide-gated channel ion channelfamily Plant Cell 15 365-379
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bonza MC Morandini P Luoni L Geisler M Palmgren MG De Michelis MI (2000) At-ACA8 encodes a plasma membrane-localizedcalcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus Plant Physiol 123 1495-1506
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Boudsocq M Sheen J (2013) CDPKs in immune and stress signaling Trends Plant Sci 18 30-40Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Willmann MR McCormack M Lee H Shan L He P Bush J Cheng SH Sheen J (2010) Differential innate immunesignalling via Ca2+ sensor protein kinases Nature 464 418-422
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boursiac Y Harper JF (2007) The origin and function of calmodulin regulated Ca2+ pjmps in plants J Bioenerg Biomembr 39 409-414
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boursiac Y Lee SM Romanowsky S Blank R Sladek C Chung WS Harper JF (2010) Disruption of the vacuolar calcium-ATPasesin Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway Plant Physiol 154 1158-1171
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bracha-Drori K Shichrur K Katz A Oliva M Angelovici R Yalovsky S Ohad N (2004) Detection of protein-protein interactions inplants using bimolecular fluorescence complementation Plant Journal 40 419-427
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Bush DS (1995) Calcium Regulation in Plant-Cells and Its Role in Signaling Annual Review of Plant Physiology and Plant MolecularBiology 46 95-122
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Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
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Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Menke FL Kang HG Chen Z Park JM Kumar D Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by theMAP kinase SIPK and mediates HR-like cell death in tobacco Mol Plant Microbe Interact 18 1027-1034
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Murata Y Mori IC Munemasa S (2015) Diverse stomatal signaling and the signal integration mechanism Annu Rev Plant Biol 66369-392
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Noel L Moores TL van Der Biezen EA Parniske M Daniels MJ Parker JE Jones JD (1999) Pronounced intraspecific haplotypedivergence at the RPP5 complex disease resistance locus of Arabidopsis Plant Cell 11 2099-2112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nomura H Komori T Uemura S Kanda Y Shimotani K Nakai K Furuichi T Takebayashi K Sugimoto T Sano S Suwastika INFukusaki E Yoshioka H Nakahira Y Shiina T (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis NatCommun 3 926
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Hayashi S Saeki M Ohta H Kinoshita K (2009) ATTED-II provides coexpressed gene networks for ArabidopsisNucleic Acids Res 37 D987-991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qudeimat E Faltusz AM Wheeler G Lang D Holtorf H Brownlee C Reski R Frank W (2008) A PIIB-type Ca2+-ATPase is essentialfor stress adaptation in Physcomitrella patens Proc Natl Acad Sci U S A 105 19555-19560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rizo J Sudhof TC (1998) C2-domains structure and function of a universal Ca2+-binding domain J Biol Chem 273 15879-15882Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rossignol P Collier S Bush M Shaw P Doonan JH (2007) Arabidopsis POT1A interacts with TERT-V(I8) an N-terminal splicingvariant of telomerase J Cell Sci 120 3678-3687
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schiott M Romanowsky SM Baekgaard L Jakobsen MK Palmgren MG Harper JF (2004) A plant plasma membrane Ca2+ pump isrequired for normal pollen tube growth and fertilization Proc Natl Acad Sci U S A 101 9502-9507
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
43
717
718
719
720
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Parsed CitationsAllen GJ Chu SP Schumacher K Shimazaki CT Vafeados D Kemper A Hawke SD Tallman G Tsien RY Harper JF Chory JSchroeder JI (2000) Alteration of stimulus-specific guard cell calcium oscillations and stomatal closing in Arabidopsis det3 mutantScience 289 2338-2342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Balagueacute C Lin B Alcon C Flottes G Malmstroumlm S Koumlhler C Neuhaus G Pelletier G Gaymard F Roby D (2003) HLM1 anessential signaling component in the hypersensitive response is a member of the cyclic nucleotide-gated channel ion channelfamily Plant Cell 15 365-379
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bonza MC Morandini P Luoni L Geisler M Palmgren MG De Michelis MI (2000) At-ACA8 encodes a plasma membrane-localizedcalcium-ATPase of Arabidopsis with a calmodulin-binding domain at the N terminus Plant Physiol 123 1495-1506
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Sheen J (2013) CDPKs in immune and stress signaling Trends Plant Sci 18 30-40Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boudsocq M Willmann MR McCormack M Lee H Shan L He P Bush J Cheng SH Sheen J (2010) Differential innate immunesignalling via Ca2+ sensor protein kinases Nature 464 418-422
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boursiac Y Harper JF (2007) The origin and function of calmodulin regulated Ca2+ pjmps in plants J Bioenerg Biomembr 39 409-414
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Boursiac Y Lee SM Romanowsky S Blank R Sladek C Chung WS Harper JF (2010) Disruption of the vacuolar calcium-ATPasesin Arabidopsis results in the activation of a salicylic acid-dependent programmed cell death pathway Plant Physiol 154 1158-1171
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bracha-Drori K Shichrur K Katz A Oliva M Angelovici R Yalovsky S Ohad N (2004) Detection of protein-protein interactions inplants using bimolecular fluorescence complementation Plant Journal 40 419-427
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Bush DS (1995) Calcium Regulation in Plant-Cells and Its Role in Signaling Annual Review of Plant Physiology and Plant MolecularBiology 46 95-122
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Capoen W Sun J Wysham D Otegui MS Venkateshwaran M Hirsch S Miwa H Downie JA Morris RJ Ane JM Oldroyd GE (2011)Nuclear membranes control symbiotic calcium signaling of legumes Proc Natl Acad Sci U S A 108 14348-14353
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen H Zou Y Shang Y Lin H Wang Y Cai R Tang X Zhou JM (2008) Firefly luciferase complementation imaging assay forprotein-protein interactions in plants Plant Physiol 146 368-376
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574
Pubmed Author and TitleCrossRef Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Clough SJ Fengler KA Yu IC Lippok B Smith RK Jr Bent AF (2000) The Arabidopsis dnd1 defense no death gene encodes amutated cyclic nucleotide-gated ion channel Proc Natl Acad Sci U S A 97 9323-9328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Creutz CE Tomsig JL Snyder SL Gautier MC Skouri F Beisson J Cohen J (1998) The copines a novel class of C2 domain-containing calcium-dependent phospholipid-binding proteins conserved from Paramecium to humans J Biol Chem 273 1393-1402
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Curran AC Hwang I Corbin J Martinez S Rayle D Sze H Harper JF (2000) Autoinhibition of a calmodulin-dependent calciumpump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain J Biol Chem 27530301-30308
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dodd AN Kudla J Sanders D (2010) The language of calcium signaling Annu Rev Plant Biol 61 593-620Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Du L Ali GS Simons KA Hou J Yang T Reddy AS Poovaiah BW (2009) Ca2+calmodulin regulates salicylic-acid-mediated plantimmunity Nature 457 1154-1158
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Frei dit Frey N Mbengue M Kwaaitaal M Nitsch L Altenbach D Haweker H Lozano-Duran R Njo MF Beeckman T Huettel BBorst JW Panstruga R Robatzek S (2012) Plasma membrane calcium ATPases are important components of receptor-mediatedsignaling in plant immune responses and development Plant Physiol 159 798-809
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127
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Geisler M Axelsen KB Harper JF Palmgren MG (2000) Molecular aspects of higher plant P-type Ca2+-ATPases Biochim BiophysActa 1465 52-78
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gottschalk A Almedom RB Schedletzky T Anderson SD Yates JR 3rd Schafer WR (2005) Identification and characterization ofnovel nicotinic receptor-associated proteins in Caenorhabditis elegans Embo J 24 2566-2578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
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Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106
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Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
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Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
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Huang LQ Berkelman T Franklin AE Hoffman NE (1993) Characterization of a Gene Encoding a Ca2+-Atpase-Like Protein in thePlastid Envelope Proceedings of the National Academy of Sciences of the United States of America 90 10066-10070
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Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
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Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
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Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591
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Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192
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Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lee SM Kim HS Han HJ Moon BC Kim CY Harper JF Chung WS (2007) Identification of a calmodulin-regulated autoinhibitedCa2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis FEBS Lett 581 3943-3949
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Lewis RS (2001) Calcium signaling mechanisms in T lymphocytes Annu Rev Immunol 19 497-521Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
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Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456
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Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980
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Menke FL Kang HG Chen Z Park JM Kumar D Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by theMAP kinase SIPK and mediates HR-like cell death in tobacco Mol Plant Microbe Interact 18 1027-1034
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Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615
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Capoen W Sun J Wysham D Otegui MS Venkateshwaran M Hirsch S Miwa H Downie JA Morris RJ Ane JM Oldroyd GE (2011)Nuclear membranes control symbiotic calcium signaling of legumes Proc Natl Acad Sci U S A 108 14348-14353
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Chen H Zou Y Shang Y Lin H Wang Y Cai R Tang X Zhou JM (2008) Firefly luciferase complementation imaging assay forprotein-protein interactions in plants Plant Physiol 146 368-376
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Choi HW Lee DH Hwang BK (2009) The Pepper Calmodulin Gene CaCaM1 Is Involved in Reactive Oxygen Species and NitricOxide Generation Required for Cell Death and the Defense Response Molecular Plant-Microbe Interactions 22 1389-1400
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Church DL Lambie EJ (2003) The promotion of gonadal cell divisions by the Caenorhabditis elegans TRPM cation channel GON-2is antagonized by GEM-4 copine Genetics 165 563-574
Pubmed Author and TitleCrossRef Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Google Scholar Author Only Title Only Author and Title
Clark RM Schweikert G Toomajian C Ossowski S Zeller G Shinn P Warthmann N Hu TT Fu G Hinds DA Chen H Frazer KAHuson DH Scholkopf B Nordborg M Ratsch G Ecker JR Weigel D (2007) Common sequence polymorphisms shaping geneticdiversity in Arabidopsis thaliana Science 317 338-342
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Clough SJ Fengler KA Yu IC Lippok B Smith RK Jr Bent AF (2000) The Arabidopsis dnd1 defense no death gene encodes amutated cyclic nucleotide-gated ion channel Proc Natl Acad Sci U S A 97 9323-9328
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Creutz CE Tomsig JL Snyder SL Gautier MC Skouri F Beisson J Cohen J (1998) The copines a novel class of C2 domain-containing calcium-dependent phospholipid-binding proteins conserved from Paramecium to humans J Biol Chem 273 1393-1402
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Curran AC Hwang I Corbin J Martinez S Rayle D Sze H Harper JF (2000) Autoinhibition of a calmodulin-dependent calciumpump involves a structure in the stalk that connects the transmembrane domain to the ATPase catalytic domain J Biol Chem 27530301-30308
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Dodd AN Kudla J Sanders D (2010) The language of calcium signaling Annu Rev Plant Biol 61 593-620Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Du L Ali GS Simons KA Hou J Yang T Reddy AS Poovaiah BW (2009) Ca2+calmodulin regulates salicylic-acid-mediated plantimmunity Nature 457 1154-1158
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Frei dit Frey N Mbengue M Kwaaitaal M Nitsch L Altenbach D Haweker H Lozano-Duran R Njo MF Beeckman T Huettel BBorst JW Panstruga R Robatzek S (2012) Plasma membrane calcium ATPases are important components of receptor-mediatedsignaling in plant immune responses and development Plant Physiol 159 798-809
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Geisler M Axelsen KB Harper JF Palmgren MG (2000) Molecular aspects of higher plant P-type Ca2+-ATPases Biochim BiophysActa 1465 52-78
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gottschalk A Almedom RB Schedletzky T Anderson SD Yates JR 3rd Schafer WR (2005) Identification and characterization ofnovel nicotinic receptor-associated proteins in Caenorhabditis elegans Embo J 24 2566-2578
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang LQ Berkelman T Franklin AE Hoffman NE (1993) Characterization of a Gene Encoding a Ca2+-Atpase-Like Protein in thePlastid Envelope Proceedings of the National Academy of Sciences of the United States of America 90 10066-10070
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lee SM Kim HS Han HJ Moon BC Kim CY Harper JF Chung WS (2007) Identification of a calmodulin-regulated autoinhibitedCa2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis FEBS Lett 581 3943-3949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lewis RS (2001) Calcium signaling mechanisms in T lymphocytes Annu Rev Immunol 19 497-521Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Menke FL Kang HG Chen Z Park JM Kumar D Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by theMAP kinase SIPK and mediates HR-like cell death in tobacco Mol Plant Microbe Interact 18 1027-1034
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Murata Y Mori IC Munemasa S (2015) Diverse stomatal signaling and the signal integration mechanism Annu Rev Plant Biol 66369-392
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Noel L Moores TL van Der Biezen EA Parniske M Daniels MJ Parker JE Jones JD (1999) Pronounced intraspecific haplotypedivergence at the RPP5 complex disease resistance locus of Arabidopsis Plant Cell 11 2099-2112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nomura H Komori T Uemura S Kanda Y Shimotani K Nakai K Furuichi T Takebayashi K Sugimoto T Sano S Suwastika INFukusaki E Yoshioka H Nakahira Y Shiina T (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis NatCommun 3 926
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Hayashi S Saeki M Ohta H Kinoshita K (2009) ATTED-II provides coexpressed gene networks for ArabidopsisNucleic Acids Res 37 D987-991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qudeimat E Faltusz AM Wheeler G Lang D Holtorf H Brownlee C Reski R Frank W (2008) A PIIB-type Ca2+-ATPase is essentialfor stress adaptation in Physcomitrella patens Proc Natl Acad Sci U S A 105 19555-19560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rizo J Sudhof TC (1998) C2-domains structure and function of a universal Ca2+-binding domain J Biol Chem 273 15879-15882Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rossignol P Collier S Bush M Shaw P Doonan JH (2007) Arabidopsis POT1A interacts with TERT-V(I8) an N-terminal splicingvariant of telomerase J Cell Sci 120 3678-3687
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schiott M Romanowsky SM Baekgaard L Jakobsen MK Palmgren MG Harper JF (2004) A plant plasma membrane Ca2+ pump isrequired for normal pollen tube growth and fertilization Proc Natl Acad Sci U S A 101 9502-9507
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Forde BG Roberts MR (2014) Glutamate receptor-like channels in plants a role as amino acid sensors in plant defenceF1000Prime Rep 6 37
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Gao X Chen X Lin W Chen S Lu D Niu Y Li L Cheng C McCormack M Sheen J Shan L He P (2013) Bifurcation of ArabidopsisNLR immune signaling via Ca2+-dependent protein kinases PLoS Pathog 9 e1003127
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George L Romanowsky SM Harper JF Sharrock RA (2008) The ACA10 Ca2+-ATPase regulates adult vegetative development andinflorescence architecture in Arabidopsis Plant Physiol 146 716-728
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Gottschalk A Almedom RB Schedletzky T Anderson SD Yates JR 3rd Schafer WR (2005) Identification and characterization ofnovel nicotinic receptor-associated proteins in Caenorhabditis elegans Embo J 24 2566-2578
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Gou M Zhang Z Zhang N Huang Q Monaghan J Yang H Shi Z Zipfel C Hua J (2015) Opposing Effects on Two Phases ofDefense Responses from Concerted Actions of HEAT SHOCK COGNATE70 and BONZAI1 in Arabidopsis Plant Physiol 169 2304-2323 httpsplantphysiolorgDownloaded on January 22 2021 - Published by
Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang LQ Berkelman T Franklin AE Hoffman NE (1993) Characterization of a Gene Encoding a Ca2+-Atpase-Like Protein in thePlastid Envelope Proceedings of the National Academy of Sciences of the United States of America 90 10066-10070
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lee SM Kim HS Han HJ Moon BC Kim CY Harper JF Chung WS (2007) Identification of a calmodulin-regulated autoinhibitedCa2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis FEBS Lett 581 3943-3949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lewis RS (2001) Calcium signaling mechanisms in T lymphocytes Annu Rev Immunol 19 497-521Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Menke FL Kang HG Chen Z Park JM Kumar D Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by theMAP kinase SIPK and mediates HR-like cell death in tobacco Mol Plant Microbe Interact 18 1027-1034
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Murata Y Mori IC Munemasa S (2015) Diverse stomatal signaling and the signal integration mechanism Annu Rev Plant Biol 66369-392
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Noel L Moores TL van Der Biezen EA Parniske M Daniels MJ Parker JE Jones JD (1999) Pronounced intraspecific haplotypedivergence at the RPP5 complex disease resistance locus of Arabidopsis Plant Cell 11 2099-2112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nomura H Komori T Uemura S Kanda Y Shimotani K Nakai K Furuichi T Takebayashi K Sugimoto T Sano S Suwastika INFukusaki E Yoshioka H Nakahira Y Shiina T (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis NatCommun 3 926
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Hayashi S Saeki M Ohta H Kinoshita K (2009) ATTED-II provides coexpressed gene networks for ArabidopsisNucleic Acids Res 37 D987-991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qudeimat E Faltusz AM Wheeler G Lang D Holtorf H Brownlee C Reski R Frank W (2008) A PIIB-type Ca2+-ATPase is essentialfor stress adaptation in Physcomitrella patens Proc Natl Acad Sci U S A 105 19555-19560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rizo J Sudhof TC (1998) C2-domains structure and function of a universal Ca2+-binding domain J Biol Chem 273 15879-15882Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rossignol P Collier S Bush M Shaw P Doonan JH (2007) Arabidopsis POT1A interacts with TERT-V(I8) an N-terminal splicingvariant of telomerase J Cell Sci 120 3678-3687
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schiott M Romanowsky SM Baekgaard L Jakobsen MK Palmgren MG Harper JF (2004) A plant plasma membrane Ca2+ pump isrequired for normal pollen tube growth and fertilization Proc Natl Acad Sci U S A 101 9502-9507
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Granqvist E Sun J Op den Camp R Pujic P Hill L Normand P Morris RJ Downie JA Geurts R Oldroyd GE (2015) Bacterial-induced calcium oscillations are common to nitrogen-fixing associations of nodulating legumes and nonlegumes New Phytol 207551-558
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Harper JF Hong B Hwang I Guo HQ Stoddard R Huang JF Palmgren MG Sze H (1998) A novel calmodulin-regulated Ca2+-ATPase (ACA2) from Arabidopsis with an N-terminal autoinhibitory domain J Biol Chem 273 1099-1106
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Heinrich C Keller C Boulay A Vecchi M Bianchi M Sack R Lienhard S Duss S Hofsteenge J Hynes NE (2010) Copine-IIIinteracts with ErbB2 and promotes tumor cell migration Oncogene 29 1598-1610
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hua J Grisafi P Cheng SH Fink GR (2001) Plant growth homeostasis is controlled by the Arabidopsis BON1 and BAP1 genesGenes Dev 15 2263-2272
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Huang LQ Berkelman T Franklin AE Hoffman NE (1993) Characterization of a Gene Encoding a Ca2+-Atpase-Like Protein in thePlastid Envelope Proceedings of the National Academy of Sciences of the United States of America 90 10066-10070
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Hubbard KE Siegel RS Valerio G Brandt B Schroeder JI (2012) Abscisic acid and CO2 signalling via calcium sensitivity priming inguard cells new CDPK mutant phenotypes and a method for improved resolution of stomatal stimulus-response analyses Ann Bot109 5-17
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim MC Panstruga R Elliott C Muller J Devoto A Yoon HW Park HC Cho MJ Schulze-Lefert P (2002) Calmodulin interacts withMLO protein to regulate defence against mildew in barley Nature 416 447-451
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kim TH Bohmer M Hu H Nishimura N Schroeder JI (2010) Guard cell signal transduction network Advances in understandingabscisic acid CO2 and Ca2+ signaling Annu Rev Plant Bio 61561-591
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Krebs M Held K Binder A Hashimoto K Den Herder G Parniske M Kudla J Schumacher K (2012) FRET-based geneticallyencoded sensors allow high-resolution live cell imaging of Ca2+ dynamics Plant J 69 181-192
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Kudla J Batistic O Hashimoto K (2010) Calcium signals the lead currency of plant information processing Plant Cell 22 541-563Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lecourieux D Ranjeva R Pugin A (2006) Calcium in plant defence-signalling pathways New Phytol 171 249-269Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lee SM Kim HS Han HJ Moon BC Kim CY Harper JF Chung WS (2007) Identification of a calmodulin-regulated autoinhibitedCa2+-ATPase (ACA11) that is localized to vacuole membranes in Arabidopsis FEBS Lett 581 3943-3949
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Lewis RS (2001) Calcium signaling mechanisms in T lymphocytes Annu Rev Immunol 19 497-521Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Li Y Gou M Sun Q Hua J (2010) Requirement of calcium binding myristoylation and protein-protein interaction for the CopineBON1 function in Arabidopsis J Biol Chem 285 29884-29891
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Li Y Yang S Yang H Hua J (2007) The TIR-NB-LRR gene SNC1 is regulated at the transcript level by multiple factors Mol PlantMicrobe Interact 20 1449-1456
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Ma W Berkowitz GA (2007) The grateful dead calcium and cell death in plant innate immunity Cell Microbiol 9 2571-2585Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Melotto M Underwood W Koczan J Nomura K He SY (2006) Plant stomata function in innate immunity against bacterial invasionCell 126 969-980
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Menke FL Kang HG Chen Z Park JM Kumar D Klessig DF (2005) Tobacco transcription factor WRKY1 is phosphorylated by theMAP kinase SIPK and mediates HR-like cell death in tobacco Mol Plant Microbe Interact 18 1027-1034
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Monaghan J Matschi S Shorinola O Rovenich H Matei A Segonzac C Malinovsky FG Rathjen JP MacLean D Romeis T ZipfelC (2014) The calcium-dependent protein kinase CPK28 buffers plant immunity and regulates BIK1 turnover Cell Host Microbe 16605-615
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Murata Y Mori IC Munemasa S (2015) Diverse stomatal signaling and the signal integration mechanism Annu Rev Plant Biol 66369-392
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Noel L Moores TL van Der Biezen EA Parniske M Daniels MJ Parker JE Jones JD (1999) Pronounced intraspecific haplotypedivergence at the RPP5 complex disease resistance locus of Arabidopsis Plant Cell 11 2099-2112
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Nomura H Komori T Uemura S Kanda Y Shimotani K Nakai K Furuichi T Takebayashi K Sugimoto T Sano S Suwastika INFukusaki E Yoshioka H Nakahira Y Shiina T (2012) Chloroplast-mediated activation of plant immune signalling in Arabidopsis NatCommun 3 926
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Obayashi T Hayashi S Saeki M Ohta H Kinoshita K (2009) ATTED-II provides coexpressed gene networks for ArabidopsisNucleic Acids Res 37 D987-991
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Qudeimat E Faltusz AM Wheeler G Lang D Holtorf H Brownlee C Reski R Frank W (2008) A PIIB-type Ca2+-ATPase is essentialfor stress adaptation in Physcomitrella patens Proc Natl Acad Sci U S A 105 19555-19560
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rizo J Sudhof TC (1998) C2-domains structure and function of a universal Ca2+-binding domain J Biol Chem 273 15879-15882Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Rossignol P Collier S Bush M Shaw P Doonan JH (2007) Arabidopsis POT1A interacts with TERT-V(I8) an N-terminal splicingvariant of telomerase J Cell Sci 120 3678-3687
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schiott M Romanowsky SM Baekgaard L Jakobsen MK Palmgren MG Harper JF (2004) A plant plasma membrane Ca2+ pump isrequired for normal pollen tube growth and fertilization Proc Natl Acad Sci U S A 101 9502-9507
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6
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Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202
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Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
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Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Schutze K Harter K Chaban C (2009) Bimolecular fluorescence complementation (BiFC) to study protein-protein interactions inliving plant cells Methods Mol Biol 479 189-202
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Shigaki T Hirschi KD (2006) Diverse functions and molecular properties emerging for CAX cationH+ exchangers in plants PlantBiol (Stuttg) 8 419-429
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Steinhorst L Kudla J (2013) Calcium and reactive oxygen species rule the waves of signaling Plant Physiol 163 471-485Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Sze H Liang F Hwang I Curran AC Harper JF (2000) Diversity and regulation of plant Ca2+ pumps insights from expression inyeast Annu Rev Plant Physiol Plant Mol Biol 51 433-462
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Thor K Peiter E (2014) Cytosolic calcium signals elicited by the pathogen-associated molecular pattern flg22 in stomatal guardcells are of an oscillatory nature New Phytol 204 873-881
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tidow H Poulsen LR Andreeva A Knudsen M Hein KL Wiuf C Palmgren MG Nissen P (2012) A bimodular mechanism of calciumcontrol in eukaryotes Nature 491 468-472
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Tomsig JL Sohma H Creutz CE (2004) Calcium-dependent regulation of tumour necrosis factor-alpha receptor signalling bycopine Biochem J 378 1089-1094
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wang Y Hua J (2009) A moderate decrease in temperature induces COR15a expression through the CBF signaling cascade andenhances freezing tolerance Plant J 60 340-349
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Whittaker CA Hynes RO (2002) Distribution and evolution of von Willebrandintegrin A domains widely dispersed domains withroles in cell adhesion and elsewhere Mol Biol Cell 13 3369-3387
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Wiermer M Feys BJ Parker JE (2005) Plant immunity the EDS1 regulatory node Curr Opin Plant Biol 8 383-389Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Hua J (2004) A haplotype-specific Resistance gene regulated by BONZAI1 mediates temperature-dependent growthcontrol in Arabidopsis Plant Cell 16 1060-1071
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang S Yang H Grisafi P Sanchatjate S Fink GR Sun Q Hua J (2006) The BONCPN gene family represses cell death andpromotes cell growth in Arabidopsis Plant J 45 166-179
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Yang Y Costa A Leonhardt N Siegel RS Schroeder JI (2008) Isolation of a strong Arabidopsis guard cell promoter and itspotential as a research tool Plant Methods 4 6
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhai Z Gayomba SR Jung HI Vimalakumari NK Pineros M Craft E Rutzke MA Danku J Lahner B Punshon T Guerinot ML SalthttpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
DE Kochian LV Vatamaniuk OK (2014) OPT3 Is a Phloem-Specific Iron Transporter That Is Essential for Systemic Iron Signalingand Redistribution of Iron and Cadmium in Arabidopsis Plant Cell 26 2249-2264
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
Zhu X Caplan J Mamillapalli P Czymmek K Dinesh-Kumar SP (2010) Function of endoplasmic reticulum calcium ATPase in innateimmunity-mediated programmed cell death Embo J 29 1007-1018
Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title
httpsplantphysiolorgDownloaded on January 22 2021 - Published by Copyright (c) 2020 American Society of Plant Biologists All rights reserved
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