Potato mop-top virus co-opts the stress sensor HIPP26 for long … · 3 60 (reviewed in Tilsner et...

1

1

Short title PMTV co-opts HIPP26 for systemic infection 2

Corresponding author Professor Lesley Torrance University of St Andrews and The James 3

Hutton Institute Scotland UK 4

Title Potato mop-top virus co-opts the stress sensor HIPP26 for long-distance movement 5

Graham H Cowan1 Alison G Roberts1 Susan Jones1 Pankaj Kumar2 6

Pruthvi B Kalyandurg 3 Jose F Gil3 Eugene I Savenkov3 Piers A Hemsley15 and Lesley 7

Torrance1 2 8

1 The James Hutton Institute Invergowrie Scotland UK 9

2School of Biology The University of St Andrews St Andrews Fife UK 10

3Department of Plant Biology Uppsala BioCenter Linnean Centre of Plant Biology Swedish 11

University of Agricultural Sciences Uppsala Sweden 12

5Division of Plant Sciences University of Dundee at the James Hutton Institute Invergowrie 13

Scotland UK 14

Summary sentence After a virus movement protein interacts with the HIPP26 stress sensor 15

the complex re-localises to the nucleus activating the drought stress response and thereby 16

facilitating virus long distance movement 17

Footnotes 18

Author contributions LT conceived the research plans LT PH and EIS designed the 19

research GC SJ PK PBK JFG AR and PH performed the research GC LT AR PH SJ 20

and EIS analyzed the data and LT AR PH and EIS wrote the paper with contributions from 21

all authors 22

Funding The work of LT GC SJ and AR is funded by the Scottish Governmentrsquos Rural and 23

Environmental Science and Analytical Services (RESAS) Division PH by the BBSRC (grant 24

BBM0249111) and The Royal Society and EIS by the Swedish Research Council Formas 25

and the Carl Tryggers Foundation 26

27

Corresponding author email LesleyTorrancehuttonacuk lt27st-andrewsacuk 28

Plant Physiology Preview Published on January 26 2018 as DOI101104pp1701698

Copyright 2018 by the American Society of Plant Biologists

wwwplantphysiolorgon September 4 2020 - Published by Downloaded from Copyright copy 2018 American Society of Plant Biologists All rights reserved

2

The authors responsible for distribution of materials integral to the findings presented in this 29

article are Lesley Torrance (LesleyTorrancehuttonacuk) and Eugene Savenkov 30

(eugenesavenkovsluse) 31

Abstract 32

Virus movement proteins facilitate virus entry into the vascular system to initiate systemic 33

infection The potato mop-top virus (PMTV) movement protein TGB1 is involved in long-34

distance movement of both viral ribonucleoprotein complexes and virions Here our analysis 35

of TGB1 interactions with host Nicotiana benthamiana proteins revealed an interaction with 36

a member of the heavy metal-associated isoprenylated plant protein family HIPP26 which 37

acts as a plasma membrane-to-nucleus signal during abiotic stress We found that knockdown 38

of NbHIPP26 expression inhibited virus long-distance movement but did not affect cell-to-39

cell movement Drought and PMTV infection upregulated NbHIPP26 gene expression and 40

PMTV infection protected plants from drought In addition NbHIPP26 promoter-reporter 41

fusions revealed vascular tissue-specific expression Mutational and biochemical analysis 42

indicated that NbHIPP26 sub-cellular localisation at the plasma membrane and 43

plasmodesmata was mediated by lipidation (S-acylation and prenylation) as non-lipidated 44

NbHIPP26 was predominantly in the nucleus Notably co-expression of NbHIPP26 with 45

TGB1 resulted in a similar nuclear accumulation of NbHIPP26 TGB1 interacted with the C-46

terminal CVVM (prenyl) domain of NbHIPP26 and bimolecular fluorescence 47

complementation revealed that the TGB1-HIPP26 complex localized to microtubules and 48

accumulated in the nucleolus with little signal at the plasma membrane or plasmodesmata 49

These data support a mechanism where interaction with TGB1 negates or reverses NbHIPP26 50

lipidation thus releasing membrane-associated NbHIPP26 and re-directing it via 51

microtubules to the nucleus thereby activating the drought stress response and facilitating 52

virus long-distance movement 53

54

INTRODUCTION 55

The systemic infection of plants by viruses results in major economic losses in yield andor 56

quality of harvested tissues (Pennazio et al 1996 Waterworth and Hadidi 1998) To 57

achieve a systemic infection viruses must move from the initial point of entry to 58

neighbouring cells and then long distance in the vasculature to other leaves and organs 59


3

(reviewed in Tilsner et al 2014) Although there has been much research on the mechanisms 60

of virus cell-to-cell movement there is much less knowledge of the molecular details of how 61

viruses access the vasculature and the phloem mass transport pathway for long distance 62

movement A better understanding of this process is needed to develop new interventions for 63

disease control 64

The plant vasculature is a major conduit not only for nutrient and water transfer but also for 65

the perception and response to abiotic and biotic stresses (Kehr and Buhtz 2008) Plant 66

proteins destined for phloem transport are made in or transferred to companion cells prior to 67

transfer into sieve elements through specialised plasmodesmata (PD) called pore 68

plasmodesmal units (PPU) (reviewed in Lough and Lucas 2006) The phloem contains many 69

different macromolecules including protein chaperones calcium sensors RNA binding 70

proteins (Giavalisco et al 2006) and different species of RNAs (including mRNAs miRNAs 71

and siRNAs) (Buhtz et al 2008 2010 reviewed by Turgeon and Wolf 2009) These 72

phloem-mobile signalling molecules enable plants to coordinate their growth and 73

development and respond to stress (Kehr and Buhtz 2008) A recent report on systemic 74

movement of Tobacco mosaic virus (TMV) suggests that interaction with phloem-specific 75

transcriptional regulators plays a major role in virus phloem loading in older tissues 76

suggesting that TMV reprogramming of gene expression enhances systemic spread (Collum 77

et al 2016) 78

In this paper we studied the long-distance movement of Potato mop-top virus (PMTV) the 79

type member of the genus Pomovirus PMTV is one of only a few plant viruses where in 80

addition to movement as encapsidated virions the genome components can also move as 81

ribonucleoprotein (vRNP) complexes and the largest movement protein TGB1 plays a key 82

role in the long-distance movement of both (Torrance et al 2011) Previous work has 83

shown that the N-terminal domain of TGB1 contains two nucleolar localisation signals and 84

TGB1 associates with importin-α which is required for nuclearnucleolar accumulation and 85

virus long distance movement (Lukhovitskaya et al 2015) The nucleolus is a distinct region 86

in the nucleus that has recently been shown to play a role in long distance movement of some 87

plant viruses (reviewed in Taliansky et al 2010) For example Groundnut rosette virus 88

(GRV) does not encode a coat protein but encodes the protein ORF3 that interacts with a 89

nucleolar component fibrillarin to form movement competent vRNP for long distance 90

transport (Kim et al 2007) 91


4

Here we describe the association of a virus movement protein PMTV TGB1 with a vascular-92

expressed plant stress sensor HIPP26 HIPP26 is one member of a family of proteins that 93

contain heavy metal binding and C-terminal isoprenylation motifs (heavy metal-associated 94

isoprenylated plant protein HIPP) HIPPs are unique to vascular plants and form a large and 95

diverse family in Arabidopsis (Arabidopsis thaliana Dykema et al 1999 Barth et al 2009 96

Tehseen et al 2010 de Abreu-Neto et al 2013) They have been shown to act in heavy 97

metal homeostasis and in regulating the transcriptional response to abiotic stress (de Abreu-98

Neto et al 2013 Barth et al 2009 Gao et al 2009) and pathogens (de Abreu-Neto et al 99

2013) Our results show that PMTV TGB1 interacts with HIPP26 and the complex 100

accumulates in the nucleus which leads to the activation of the drought stress response 101

pathway in vascular tissues which in turn facilitates the access of PMTV to the transport 102

phloem for long distance movement 103

104

RESULTS 105

PMTV TGB1 interacts with tobacco (Nicotiana benthamiana) HIPP26 106

To investigate interactions of TGB1 with host factors an N benthamiana cDNA library 107

(Hybrigenics Services SAS) was screened by the yeast two-hybrid (Y2H) system and 50 108

clones were identified as interacting with TGB1 The results were returned with each 109

interacting clone provided with a predicted biological score (Formstecher et al 2005) ranked 110

in categories A to F with A as the highest level of confidence in the specificity of the 111

interaction Twenty-nine clones were ranked A and by using BLAST (Altschul et al 1990) 112

they were all identified as N benthamiana homologues of Arabidopsis HIPP26 A 113

representative clone A44 which contained the near complete sequence (missing 33 5rsquo 114

proximal base pairs coding for 11 N-terminal amino acid residues) of the N benthamiana 115

homologue of Arabidopsis HIPP26 was obtained from Hybrigenics In addition the 116

complete open reading frame (ORF) was cloned from N benthamiana plants and sequenced 117

The cDNA sequence of NbHIPP26 ORF was used to search the predicted proteins from the 118

draft N benthamiana genome (assembly v101) using Blast through the Sol Genomics 119

Network web server (httpsolgenomicsnet) (Fernandez-Pozo et al 2014) This revealed 120

significant matches on two different scaffolds (Niben101Scf07109g050041 and 121

Niben101Scf02621g040261) that have not yet been assigned to any chromosomes The two 122


5

proteins only differed in one amino acid residue at position 16 near the N-terminus which 123

was either a serine or glycine Further genome assembly and annotation is required to 124

confirm whether the proteins are coded by alleles or distinct genes In this work we have 125

investigated the protein containing S16 since serine was the most common residue at that 126

position in the interacting clones 127

HIPPs have been variously clustered into five (de Abreu-Neto et al 2013) six (Barth et al 128

2009) and seven major groups (Tehseen et al 2010) The phylogeny created from the 129

alignment of the interacting clone with full length Arabidopsis HIPPs reveals five clusters 130

similar to those identified by de Abreu-Neto and co-workers (de Abreu-Neto et al 2013) 131

(Fig 1A Supplemental Fig 1) In this phylogeny clone A44 clusters with AtHIPP26 in HIPP 132

group II which features proteins with a single heavy metal associated (HMA) domain 133

Sequence alignments of NbHIPP26 with homologues from Arabidopsis tomato (Solanum 134

lycopersicum) and potato (Solanum tuberosum) revealed a high level of conservation with 135

843 sequence identity between N benthamiana and Arabidopsis 954 identity between 136

N benthamiana and potato and 948 between N benthamiana and tomato sequences (Fig 137

1B and C) 138

Analysis revealed that the C152VVM155 sequence at the C-terminus of NbHIPP26 represents a 139

CaaX amino acid motif (a = I V L A S T X = I L M Q S A) characteristic of proteins 140

that are post-translationally lipid modified by the addition of a prenyl group to enable 141

membrane association (reviewed in Crowell 2000) AtHIPP7 was previously shown to be 142

prenylated in vitro through a similar CTVM motif (Dykema et al 1999) while mammalian N-143

Ras is known to be prenylated at an identical CVVM motif in vivo and the CVVM motif 144

alone fused to GFP is sufficient to target GFP to membranes (Choy et al 1999) Plant 145

proteins in the HIPP family all contain a CaaX motif and are all thought to be prenylated (de 146

Abreu-Neto et al 2013) Furthermore a strong prenylation prediction was obtained when 147

the NbHIPP26 sequence was analysed using Prenylation Prediction Suite software (Score = 148

2514 P = 0000554 Maurer-Stroh and Eisenhaber 2005) These data combined indicate that 149

NbHIPP26 is likely prenylated and we therefore term the HIPP26 CVVM motif a prenylation 150

(or prenyl) motif 151

The full length NbHIPP26 ORF was cloned and tested again in Y2H confirming the 152

interaction with PMTV TGB1 (Fig 2A) In addition the movement proteins of four other 153

viruses were tested to investigate whether the interaction with PMTV TGB1 was specific or 154


6

whether other virus movement proteins might also interact with NbHIPP26 These proteins 155

were chosen to represent common types of virus movement proteins (Tilsner et al 2014 156

Verchot-Lubicz et al 2010) including a) beet virus Q (BVQ) TGB1 representing another 157

pomovirus b) barley stripe mosaic virus (BSMV) TGB1 a lsquohordei-typersquo TGB1 protein with 158

a helicase domain and large N-terminal (RNA binding) extension which does not share 159

similarity with the N-terminal domain of PMTV TGB1 c) potato virus X (PVX) TGB1 160

representing a lsquopotex-typersquo TGB which contains a helicase domain but not an N-terminal 161

extension and d) tobacco mosaic virus (TMV) 30K movement protein representing the 162

extensively studied 30K family These were cloned into the Y2H system but while PMTV 163

TGB1 interacted with NbHIPP26 no interactions were obtained with the other virus 164

movement proteins (Fig 2A) The BSMV BVQ TMV and PVX movement proteins were 165

detected by immunoblot in the yeast clones confirming that the proteins were expressed in 166

yeast 167

TGB1 interacts with the C-terminus of NbHIPP26 168

To identify key residues involved in the interactions mutations were introduced into PMTV 169

TGB1 and the predicted functional domains of NbHIPP26 The TGB1 mutants were 170

previously shown to produce N-terminally truncated TGB1 proteins lacking amino acid 171

residues 1-84 and 1-149 (TGB1 Δ84 and Δ149 respectively Wright et al 2010 172

Lukhovitskaya et al 2015) In NbHIPP26 the prenyl motif C152VVM155 was altered by 173

replacing C152 with glycine which would interrupt any possible prenylation and was referred 174

to as PrenylM The HMA domain (M36DCEGC41) was altered by replacing the C3841 residues 175

with glycine residues to give HMAM (Fig 2B) 176

In Y2H assays TGB1 Δ84 interacted with NbHIPP26 but the truncated protein TGB1 Δ149 177

did not (Fig 2C) suggesting that some or all of the interacting surface is within the TGB1 178

region spanning amino acid residues 85 to 149 This idea was further supported by the lack 179

of interaction between NbHIPP26 and BVQ TGB1 the movement protein of a related virus 180

which showed very little similarity in the TGB185-149 fragment (Fig 2D) In general the C-181

terminal part of pomoviral TGB1 proteins such as those from PMTV and BVQ was well 182

conserved and comprised the helicase domain whereas the N-termini of the proteins showed 183

very little similarity The PrenylM altered protein did not interact with TGB1 whereas HMAM 184

retained the interaction (Fig 2C) indicating that the site containing the CVVM motif was 185

important for binding to TGB1 186


7

GFP-NbHIPP26 localises in several sub-cellular domains 187

To investigate the localisation of NbHIPP26 the protein was expressed as a translational 188

fusion to GFP in N benthamiana epidermal cells Green fluorescence was observed in the 189

plasma membrane small motile vesicles (approx 2 microm diameter Supplemental Movie S1) 190

the nucleoplasm and the nucleoli (Fig 3A) Co-expression with mRFP-fibrillarin (a marker 191

for the nucleolus Goodin et al 2007) showed precise co-localisation (Fig 3B) Expression 192

of GFP-NbHIPP26 in N benthamiana plants transformed with the mOrange-LTi6b plasma 193

membrane marker (Wright et al 2017) showed strong co-localisation of GFP and mOrange 194

signal as well as punctate spots of GFP-NbHIPP26 around the cell periphery which 195

resembled PD (Fig 3C) To determine whether these spots were PD GFP-NbHIPP26 was co-196

expressed with the PD-localised TMV 30K movement protein fused to mRFP Both 197

fluorescent proteins co-localised in some punctate spots at the periphery (Fig 3D) showing 198

that NbHIPP26 localises to a population of PDs Thus the results show that NbHIPP26 has 199

several subcellular localisations including PD plasma membrane motile vesicles 200

nucleoplasm and nucleolus 201

The TGB1-NbHIPP26 complex accumulates in the nucleolus 202

To investigate the TGB1-NbHIPP26 interaction in plant cells BiFC experiments were 203

conducted using split YFP constructs (Martin et al 2009) NbHIPP26 and TGB1 genes were 204

cloned to allow expression of NbHIPP26 fused to the N-terminal domain of YFP (Y-205

NbHIPP26) and TGB1 fused to the C-terminal domain (TGB1-FP) When these constructs 206

were co-expressed in N benthamiana epidermal cells yellow fluorescence was seen labelling 207

microtubules and nucleoplasm with particularly strong yellow fluorescence in the nucleolus 208

(Fig 3E and F) GFP-HIPP26 never localised to microtubules when expressed alone The 209

TGB1 N-terminally-truncated derivatives ∆84 and ∆149 were also tested in the BiFC system 210

and the results showed that yellow fluorescence was reconstituted in the nucleus and 211

nucleolus but not on microtubules with the combination ∆84-FP and Y-NbHIPP26 (Fig 3G) 212

but no complementation was observed in tests with ∆149-FP and Y-NbHIPP26 (data not 213

shown no fluorescence was visible) No complementation (no fluorescence) was observed in 214

control experiments with Y-NbHIPP26 or TGB1-FP and the corresponding empty vectors 215

encoding Y- or FP fragments Both HIPP26 and TGB1 localise in the nucleus (Barth et al 216

2009 Lukhovitskaya et al 2015 respectively) however the BiFC data show that the TGB1-217

NbHIPP26 complex also localises to microtubules This result taken together with the 218


8

absence of visible fluorescence at the plasma membrane and PD indicated that the interaction 219

with TGB1 caused a microtubule-guided re-localisation of HIPP26 from the plasma 220

membrane to the nucleus and nucleolus This result recapitulates the TGB1 distribution 221

pattern seen previously with YFP-TGB1 behind the leading edge of the virus infection 222

(Wright et al 2010) 223

To support the BiFC data GFP-HIPP26 was also co-expressed in epidermal cells with RFP-224

TGB1 In these experiments green and red fluorescence co-localized precisely in the 225

nucleoplasm and nucleolus and on microtubules (Fig 3H I and J) We also expressed GFP-226

HIPP26 in PMTV-infected cells and in these cells GFP fluorescence was localised as 227

described before when expressed in non-infected cells but it also accumulated on 228

microtubules (Fig 3K) Note that GFP-HIPP26 was never seen on microtubules when 229

expressed alone Thus we believe these data support the BiFC results and provide 230

confirmatory evidence of the interaction between HIPP26 and TGB1 in plants 231

NbHIPP26 is an S-acylated protein 232

Proteomic studies have revealed that many Arabidopsis proteins involved in abiotic or biotic 233

stresses can be reversibly lipid modified by S-acylation (Hemsley et al 2013) S-acylation 234

involves the post-translational addition of fatty acids predominantly stearic or palmitic acid 235

to cysteine residues within proteins through a reversible thioester linkage (Sorek et al 2007) 236

Various Arabidopsis HIPP family members were proposed as S-acylated based on proteomics 237

data (Hemsley et al 2013) We therefore tested whether GFP-NbHIPP26 expressed in N 238

benthamiana was S-acylated using a modified Acyl-RAC assay (Hemsley et al 2008 239

Forrester et al 2011) The results revealed that NbHIPP26 was indeed S-acylated (Figure 4) 240

Substitution of candidate S-acylated cysteine resides by glycine alanine or serine is the most 241

common way to identify S-acylation sites NbHIPP26 contains four cysteine residues one as 242

the prenyl acceptor in the CVVM motif two in the HMA domain presumed to act as metal 243

ion coordinators and one of unknown function at the N-terminus (C13) On the basis that C13 244

is the only cysteine residue with no proposed function we replaced it with glycine (C13G) 245

Subsequent quantification of S-acylation assays comparing the C13G and PrenylM NbHIPP26 246

to wild type (Figs 2B and 4A) indicated that the C13G altered protein showed almost total 247

loss of S-acylation signal and C13 was therefore the likely site of S-acylation The C13G 248

altered protein is henceforth referred to as S-AcylM Prior membrane association is thought 249

to be required for maximal S-acylation efficiency by the integral membrane S-acylating 250


9

enzymes The small but apparent (n = 2 P = 0103) decrease in the S-acylation state of 251

NbHIPP26 altered protein PrenylM compared with wild type (Figure 4B) indicated that non-252

prenylated NbHIPP26 was still a substrate for S-acylation This suggests that HIPP26 can 253

interact with membranes through means other than prenylation and S-acylation 254

255

NbHIPP26 proteins altered in the lipidation domains show depletion from membranes 256

and nuclear enrichment 257

To determine whether lipidation is needed for NbHIPP26 binding to the plasma membrane 258

and PD the subcellular localisations of GFP-tagged NbHIPP26 wild type and altered proteins 259

S-AcylM PrenylM S-AcylM PrenylM and HMAM were investigated (Fig 5A-D) Expression 260

of HMAM gave a phenotype essentially the same as wild type (compare Fig 5A with Fig 261

3A) In comparison to the wild type GFP-HIPP26 and HMAM GFP fluorescence in the cells 262

expressing each of the other three altered proteins was observed to accumulate more strongly 263

in the nucleus (Fig 5B-D) In addition GFP labelling of the plasma membrane was reduced 264

and substantial fluorescence was observed in the cytosol and in some motile vesicles The 265

phenotypes of the three altered proteins were similar except that there were fewer motile 266

vesicles and more cytosolic labelling in cells expressing the double modified protein S-267

AcylM PrenylM These experiments indicated that NbHIPP26 binding to the plasma 268

membrane and PD was weaker in the altered (non-lipidated) proteins suggesting that 269

disruption of the lipid anchors results in release of the proteins to the cytoplasm and allowing 270

the subsequent marked increase in nuclear accumulation 271

In order to study the association of the wild type and altered NbHIPP26 proteins with the 272

plasma membrane in more detail cells expressing the GFP fusion proteins were plasmolysed 273

to pull the plasma membrane away from the cell wall (Fig 5E-H) It is known that as the 274

protoplast retracts away from the wall the plasma membrane is stretched into thin Hechtian 275

strands where it appears as thin tubules with associated small blebs and vesicles along the 276

strands The wild-type NbHIPP26 protein was associated with a profusion of these extra-277

protoplast structures (Fig 5E) In cells expressing either PrenylM or S-AcylM GFP 278

fluorescence was associated with some membrane-derived structures external to the 279

protoplast (eg Hechtian strands vesicles and membrane blebs) which formed as the plasma 280

membrane pulled off the cell wall as the protoplast retracted The amount of apoplastic 281

fluorescence was reduced compared to wild type (Fig 5F and G) and expression of S-AcylM 282


10

PrenylM showed almost no association with remnants of plasma membrane in the region 283

between the protoplast and cell wall (Fig 5H) These results suggest that lipidation is 284

required to retain NbHIPP26 in association with membranes supporting a mechanism 285

whereby interaction with TGB1 disturbs or disrupts NbHIPP26 lipidation resulting in 286

dissociation of the NbHIPP26-TGB1 complex from the membranes at the cell periphery 287

NbHIPP26 is expressed specifically in the vascular parenchyma and induced by drought 288

and PMTV infection 289

Analysis of the nucleotide sequence up to 2 kb upstream from the NbHIPP26 ORF showed 290

the presence of multiple stress responsive elements (Supplemental Fig 2) A region of 291

approx 1 kb upstream of the predicted translational start site of NbHIPP26 thought to 292

encompass the promoter sequence was cloned and fused to the uidA gene and the resulting 293

construct used to transform N benthamiana plants Seven independent transgenic lines were 294

tested for -glucoronidase (GUS) expression and staining was visibly concentrated in the 295

vascular tissue of all plants In whole plants GUS staining was seen predominantly in the 296

vasculature of lower leaves stems and roots (Fig 6A B and C) At low magnification (Fig 297

6D and E) GUS activity was seen in all vein classes of mature N benthamiana leaves 298

Transverse sections of major veins (Fig 6F and G) showed GUS activity to be concentrated 299

in the periphery of cells within the vascular bundles Vascular parenchyma cells (associated 300

with both phloem and xylem tissue) showed blue staining with the strongest promoter 301

activity associated with clusters of phloem cells (Fig 6F areas outlined in red and Fig 6G) 302

Phloem tissue was identified as clusters of cells containing small cells (sieve elements) that 303

lie immediately adaxial and abaxial to the xylem tissue These results are supported by 304

experiments using HIPP26prouidA constructs in Arabidopsis which also show strong 305

vascular expression of GUS activity (Barth et al 2009) and are consistent with previous 306

reports that stress-related genes are preferentially expressed in the vascular tissues (reviewed 307

in Turgeon and Wolf 2009) 308

NbHIPP26 expression was measured in different tissues by reverse transcription quantitative 309

PCR (RT-qPCR) and was shown to be most strongly expressed in root tissues followed by 310

the hypocotyl but was detected in all tissues sampled (Figure 6H) 311

To establish whether NbHIPP26 is induced by drought N benthamiana plants were 312

subjected to drought stress by removing whole plants from the growing medium and 313

exposing them to air at room temperature for 30-240 min during which NbHIPP26 314


11

expression was measured by RT-qPCR Relative NbHIPP26 expression was compared 315

against the endogenous genes PP2A and F-BOX and was shown to increase over time in 316

response to drought stress (Fig 7A) 317

To determine whether NbHIPP26 is also induced by virus infection gene expression levels 318

were investigated in PMTV-infected N benthamiana Total RNA was obtained from upper 319

non-inoculated leaves Relative expression levels of NbHIPP26 were compared by RT-qPCR 320

against three endogenous genes (PP2A CDC2 and F-BOX) in both virus-infected and mock-321

inoculated plants at different time points post inoculation Relative NbHIPP26 expression 322

increased in approx 71 of PMTV-infected plants tested between 10 and 14 days post 323

inoculation (dpi) compared to mock inoculated plants (Fig 7B shows a representative 324

experiment at 12 dpi) 325

PMTV infection protects N benthamiana from drought stress 326

Plants infected with some plant viruses are known to be drought tolerant (Xu et al 327

2008) Therefore we investigated whether infection with PMTV influenced drought 328

tolerance N benthamiana plants were manually inoculated with PMTV or mock inoculated 329

and then maintained in a glasshouse for 14 days to allow systemic spread Water was then 330

withheld and plants were monitored for visible drought response After a further 13 days 331

non-infected plants showed signs of wilting but in marked contrast the infected plants did not 332

wilt at this time point (cf Fig 8A and 8B) We then tested whether PMTV could protect 333

plants from drought by withholding water until both mock- (Fig 8CD and E) and PMTV-334

infected plants (Fig 8FG and H) were completely wilted and then re-watering the plants 335

The results were similar in two independent experiments the numbers of plants surviving (as 336

judged by the presence of new green shoots)number tested were experiment one 210 337

mock-infected 58 PMTV-infected experiment two 25 mock-infected 710 PMTV-338

infected Thus fewer mock-inoculated plants survived 27 compared with plants infected 339

with PMTV 67 340

341

Knock down of NbHIPP26 expression inhibits PMTV long distance movement 342

To examine the role of HIPP26 in PMTV movement a tobacco rattle virus (TRV)-based 343

system was used to knock down expression of HIPP26 in N benthamiana plants A 120 bp 344

fragment of NbHIPP26 was cloned into TRV in an anti-sense orientation (TRVHIPP26) and 345


12

the results showed that expression of the targeted HIPP26 gene was specifically reduced in 346

TRV-RNA1 + TRVHIPP26 inoculated plants compared to the empty vector controls (TRV-347

RNA1 + TRV00) (Fig 9A) The NbHIPP26-silenced and control plants were challenged 348

with PMTV and analysed two weeks post inoculation RT-qPCR of RNA samples isolated 349

from the upper non-inoculated leaves of PMTV-inoculated plants revealed accumulation of 350

all three genomic RNA components (Fig 9B) However accumulation of all three virus 351

genomic components in upper leaves of the plants silenced for NbHIPP26 was markedly 352

decreased as compared to TRV00 control plants (Fig 9B) Quantitative analysis of PMTV 353

accumulation by ELISA (to detect virus particles) two weeks post inoculation revealed 354

similar levels of PMTV accumulation in inoculated leaves of plants silenced for NbHIPP26 355

and in non-silenced plants inoculated with TRV00 (Fig 9C) indicating that the cell-to-cell 356

movement of virus particles and accumulation in inoculated leaves was not affected 357

However in the upper leaves of plants silenced for NbHIPP26 PMTV particles accumulated 358

in much lower amounts as compared to the control plants (absorbance values were ~30 of 359

control Plt005 Studentrsquos two tailed t test Fig 9C) Seven of thirty-six (19) NbHIPP26-360

silenced plants challenged with PMTV over six experiments were completely negative for the 361

presence of virus particles in ELISA while all control plants were infected with PMTV 362

As PMTV moves long distance both as virus particles and vRNPs we next asked 363

whether systemic movement of vRNPs was affected in the plants silenced for NbHIPP26 To 364

address this question NbHIPP26-silenced and control plants were inoculated with RNA-Rep 365

and RNA-TGB in vitro generated transcripts and analysed two weeks post inoculation RT-366

qPCR of RNA samples isolated from the upper non-inoculated leaves of plants challenged 367

with these two genomic components revealed accumulation of RNA-Rep and RNA-TGB 368

(Fig 9D) However accumulation of these two genomic components in upper leaves of the 369

plants silenced for NbHIPP26 was markedly decreased as compared to TRV00 control plants 370

(Plt005 Fig 9D) Hence collectively the results show that systemic movement of both 371

virus particles and vRNPs but not cell-to-cell movement in N benthamiana plants silenced 372

for NbHIPP26 was inhibited Overall the results show that the interaction of PMTV TGB1 373

with NbHIPP26 is essential for efficient systemic movement of the virus 374

375

376

DISCUSSION 377


13

TGB1 plays a central role in the long-distance movement of PMTV TGB1 accumulates in 378

the nucleolus and we previously showed that nucleolar accumulation by an importin-α-379

dependent mechanism contributes to long distance movement (Lukhovitskaya et al 2015) 380

Here we further investigated the interactions of TGB1 with plant proteins and the role of 381

TGB1 nucleolar accumulation in systemic virus movement Y2H experiments showed that 382

TGB1 interacted with the N benthamiana homologue of the Arabidopsis protein HIPP26 383

and sequence analysis revealed a high level of sequence identity (84) between the two 384

proteins HIPP26 is a member of a family of proteins that contain heavy metal-binding and 385

C-terminal isoprenylation motifs 386

Previously NbHIPP26 was found to be present in the PD proteome (Fernandez-Calvino et al 387

2011) and the plasma membrane proteome (Marmagne et al 2007) and YFP-TGB1 388

accumulated in PD when expressed from a virus clone (Wright et al 2010) HIPP proteins 389

like many membrane associated proteins are modified post-translationally by the attachment 390

of lipid moieties HIPP26 contains a C-terminal motif CVVM identical to the known 391

prenylation motif of N-Ras and we showed that NbHIPP26 is lipid-modified by S-acylation 392

through residue C13 Both modifications were shown to be important for the observed 393

localisation of NbHIPP26 at PD and the plasma membrane Mutation of both S-acylation and 394

CVVM lipidation sites led to a marked accumulation of HIPP26 in the nucleus and nucleolus 395

and loss from the cell periphery S-acylation provides a membrane association strength many 396

times greater than a prenyl group (more akin to a transmembrane domain) but critically is 397

reversible This provides a mechanism to change the membrane association of proteins as 398

has been shown for active and inactive type-I ROP GTPases (Sorek et al 2017) While lipid 399

modifications usually promote protein-membrane interaction they can also mediate protein-400

protein interactions as illustrated by the prenyl group-mediated interaction of K-Ras with 401

Galectin-1 and -3 (Ashery et al 2006) Given that TGB1 binding to NbHIPP26 requires an 402

intact prenylation site it is possible that TGB1 binds NbHIPP26 at least partly through the 403

prenyl moiety This may mask the ability of the prenyl group to interact with the membrane 404

and promote dissociation of NbHIPP26 from the plasma membrane environment Consistent 405

with this idea BiFC showed that on interaction with TGB1 the NbHIPP26-TGB1 complex 406

localised predominantly to microtubules and the nucleolus with a small quantity in the 407

nucleoplasm The following model is consistent with our data TGB1 binds NbHIPP26 most 408

likely at PD through the NbHIPP26 prenyl group leading to NbHIPP26 conformational 409

change and exposure of the S-acyl thioester The thioester is now accessible for cleavage and 410


14

releases NbHIPP26 from the plasma membrane allowing trafficking of the NbHIPP26-TGB1 411

complex to the nucleus via microtubules 412

N benthamiana plants expressing HIPP26 promoter-reporter fusions revealed vascular tissue 413

specific expression similar to that found for AtHIPP26 suggesting a similar role for 414

NbHIPP26 in drought tolerance In addition PMTV infection helps to protect N 415

benthamiana plants against drought presumably due to the increased expression of 416

NbHIPP26 during the systemic infection process 417

VIGS of NbHIPP26 expression resulted in decreased accumulation of PMTV in upper (non-418

inoculated) leaves In many of the NbHIPP26 knock down plants there was approx 50 less 419

virus than in non-inoculated plants and in 19 of plants no particles were detected whereas 420

particles were detected in all of the non-silenced control plants 421

The eukaryotic cell nucleolus contains the factors needed for its main function in ribosome 422

subunit production and this is where ribosomal RNA transcription processing and 423

ribosomal RNP assembly occur In mammalian cells major changes occur in the organisation 424

and protein composition of the nucleolus in response to stress where it plays a key role in 425

responding to abiotic and biotic stress signalling (Boulon et al 2010) Several positive-strand 426

RNA viruses encode proteins that localize to the nucleolus (Taliansky et al 2010) including 427

the GRV ORF3 movement protein The GRV ORF3 movement protein enters the nucleolus 428

and associates with the nucleolar protein fibrillarin before the fibrillarin-ORF3 complex 429

returns to the cytoplasm enabling the formation of vRNP needed for long distance movement 430

(Kim et al 2007) 431

The TMV replication protein interacts with three vascular-expressed auxinindole acetic acid 432

(AuxIAA) transcriptional regulators including IAA26 whichis expressed in phloem 433

companion cells (Padmanabhan et al 2006) These AuxIAA proteins are thought to 434

function in regulation of genes involved in phloem loading Recently it has been shown that 435

TMV infection inhibits nuclear localisation of AuxIAA proteins leading to transcriptional 436

reprogramming of mature vascular tissue which correlates with enhanced TMV movement 437

and spread in the phloem of older leaves (Collum et al 2016) 438

Our results indicate that the nuclear and nucleolar association of PMTV TGB1 has a different 439

function in virus long-distance movement to that described for other viruses They suggest 440

that nuclear association of plant RNA virus components can manipulate transcriptional 441


15

regulation and re-programme gene expression in vascular tissue and that this may be a more 442

general strategy used by plant viruses to facilitate long-distance movement than previously 443

supposed (Solovyev and Savenkov 2014 Collum et al 2016) 444

However another consideration is whether the interaction of TGB1 with NbHIPP26 may 445

have a role in pathogenicity As stated before HIPPs are a large family of plant proteins that 446

play roles in response to both abiotic and biotic stresses (de Abreu-Neto et al 2013) For 447

example the cytoplasmically-located rice protein OsHIPP05 is a recessive susceptibility 448

factor in rice blast disease the resistant allele carries deletions in proline-rich motifs thought 449

to be targeted by pathogen protein(s) to facilitate infection (Fukuoka et al 2009) While we 450

cannot completely rule out that the interaction of NbHIPP26 with PMTV-TGB1 may be 451

suppressing a putative role of HIPP26 in host defence (eg by transporting HIPP26 on 452

microtubules for degradation) we think that this is unlikely since logically knock down of 453

NbHIPP26 would result in increased spread of infection and not inhibition as we observed 454

Our results showed that NbHIPP26 moves as a complex with TGB1 and accumulates in the 455

nucleus By analogy to AtHIPP26 which interacts in the nucleus with the transcriptional 456

activator ZFHD1 (Tran et al 2004 2007 Barth et al 2009) we suggest that the 457

Nbenthamiana TGB1 interaction leads to the promotion of drought tolerance by activating 458

expression of dehydration-inducible genes Loss of function of HIPP26 in an Arabidopsis 459

mutant inhibits expression of stress-responsive genes regulated by ZFHD1 and Barth et al 460

(2009) proposes a mechanism where the heavy metal carried by HIPP26 inactivates or 461

displaces the ZF-HD repressor and allows activation of the stress-inducible genes Our data 462

suggests a model where once the PMTV infection reaches the vascular tissue TGB1 binds to 463

NbHIPP26 The NbHIPP26TGB1 complex is then transferred from the plasma membrane or 464

PDs via microtubule-directed transfer to the nucleus and nucleolus where HIPP26 465

accumulation leads to upregulation of dehydration-responsive gene expression in the 466

vasculature and establishment of a drought-tolerant state in the plant The changes in 467

vascular gene expression also allow virions or RNPs to enter the sieve elementcompanion 468

cell complex for long distance movement (Fig 10) The PMTV genome is tripartite and 469

intact virus particles are essential for vector transmission (Cowan et al 1997 Reavy et al 470

1998) A mechanism allowing systemic movement of virions or RNPs (including all genome 471

components) ensures genome integrity for successful future vector-based transmission of 472

fully infective virus from tissues far removed from the initial infection site 473


16

474

475

MATERIALS AND METHODS 476

Yeast Two-Hybrid Analysis 477

Yeast two-hybrid screening was performed by Hybrigenics Services SAS Paris France 478

(httpwwwhybrigenics-servicescom) The Hybrigenics system employs plasmids pB27 479

(containing the LexA binding domain BD) and pP6 (containing the P6 activation domain 480

AD) derived from the original pBTM116 (Vojtek and Hollenberg 1995) and pGADGH 481

(Bartel et al 1993) plasmids respectively The coding sequence for the full-length protein 482

TGB1 (GenBank accession number AJ277556) was PCR-amplified and cloned into pB27 as 483

a C-terminal fusion to LexA The construct was confirmed by sequencing the entire insert 484

and used as a bait to screen a randomly-primed tobacco (Nicotiana benthamiana) cDNA 485

library constructed into pP6 One hundred twenty-five million clones (12-fold the complexity 486

of the library) were screened using a mating approach with YHGX13 (Y187 ade2-101loxP-487

kanMX-loxP mata) and L40∆Gal4 (mata) yeast strains as previously described (Fromont-488

Racine et al 1997) One hundred eighty-six colonies were selected on a medium lacking 489

tryptophan leucine and histidine The prey fragments of the positive clones were amplified 490

by PCR and sequenced at their 5rsquo and 3rsquo junctions The resulting sequences were used to 491

identify the corresponding interacting proteins in the GenBank database (NCBI) using a fully 492

automated procedure 493

A confidence score (PBS for Predicted Biological Score) was attributed to each 494

interaction as previously described (Formstecher et al 2005) The PBS relies on two 495

different levels of analysis Firstly a local score takes into account the redundancy and 496

independence of prey fragments as well as the distribution of reading frames and stop codons 497

in overlapping fragments Secondly a global score takes into account the interactions found 498

in all the screens performed at Hybrigenics using the same library This global score 499

represents the probability of an interaction being nonspecific For practical use the scores 500

were divided into four categories from A (highest confidence) to D (lowest confidence) A 501

fifth category (E) specifically flags interactions involving highly connected prey domains 502

previously found several times in screens performed on libraries derived from the same 503

organism Finally several of these highly connected domains were confirmed as false-504

positives of the technique and were tagged as F The PBS scores have been shown to 505


17

positively correlate with the biological significance of interactions (Rain et al 2001 Wojcik 506

et al 2002) 507

Plasmids of representative interacting clone pP6-A44 were obtained from Hybrigenics and 508

tested again at the James Hutton Institute The interaction between Gal4 and LexA protein 509

fusions was examined in the yeast L40 strain [MATa his3D200 trp1-901 leu 2-3112 ade2 510

lys2-801am URA3(lexAop)8-lacZ LYS2(lexAop)4-HIS3] Yeast cells were co-511

transformed by the small-scale lithium acetate yeast transformation method as described in 512

Clontechrsquos Yeast Protocols Handbook Transformants were selected on synthetic dropout 513

minimal medium base containing 2 (wv) glucose and dropout supplements lacking Leu 514

and Trp (+H) or lacking Leu Trp and His (-H) In experiments with LexA-IMP and LexA-515

PVXTGB1 a small amount of autoactivation was observed which was suppressed by the 516

addition of 10 and 50 mM 3-AT respectively 517

Primer sequences used for cloning are given in the Supplemental Table The coding sequence 518

for the NbImportin-α 1 (ABMO5487 GenBank Accession EF1372531) was PCR amplified 519

using primers IMPFOR and IMPREV (Lukhovitskaya et al 2015) with template plasmid 520

eGFP-NbIMP-α1 and cloned into pP6 (Hybrigenics) The virus movement proteins were 521

cloned into the bait vector pB27 PVX TGB1 was PCR amplified from pPVX201 522

(Baulcombe et al 1995) using primers PVXTGB1FOR and PVXTGB1REV TMV 30K was 523

PCR amplified from TMV-30B (Shivprasad et al 1999) using primers TMV30KFOR and 524

TMV30KREV and BSMV TGB1 was PCR amplified from a BSMV RNA β cDNA clone 525

(Torrance et al 2006) using primers BSMVTGB1FOR and BSMVTGB1REV 526

Sequence alignments and phylogenies 527

To place the clone A44 (NbHIPP26) within the full family repertoire of HIPP proteins 528

previously identified in Arabidopsis (Arabidopsis thaliana) the full length protein sequences 529

of 45 HIPP proteins identified by de Abreu-Neto (de Abreu-Neto et al 2013) were 530

downloaded from GenBank (Release 205)(Benson et al 2013) A multiple alignment of 531

these 45 proteins along with the clone sequence A44 was made using MUSCLE with default 532

parameters (Edgar 2004) and a phylogeny was created using the neighbour joining method 533

within MEGA (v606) The phylogenetic tree was rendered as a mid-point rooted tree with 534

proportional branch lengths using FigureTree (v142) (Rambault 2012) 535


18

To assess the percentage sequence similarity between the NbHIPP26 protein and its 536

homologues in potato (Solanum tuberosum) tomato (Solanum lycopersicum) and 537

Arabidopsis a multiple sequence alignment was made using MUSCLE with default 538

parameters (Edgar 2004) A sequence identity matrix of protein-protein pairs was calculated 539

from the alignment 540

Plant material 541

N benthamiana plants were grown in a glasshouse (21degC day and 16degC night 18 h day 542

length with supplementary lighting of 250 W m-2) N benthamiana plants expressing 543

mRFPAtFib1 fusions (RFP-labelled fibrillarin) were given by Michael Goodin N 544

benthamiana plants expressing mOrange-LTi6b (mOrange labelled plasma membrane) were 545

created at the James Hutton Institute (Wright et al 2017) 546

To check gene expression N benthamiana plants (35 days post germination) were removed 547

from growth medium The roots were rinsed free of compost and plants left in air on filter 548

paper for the designated time Tissues were then processed for RT-qPCR as described below 549

To test the effect of PMTV infection In two independent experiments N benthamiana plants 550

were mechanically inoculated with PMTV or water (mock) and maintained in the glasshouse 551

(as described above) The upper non-inoculated leaves were tested by ELISA 12-14 days post 552

inoculation (dpi) to confirm systemic infection Water was then withheld until the plants 553

reached wilting point and then watering was restored 554

Cloning of plasmids and mutants 555

All of the plasmids were constructed so that fluorescent protein expression was driven by the 556

35S promoter Primer sequences are provided in the Supplemental Table 557

Constructs used for confocal microscopy The NbHIPP26 encoding sequence was amplified 558

from N benthamiana total RNA using attB adapter flanked primers HIPP26FOR and 559

HIPP26REV and recombined into pDONR207 using Gateway BP Clonase II This entry 560

clone was recombined with pB7WGF2 (Karimi et al 2002) using Gateway LR Clonase II to 561

create plasmid eGFP-HIPP26 562

563

Constructs used for BiFC pDONR207 entry clone constructs containing the sequences 564

coding for NbHIPP26 and TGB1 were recombined into pSITE-cEYFP-N1 (Martin et al 565


19

2009) using Gateway LR Clonase II pDONR207 constructs containing the sequences coding 566

for TGB1 NbHIPP26C174G and TGB1∆84 were recombined into pSITE-nEYFP-C1 (Martin 567

et al 2009) using Gateway LR Clonase II (Invitrogen) 568

569

Construction of HIPP 26 mutants Mutations in the NbHIPP26 sequence were made using 570

the QuikChange Site-Directed Mutagenesis kit (Stratagene) The Cys152 codon was changed 571

to GGA (glycine codon) using primers CVVMFOR and CVVMREV to create 572

NbHIPP26C152G The cysteine38 and cysteine41 codons within the HMA domain were 573

changed to GGA using primers HMAmutFOR and HMAmutREV to create 574

NbHIPP26G38CG41C The Cys13 codon was mutated to GGA using primers 575

HIPP26cysmutFOR and HIPP26cysmutREV to create NbHIPP26C13G A double mutant was 576

created where the Cys13 and Cys152 codons were both mutated to GGA using primers 577

HIPP26cysmutFOR and HIPP26cysmutREV with template GFP-NbHIPP26C152G 578

579

Live cell imaging of fluorescent proteins 580

Unless otherwise stated all constructs were delivered into N benthamiana leaves by agro-581

infiltration through the abaxial stomata Agrobacterium tumefaciens (strain LB4404) cultures 582

carrying the plasmid constructs used for live cell imaging were grown overnight (16 h) at 583

28oC in Luria Bertani (LB) media supplemented with Rifampicin (50 mgL) and 584

Spectinomycin (100 mgL) Cells were collected by centrifugation at 3500 rpm for 15 min 585

and then resuspended in infiltration buffer (10 mM MgCl2 10 mM MES 150 μM 586

Acetosyringone) to a final OD600 of 01 before infiltration into the abaxial side of N 587

benthamiana leaves All confocal imaging was conducted using a Leica TCS-SP2 AOBS 588

spectral confocal scanner (Leica Microsystems GmbH Heidelberg Germany) Unless 589

otherwise stated images were obtained using a Leica HCX APO x6309W water-dipping 590

lens and whole lesions using a HCX PL Fluotar times16005 lens GFP and YFP were imaged 591

sequentially GFP excitation at 488 nm emission at 490 to 510 nm YFP excitation at 514 592

nm emission at 535 to 545 nm GFP and mRFP were imaged sequentially GFP excitation at 593

488 nm emission at 500 to 530 nm mRFP excitation at 561 nm emission at 590 to 630 nm 594

Images of GUS localisation were taken on a Zeiss AxioImager microscope (Carl Zeiss Ltd 595

Cambridge UK) equipped with a Zeiss Axiocam HRC camera Images were prepared using 596

Adobe Photoshop CS5 597

598


20

Reverse transcription quantitative real-time polymerase chain reaction (RT-qPCR) 599

Five-week-old seedlings of N benthamiana were manually inoculated with leaf extracts from 600

PMTV-infected N benthamiana or mock-inoculated plants and grown in a Snijders growth 601

cabinet providing a 12 h day (with a light intensity setting of 375 micromol m-2 s-1) 12 h night at 602

constant 14oC Total RNA was isolated from leaf samples using the RNeasy Plant Minikit 603

with the on-column DNaseI treatment for removal of genomic DNA contamination according 604

to the manufacturers protocol (Qiagen) Reverse transcription of 5microg of total RNA was 605

performed using Ready-To-Go-You-Prime-First Strand Beads (GE Healthcare) according to 606

the manufacturers protocol cDNA was used as a template for real-time PCR using the 607

Universal Probe Library System ((httpswwwroche-applied-608

sciencecomsisrtpcruplindexjsp) Reactions were performed in 25 μl containing 1 x 609

FastStart TaqManreg Probe Master (supplemented with ROX reference dye) Gene-specific 610

primers and probes were used at a concentration of 02 μM and 01 μM respectively 611

Thermal cycling conditions were 95 degC for 10 min followed by 40 cycles (15 s at 94 degC 60 s 612

at 60 degC) Relative expression levels were calculated and the primers validated using the 613

ΔΔCt method (Livak 1997) using data obtained from the reference controls PROTEIN 614

PHOSPHATASE 2A (PP2A Accession TC21939) F-Box protein (F-BOX Accession 615

Nibenv03Ctg24993647) CYCLIN-DEPENDENT KINASE 2 (CDC2 Accession Q40482) 616

The Universal ProbeLibrary (UPL) primer pairs and probe sequences were as follows 617

cdc2FORcdc2REV with UPL probe number 19 F-BOXFORF-BOXREV with UPL probe 618

number 1 PP2AFORPP2AREV with UPL probe number 22 HIPP26QFORHIPP26QREV 619

with UPL probe number 112 Primer sequences are provided in the Supplemental Table 620

621

Tobacco rattle virus-mediated VIGS 622

To facilitate the use of tobacco rattle virus (TRV)-mediated VIGS the 012 kb fragment of 623

NbHIPP26 cDNA was cloned into TRV00 (TRV RNA2 infectious cDNA clone) in an anti-624

sense orientation Two weeks post TRV-RNA1 + TRVHIPP26 infection reverse 625

transcription (RT)-PCR tests were carried out on upper (non-inoculated infected) leaves using 626

primers designed to amplify endogenous NbHIPP26RNA transcripts 627

Agrobacterium tumefaciens (strain C58C1) cultures carrying the TRV-RNA1 plasmid and 628

TRV-RNA2 cassettes were grown overnight collected by centrifugation at 3500 rpm for 10 629

min and then resuspended in infiltration buffer to a final OD600 of 05 Equal volumes of 630


21

TRV-RNA2-expressing cells and the TRV-RNA1 cultures were mixed and infiltrated into 631

the abaxial side of two N benthamiana leaves on seedlings at the four-leaf stage Plants were 632

grown for two weeks prior to challenging with PMTV The uppermost leaves were collected 633

and assayed for NbHIPP26 silencing by RT-qPCR 634

635

RT-qPCR for PMTV Results were obtained from two RT-qPCR experiments (three plants 636

per treatment either TRVHIPP26 or TRV00) Reverse transcription of 1microg of total RNA 637

was performed using an iScript cDNA synthesis kit (Bio-Rad) according to the 638

manufacturerrsquos instructions Four microl of ten-fold diluted cDNA was used as a template for 639

real-time PCR Reactions were performed in a 20 microl reaction containing 2x DyNAmo Flash 640

SYBR Green master mix supplemented with ROX passive reference dye along with 02 microM 641

primers specific to the respective gene of interest Data analysis was performed using the 642

Bio-Rad CFX manager 31 (Bio-Rad) 643

In order to obtain a standard curve ten-fold serial dilutions for respective plasmids of RNA-644

Rep RNA-CP and RNA-TGB were made from 100 pgmicrol to 001 pgmicrol Four microl of the 645

diluted plasmid was used as a template for the real-time PCR Total number of viral copies 646

was calculated using the formula of ldquoNo of molecules = (ng of dsDNA) x (no of 647

moleculesmole) x (1Number of bases) x (1660 g) x (110^9 ngg)rdquo The Avogadro constant 648

of 6023 x 10^23 moleculesmole was used to calculate the number of copies and the formula 649

weight of base pairs in dsDNA was considered as 660 g See also copy number calculator for 650

real-time PCR httpscienceprimercomcopy-number-calculator-for-realtime-pcr 651

652

S-acylation assays 653

Protein S-acylation state was assessed as described by Hemsley et al 2008 and Forrester et 654

al 2011 with some modifications GFP-tagged NbHIPP26 constructs were transiently 655

expressed in N benthamiana using agro-infiltration along with the p19 silencing suppressor 656

(Voinnet et al 2003) at OD600 of 01 for both Flash frozen tissue was ground in liquid 657

nitrogen to a fine powder and solubilised in four volumes of TENS buffer (100 mM TrisHCl 658

pH72 5 mM EDTA 150 mM NaCl 25 SDS) with protease inhibitors (Sigma P9599 5 ul 659

ml-1) and 25 mM N-ethylmaleimide (NEM) at 20degC for 10 minutes with constant mixing 660

Lysates were clarified for 5 minutes at 3000 x g passed through two layers of miracloth and 661


22

re-centrifuged at 16000 x g for 10 minutes all at 20degC The clarified supernatant was 662

retained and protein concentration was calculated using a BCA assay One mg of protein was 663

diluted to 1 ml final volume in TENS buffer with protease inhibitors and 25 mM NEM and 664

incubated with constant mixing for 1 hour at 20degC Samples were precipitated with 665

chloroformmethanol (Wessel and Flugge 1984) briefly air dried and resuspended in 1 ml 666

(100 mM TrisHCl pH72 5 mM EDTA 150 mM NaCl 6M Urea 2 SDS) with heating to 667

37degC and gentle mixing Samples were then split into two 05-ml aliquots in 15-ml 668

microfuge tubes One aliquot was treated with 05 ml of 1M hydroxylamineHCl (pH72 with 669

10M NaOH made fresh) while the other (negative control) was treated with 05 ml of 1M 670

NaCl After brief mixing 100 microl was removed from each as a loading control and incubated 671

at 20degC for 1 hour before precipitation with chloroformmethanol To the remainder 40 microl of 672

50 thiopropyl sepharose 6B bead suspension in TENS buffer was added to capture S-673

acylated proteins and gently mixed at 20degC for 1 hour Beads were washed 3x with 1 ml 674

TENS buffer for 5 minutes each then dried by aspiration Loading controls and bead samples 675

were resuspended in a 2x SDS-PAGE sample buffer containing 6M urea and 50 microl ml-1 -676

mercaptoethanol and heated at 37degC for 30 minutes with frequent mixing Samples were 677

subsequently run on 125 Laemli SDS-PAGE gels and blotted to a PVDF membrane 678

EGFP-HIP26 variants were detected using an anti-GFP monoclonal antibody (Abgent 679

AM1009a) Blots were imaged using GeneSys on a GBOX XT4 GelDoc system and 680

quantified using GeneTools (Syngene) 681

682

Preparation of NbHIPP26 promoter construct and production of transgenic plants 683

The 1128 bp region upstream of the translational start site of NbHIPP26 gene was amplified 684

using primers HIPProFOR and HIPProREV on genomic DNA of N benthamiana (Nb-1) 685

The resulting PCR fragment was cloned between SacII-XbaI sites of a binary vector pORE-686

R2 (Coutu et al 2007) in front of the uidA gene The resulting vector was named pORE-687

R2NbHIPP26 This vector was transferred to Agrobacterium tumefaciens (strain AGL1) 688

Wild-type N benthamiana (Sainsbury) plants were transformed and transgenic plants were 689

regenerated as described by Clemente (2006) 690

691

GUS-staining of transgenic plants 692


23

The HIPP26prouidA transgenic N benthamiana were grown in a glasshouse (21degC day and 693

16 degC night 18 h day length with supplementary lighting of 250 W m-2) Samples of leaf and 694

petiole were taken from plants 10 weeks post germination and embedded in 3 agar (Oxoid 695

No1 Oxoid Ltd Basingstoke UK) to allow suitable orientation in a further block of agar 696

mounted on a vibroslice (Model 752M Campden Instruments Ltd Loughborough UK) to 697

allow transverse sections to be cut through major veins in the leaf lamina Sections of fresh 698

tissue were cut approximately 200 microm thick and dropped immediately into GUS assay buffer 699

(28 mM NaH2PO4 72 mM Na2HPO4 [pH 72] containing 02 wv Triton X-100 2 mM 700

each of potassium hexacyanoferrate(III) and potassium hexacyanoferrate(III) trihydrate) 701

before being vacuum infiltrated and incubated overnight in the dark at 37degC Sections were 702

subsequently washed in several changes of 70 ethanol and stored in 70 ethanol before 703

being mounted in water and imaged using a Leica DMFLS light microscope Photographs 704

were collected using a Zeiss Axiocam to show the distribution of GUS stain 705

Accession Numbers 706

Arabidopsis N Benth HIPP26 Niben101Scf02621g040261 707

708

709

Supplemental Data 710

Supplemental Fig 1 Arabidopsis HIPP family phylogeny 711

Supplemental Fig 2 Cis regulatory elements upstream of the HIPP26 ORF 712

Supplemental Table List of primers and sequences 713

Supplemental Movie S1 Motile vesicles tagged with GFP-HIPP26 714

715

ACKNOWLEDGEMENTS 716

We thank Michael Goodin for the RFP-labelled fibrillarin plants and Indu Sama and Nina 717

Lukovitskaya for technical assistance with VIGS 718

719


24

Figure legends 720

Figure 1 HIPP phylogeny A Diagrammatic representation of the phylogenetic relationship 721

between A44 and five HIPP families (de Abreu-Neto et al 2013) A44 is related to 722

Arabidopsis HIPP sub family II and most closely related to HIPP26 (for full phylogeny see 723

Supplementary Fig 1) B Multiple alignments of HIPP26 proteins from Arabidopsis thaliana 724

(Arath) Nicotiana benthamiana (Benth) Solanum lycopersicum (Tomato) and Solanum 725

tuberosum (Potato) with A44 The alignments were made using MUSCLE with default 726

parameters (Edgar 2004) The line below the alignment marks conserved positions () 727

single fully conserved residue () lsquostrongrsquo group fully conserved scoring gt05 in the Gonnet 728

PAM 250 matrix () lsquoweakerrsquo group fully conserved scoring le05 in the Gonnet PAM 250 729

matrix C Percentage sequence identities for the four HIPP26 proteins and A44 shown in the 730

alignment in Fig 1B 731

732

Figure 2 Yeast two-hybrid interaction analysis A Virus movement proteins potato mop-top 733

virus (PMTV) TGB1 beet virus Q (BVQ) TGB1 tobacco mosaic virus (TMV) 30K potato 734

virus X (PVX) TGB1 and barley stripe mosaic virus (BSMV) TGB1 proteins were fused to 735

the Lex A binding domain (BD) and tested for interaction with HIPP26 fused to the p6 736

activation domain (AD) on double drop out (+H) or triple drop out (-H) media B 737

Diagrammatic representation of wild type and mutants in PMTV TGB1 (∆84 and ∆149 738

deleted by aa 1-84 and aa 1-149 respectively) and wild type and mutants of HIPP26 739

showing the position of the cysteine residues changed to glycine in putative prenylation 740

(PrenylM) S-Acylation (C13G) and heavy metal-associated (HMAM) domains C TGB1 wild 741

type and deletion mutants were tested for interaction with HIPP26 PrenylM and HMAM D 742

Amino acid sequence alignment of PMTV and BVQ N-terminal amino acids 743

744

Figure 3 Sub-cellular localisations of HIPP26 and the HIPP26-TGB1 complex A The GFP-745

HIPP26 fusion protein localised to the plasma membrane and a population of small (~2 microm 746

diameter) motile vesicles (arrowhead) A small amount of cytosolic labelling and faint 747

transvacuolar strands were seen in some cells GFP-HIPP26 was also present in the nucleus 748

both in the nucleoplasm and nucleolus and to levels greater than would be expected as a 749

result of passive diffusion from the low levels in the cytosol HIPP26 was therefore targeted 750

to the nucleus B Co-expression with an mRFP-fibrillarin (a nucleolar marker) showed 751

precise co-localisation of GFP-HIPP26 in the nucleolus (indicated by arrowhead) while (C) 752


25

co-expression and co-localisation with mOrange-LTi6b (a plasma membrane marker) showed 753

that HIPP26 was also targeted to the plasma membrane In addition to the motile vesicles 754

that were seen in the cytoplasm of the cell periphery (cf A and the Supplemental Movie) 755

small non-motile punctate were also observed at the cell periphery (D) Co-localisation of 756

these structures with an mRFP-tagged TMV 30K protein (a plasmodesmatal marker) showed 757

that HIPP26 was also associated with a population of plasmodesmata (indicated by 758

arrowheads) 759

760

EF Bimolecular fluorescence complementation (BiFC) analysis of PMTV TGB1 and 761

NbHIPP26 in Nicotiana benthamiana epidermal cells showed co-expression of Y-HIPP26 762

and TGB1-FP and re-constitution of yellow fluorescence localized to microtubules (E) to the 763

nucleoplasm and strong accumulation in the nucleolus (F) G Co-expression of Y-HIPP26 764

and ∆84TGB1-FP showed re-constitution of yellow fluorescence localized to the 765

nucleoplasm and strong accumulation in the nucleolus with no microtubule labelling No 766

complementation was obtained in control experiments where Y-HIPP26 or TGB1-FP were 767

co-expressed with the complementary empty spilt YFP plasmid (no fluorescence was visible 768

so no images are shown) HIJ Co-localisation of GFP-HIPP26 and mRFP-TGB1 (H GFP 769

I RFP and J merged) showed that both proteins localized to the nucleoplasm nucleolus and 770

microtubules as expected from previously published data and the BiFC results K 771

Expression of GFP-HIPP26 in PMTV-infected cell showed GFP fluorescence accumulated 772

on microtubules (indicated by arrow heads) Scale bars A and D = 10 microm B = 5 microm C = 10 773

microm for left panel of pairs and 5 microm for right panel of pairs E = 10 microm F = 5 microm G = 10 774

microm and J = 10 microm (used for H-J) and K = 5 microm 775

776

Figure 4 HIPP26 S-acylation A HIPP26 was S-acylated under normal plant growth 777

conditions at Cys13 S-acylation was determined by the Acyl-RAC assay Free cysteine 778

residues were blocked with N-ethylmaleimide S-acyl groups on cysteine residues within 779

proteins were cleaved by hydroxylamine (Hyd +) to reveal free sulfhydryls Sulfhydryl 780

reactive thiopropyl Sepharose beads were used to capture free sulfhydryl-containing proteins 781

Negative controls lacked hydroxylamine (Hyd -) Signal strength in experimental lanes (EX 782

Hyd+) showed the level of S-acylation Loading controls (LC) demonstrated equal loading of 783

hydroxylamine-treated and -untreated samples onto thiopropyl Sepharose beads B Relative 784

quantification of blots shown in A Mutation of the C-terminal CVVM motif cysteine 785


26

(PrenylM) had a small effect on the S-acylation state (n = 2 P= 0103 Studentrsquos one-tailed t-786

test) while changing Cys13 to Gly effectively abolished S-acylation (n = 2 P = 005) Values 787

were calculated as intensity in EX Hyd+ lanes divided by intensity in LC Hyd+ lanes Values 788

are means represented as the proportion of wild type HIPP26 S-acylation Error bars indicate 789

plusmnSD 790

Figure 5 Sub-cellular localisation of GFP-tagged NbHIPP26 modified proteins Four 791

mutants expressing proteins with altered HMA (HMAM) or lipidation domains (S-AcylM 792

PrenylM and S-AcylM PrenylM) were investigated (A-D respectively) The HMA altered 793

protein (A) was broadly similar to the wild-type HIPP26 (cf Fig 3A) showing plasma 794

membrane labelling low levels of cytosolic labelling some small motile vesicle labeling and 795

accumulation in the nucleoplasm and nucleolus In comparison all three proteins altered in 796

lipidation domains (B C and D) showed a significant increase in their nuclear accumulation 797

(in both the nucleoli and nucleoplasm) and a reduction in the amount of plasma membrane 798

labelling accompanied by an increase in the level of cytosolic fluorescence (seen most clearly 799

in transvacuolar strands arrows) Motile vesicles were seen in all three lipidation-deficient 800

proteins and the phenotypes were broadly similar although cells expressing the doubly 801

modified protein S-AcylM PrenylM (D) showed fewer vesicles and more cytosolic 802

localisation Plasmolysis of cells expressing wild-type HIPP26 (WT E) and the three altered 803

lipidation domainproteins (F-H) were also investigated Here the fluorescent cytosol was 804

contained within the protoplast which retracted from the cell walls leaving a black space in 805

the extracellular spaceapoplast Arrowheads situated in the extracellular space point to the 806

plasma membrane of the retracted protoplasts Hechtian strands (stretched extensions of 807

plasma membrane that extend from the plasmolysed protoplast to the cell wall) membrane 808

blebs and vesicles were clearly visible in the apoplast of the WT (arrows) These structures 809

were not easily detected in bright-field images but GFP fluorescence associated with the 810

remaining membrane made them clearly visible in the confocal micrographs shown here 811

There were fewer of these structures and their morphology was somewhat altered in the cells 812

expressing the proteins altered in a single lipid domain (FG arrows) and much less apparent 813

in H suggesting that removal of both lipidation domains reduced association with the plasma 814

membrane in an additive way Scale bars A-H = 10 microm 815

Figure 6 Images of glucuronidase (GUS) expression in Nicotiana benthamiana 816

transformed with the NbHIPP26 predicted promoter sequences fused to the uidA gene GUS 817

staining in mature plants (A) mature leaves (B) and roots (C) was detected throughout the 818


27

vasculature of the mature plant but was not present in young sink leaves (A) Expression was 819

particularly strong in the roots and hypocotyl and in leaves the GUS staining was more 820

evident in older leaves (A ndash C) Expression in leaf veins appeared to follow the sink-source 821

transition GUS staining was evident in tip (source) veins while the basal (sink) portion was 822

clearer (B) When studied in more detail in leaf veins GUS was specifically localised to 823

cells within the vascular bundle of all vein classes in mature leaves (D and E arrows point to 824

major vein classes and arrowheads to minor veins) At low magnification (D and E) there 825

appeared to be little or no GUS staining in ground tissues between the veins and intact leaf 826

petioles did not show staining Once cut however GUS was visible in the vascular bundles 827

of petioles Images D and E show photographs of intact mature N benthamiana leaves 828

photographed using a combination of bottom and top-lighting F and G show GUS staining 829

in transverse sections through major veins (F and G) GUS staining localised to the periphery 830

of many cells throughout the The vascular bundle (F) is delineated with a dotted black line 831

and phloem regions are outlined with red dotted lines Some GUS staining was detected in 832

cells surrounding the vascular bundles but much less than inside the vascular system (G) 833

shows an enlargement of the phloem bundle marked in F with a red arrow Blue GUS 834

staining can clearly be seen in Xylem parenchyma (XP) phloem (P) cells but is generally 835

absent from metaxylem vessels (X) and bundle sheath (BS) are shown Scale bars A = 2 836

mm B = 500 microm C = 50 microm and D = 20 microm (H) Relative NbHIPP26 expression in different 837

tissues measured by reverse transcription quantitative real-time PCR The histogram bars 838

show mean values plusmnSD (n=3 two independent experiments) 839

840

Figure 7 Relative expression of NbHIPP26 in leaves of N benthamiana (A) Relative 841

expression in N benthamiana leaves after 30-240 min of drought stress (representative 842

experiment showing means plusmnSD for each time point n = 3) and (B) in PMTV-infected 843

leaves 12 days post inoculation (representative experiment showing means plusmnSD for each 844

time point n = 8) showing that both drought stress and PMTV infection up-regulate 845

NbHIPP26 expression 846

847

Figure 8 Representative images of PMTV- or mock-inoculated N benthamiana plants after 848

drought stress (A) Mock- or (B) PMTV-inoculated plants after water was withheld for 13 849

days showing that PMTV-infected plants are more resilient to wilting compared with mock 850


28

Representative mock- (CDE) and PMTV-inoculated plants (FGH) before treatment (CF) 851

after withholding water (DG) and after re-watering (EH) showing that PMTV-infected 852

plants have produced green shoots and show recovery from drought treatment in contrast to 853

the mock-inoculated plants (E) 854

Figure 9 Effects of the knock down of NbHIPP26 expression on PMTV accumulation in 855

upper non-inoculated leaves A Gene silencing of NbHIPP26 gene in TRVHIPP26-infected 856

plants Total RNAs were extracted from the upper leaves 14 days post infiltration (dpi) and 857

were used for RT-qPCR Data are means plusmn SD from three experiments (n =14 for each 858

treatment) NbF-BOX and NbPP2A were used as normalization controls B Effect of HIPP26 859

gene silencing on viral RNA levels in upper non-inoculated leaves Total RNAs were 860

extracted from the upper leaves 14 dpi with PMTV and used for absolute RT-qPCR Ten-fold 861

serial dilutions of full-length infectious cDNA clones of RNA-Rep RNA-CP and RNA-TGB 862

were used to obtain standard curves and determine viral RNA copy number NbF-BOX and 863

NbPP2A were used as normalization controls Data are means plusmnSD from four independent 864

experiments with three or four biological replicates each Asterisks indicate statistically 865

significant differences compared with TRV00 (t test Plt005) C ELISA was used to 866

determine PMTV infection in inoculated and upper non-inoculated leaves of treated plants 867

Aabsorbance values (indicateding presence of PMTV capsid protein) in samples of 868

inoculated leaves were similar in both treatments but were gt50 greater in the upper non-869

inoculated leaves of plants inoculated with empty vector showing suppression of long 870

distance movement of PMTV particles in NbHIPP26 knock down plants D RT-qPCR 871

quantification of RNA-Rep and RNA-TGB in upper non-inoculated leaves of plants 872

challenged with these two PMTV genomic components (mean plusmn SD n = 6 two independent 873

experiments) Plt001 P=lt005 Studentrsquos two tailed test 874

875

Figure 10 Model of the mechanism of long distance movement In a PMTV infection 876

TGB1 to microtubules and traffics to the nucleus where it is localises in the nucleoplasm and 877

nucleolus (as shown in the non-vascular cell (NVC blue) HIPP26 is mainly expressed 878

inside the vascular bundle predominantly in regions of the bundle containing phloem cells 879

(phloem parenchyma companion cells and sieve elements) and also in other vascular 880

parenchyma cells which can sometimes be identified as xylem parenchyma (XP) due to their 881

proximity to metaxylem elements Once the virus reaches a vascular bundle where HIPP26 is 882


29

expressed the TGB1 binds near the CVVM (prenyl domain) of the NbHIPP26 at the plasma 883

membrane most likely in the vicinity of plasmodesmata This de-S-acylates the HIPP26 884

which releases the NbHIPP26TGB1 complex from the plasma membrane into the cytosol 885

from where it localises to microtubules and subsequently moves via importin-α-directed 886

transport to the nucleus and nucleolus where HIPP26 interacts with the transcriptional 887

activator ZFHD1 Interaction with ZFHD1 stimulates host proteins andor stress-response 888

factors such as NAC transcription factors along with molecules that increase the size 889

exclusion limit (SEL) of the plasmodesmata at the VPcompanion cell (CC) interface These 890

plasmodesmata are seen as a key control point for entry into the sieve element (SE)CC 891

complex and an important barrier to systemic spread for viruses The pore-plasmodesma 892

units (PPU) that connect CC and SE are known to have a large SEL and so the SECC 893

complex has been proposed to be a single domain where even large molecules can move 894

relatively freely into the SE It is difficult to differentiate between what may occur in the 895

phloem parenchyma and companion cells protein expression and interactions may be 896

different or the same in these cell types indicated by the question mark in the companion cell 897

nucleus The key point is that we believe the virus exploits the systemic signalling response 898

to drought in order to gain entry into the phloem Once inside the phloem tissue in the 899

presence of host proteins that gate plasmodesmata virions or RNPs can enter the SE for 900

systemic spread 901

Supplemental Fig 1 Arabidopsis HIPP family phylogeny Phylogenetic relationships 902

between A44 and 45 HIPP family proteins from Arabidopsis were previously identified by de 903

Abreu-Neto (de Abreu-Neto et al 2013) The symbols at each node denote five HIPP 904

families (de Abreu-Neto et al 2013) Diamond group I triangle group II white circle 905

group III black circle group IV square group V The phylogeny was reconstructed using 906

the neighbour-joining method in MEGA6 (Tamura et al 2013) using a bootstrap mode with 907

1000 replications The tree shown is the bootstrap consensus 908

Supplemental Fig 2 Cis regulatory elements upstream of the HIPP26 ORF Schematic 909

representation of potential cis regulatory elements involved in stress responses identified in 910

the nucleotide sequence 0-2 kbp upstream of the predicted NbHIPP26 ORF and identified 911

using the PLACE database (wwwdnaaffrcgojpPLACE) Predicted cis regulatory elements 912

are shown by coloured bars and their location with respect to the initiation codon of 913

NbHIPP26 shown on the grey bar CCATBOX1 (found in the promoter of heat shock protein 914

genes) MYB (found in the promoter of dehydration-responsive genes) ERD (Early response 915


30

to drought) W-Box (found in salicylic acid responsive promoters) MYC (found in the 916

promoter of dehydration-responsive genes) ABA responsive (found in abscisic acid and 917

stress related promoters) GT-1 Box (found in salicylic acid responsive promoters) LTRE 918

(low temperature responsive element) 919

Supplementary Table List of primers and sequences 920

Supplementary Movie Motile vesicles tagged with GFP-HIPP26 Expression of GFP-921

HIPP26 revealed GFP in small motile vesicles (approx 2 microm diameter) that move through 922

the cytoplasm and along cytoplasmic (trans-vacuolar) strands 923

924

LITERATURE CITED 925

926

Altschul SF Gish W Miller W Myers EW Lipma D (1990) Basic local alignment search 927

tool J Mol Biol 215 403-410 928

Ashery U Yizhar O Rotblat B Elad-Sfadia G Barkan B Haklai R Kloog Y (2006) 929

Spatio temporal organization of Ras signaling rasosomes and the galectin switch Cell Mol 930

Neurobiol 26 471-95 931

Bartel PL Chien CT Sternglanz R Fields S (1993) Using the two-hybrid system to detect 932

protein-protein interactions In Cellular Interactions in Development A Practical Approach 933

D A Hartley (ed) Oxford University Press Oxford pp 153-179 934

Barth O Vogt S Uhlemann R Zschiesche W Humbeck K (2009) Stress induced and 935

nuclear localized HIPP26 from Arabidopsis thaliana interacts via its heavy metal associated 936

domain with the drought stress related zinc finger transcription factor ATHB29 Plant Mol 937

Biol 69 213ndash226 938

939

Baulcombe DC Chapman S Santa Cruz S (1995) Jellyfish green fluorescent protein as a 940

reporter for virus infections Plant J Jun 7 1045-1053 941

942

Benson DA Cavanaugh M Clark K Karsch-Mizrachi I Lipman DJ Ostell J Sayers 943

EW (2013) GenBank Nucleic Acids Res Jan 41(Database issue) D36-42 944

945


31

Boulon S Westman BJ Hutten S Boisvert F-M Lamond AI (2010) The Nucleolus under 946

Stress Molecular Cell 40 216-227 947

948

Buhtz A Springer F Chappell L Baulcombe DC Kehr J (2008) Identification and 949

characterization of small RNAs from the phloem of Brassica napus Plant J 53 739ndash749 950

951

Buhtz A Pieritz Janin Springer F Kehr J (2010) Phloem small RNAs nutrient stress 952

responses and systemic mobility BMC Plant Biology 10 64 953

Choy E Chui VK Silletti J Feoktistov M Morimoto T Michaelson D Ivanov IE 954

Philips MR (1999) Endomembrane trafficking of Ras The CAAX motif targets proteins to 955

the ER and Golgi Cell 9869-80 956

Clemente T (2006) Nicotiana (Nicotiana tobacum Nicotiana benthamiana) pp 143ndash154 In 957

Wang K (ed) Methods in Molecular Biology vol 343 Agrobacterium Protocols 2e 958

volume 1 Humana Press Inc Totowa New Jersey 959

Collum TD Padmanabhan MS Hsieh Y-C Culver JN (2016) Tobacco mosaic virus-960

directed reprogramming of auxinindole acetic acid protein transcriptional responses 961

enhances virus phloem loading Proc Natl Acad Sci USA 10 113 (19)E2740-9 962

963

Cowan GH Torrance L Reavy B (1997) Detection of potato mop-top virus capsid 964

readthrough protein in virus particles J Gen Virol 78 1779-1783 965

966

Coutu C Brandle J Brown D Brown K Miki B Simmonds J Hegedus DD (2007) 967

pORE A modular binary vector series suited for both monocot and dicot plant 968

transformation Transgenic Res 16 771ndash781 969

Crowell DN (2000) Functional implications of protein isoprenylation in plants Progress in 970

Lipid Research 39 393-408 971

de Abreu-Neto JB Turchetto-Zolet AC de Oliveira LF Zanettini MH Margis-Pinheiro 972

M (2013) Heavy metal-associated isoprenylated plant protein (HIPP) characterization of a 973

family of proteins exclusive to plants FEBS J 280 1604-16 974

975


32

Dykema PE Sipes PR Marie A Biermann BJ Crowell DN Randall SK (1999) A new 976

class of proteins capable of binding transition metals Plant Mol Biol 41 139ndash150 977

978

Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high 979

throughput Nucleic Acids Res 32(5) 1792ndash1797 980

Fernandez-Calvino L Faulkner C Walshaw J Saalbach G Bayer E Benitez-Alfonso 981

Y Maule A (2011) Arabidopsis Plasmodesmal Proteome PLoS ONE 6(4) e18880 982

Fernandez-Pozo N Menda N Edwards JD Saha S Tecle IY Strickler SR Bombarely 983

A Fisher-York T Pujar A Foerster H Yan A Mueller LA The Sol Genomics Network 984

(SGN)mdashfrom genotype to phenotype to breeding Nucleic Acids Res (28 January 2015) 43 985

(D1) D1036-D1041 986

Forrester MT Hess DT Thompson JW Hultman R Moseley MA Stamler JSCasey PJ 987

(2011) Site-specific analysis of protein S-acylation by resin-assisted capture M J Lipid Res 988

52393-8 989

Formstecher E Aresta S Collura V Hamburger A Meil A Trehin A Reverdy C Betin 990

V Maire S Brun C Jacq B Arpin M Bellaiche Y Bellusci S Benaroch P Bornens M 991

Chanet R Chavrier P Delattre O Doye V Fehon R Faye G Galli T Girault JA Goud 992

B de Gunzburg J Johannes L Junier MP Mirouse V Mukherjee A Papadopoulo D 993

Perez F Plessis A Rosseacute C Saule S Stoppa-Lyonnet D Vincent A White M Legrain P 994

Wojcik J Camonis J Daviet L (2005) Protein interaction mapping a Drosophila case 995

study Genome Res 15 376-84 996

997

Fromont-Racine M Rain JC Legrain P (1997) Toward a functional analysis of the yeast 998

genome through exhaustive two-hybrid screens Nat Genet 16 277-82 999

1000

Fukuoka S Saka N Koga H Ono K Shimizu T Ebana K Hayashi N Takahashi A 1001

Hirochiko H Okuno K Yano M (2009) Loss of function of a proline-containing protein 1002

confers durable disease resistance in rice Science 325 998ndash1001 1003

1004


33

Gao W Xiao S Li H-Y Tsao S-W Chye M-L (2009) Arabidopsis thaliana acyl-CoA-1005

binding protein ACBP2 interacts with heavy-metal-binding farnesylated protein AtFP6 New 1006

Phytologist 181 89ndash102 1007

1008

Giavalisco P Kapitza K Kolasa A Buhtz A Kehr J (2006) Towards the proteome of 1009

Brassica napus phloem sap Proteomics 6 896ndash909 1010

1011

Goodin MM Chakrabarty R Banerjee R Yelton S DeBolt S (2007) New gateways to 1012

discovery Update on live-cell imaging in plants Plant Physiol 145 1100-1109 1013

1014

Hemsley PA Taylor L Grierson CS (2008) Assaying protein palmitoylation in plants 1015

Plant Methods 11 42 1016

1017

Hemsley PA (2015) The importance of lipid modified proteins in plants New Phytologist 1018

205 476-89 1019

Karimi M Inzeacute D Depicker A (2002) Gateway vectors for agrobacterium-mediated plant 1020

transformation Trends Plant Sci 7 193-195 1021

Kehr J Buhtz A (2008) Long distance transport and movement of RNA through the phloem 1022

J Exp Bot 59 85ndash92 1023

1024

Kim SH Ryabov EV Kalinina NO Rakitina DV Gillespie T MacFarlane S Taliansky 1025

M (2007) Cajal bodies and the nucleolus are required for a plant virus systemic infection 1026

EMBO J 26 2169-2179 1027

1028

Livak KJ (1997) Relative quantitation of gene expression Foster City CA USA PE 1029

Applied Biosystems 11ndash15 1030

1031

Lough TJ Lucas WJ (2006) Integrative Plant Biology role of the phloem in long-distance 1032

macromolecular trafficking Annu Rev Plant Biol 57 203-232 1033


34

Lukhovitskaya NI Cowan GH Vetukuri RR Tilsner J Torrance L Savenkov EI 1034

(2015) Importin-α-guided accumulation of TGB1 in the nucleolus in virus systemic 1035

movement in Nicotiana benthamiana Plant Physiol 167 738-752 1036

Martin K Kopperud K Chakrabarty R Banerjee R Brooks R Goodin MM (2009) 1037

Transient expression in Nicotiana benthamiana fluorescent marker lines provides enhanced 1038

definition of protein localization movement and interactions in planta Plant J 59 150ndash162 1039

1040

Marmagne A Ferro M Meinnel T Christophe Bruley C Kuhn L Garin J Barbier-1041

Brygoo H Ephritikhine G (2007) A high content in lipid-modified peripheral proteins and 1042

integral receptor kinases features in the Arabidopsis plasma membrane proteome Mol Cell 1043

Proteomics 6 1980-96 1044

Maurer-Stroh S Eisenhaber F (2005) Refinement and prediction of protein prenylation 1045

motifs Genome Biol 26 (6)R55 1046

Padmanabhan MS Shiferaw H Culver JN (2006) The Tobacco mosaic virus replicase 1047

protein disrupts the localization and function of interacting AuxIAA proteins Mol Plant 1048

Microbe Interact 19864-73 1049

Pennazio S Roggero P Conti M (1996) Yield losses in virus-infected crops Arch 1050

Phytopathol Plant Prot 30 283-296 1051

Rambault A (2012) Figtree 142 httptreebioedacuksoftwarefigtree 1052

Rain JC Selig L De Reuse H Battaglia V Reverdy C Simon S Lenzen G Petel F 1053

Wojcik J Schaumlchter V Chemama Y Labigne A Legrain P (2001) The protein-protein 1054

interaction map of Helicobacter pylori Nature 409 211-215 1055

1056

Reavy B Arif M Cowan GH Torrance L (1998) Association of sequences in the coat 1057

proteinreadthrough domain of potato mop-top virus with transmission by Spongospora 1058

subterranean J Gen Virol 79 2343-2347 1059

1060


35

Shivprasad S Pogue GP Lewandowski DJ Hidalgo J Donson J Grill LK Dawson WO 1061

(1999) Heterologous sequences greatly affect foreign gene expression in tobacco mosaic 1062

virus-based vectors Virology 255 312ndash323 1063

1064

Solovyev AG Savenkov EI (2014) Factors involved in systemic transport of plant RNA 1065

viruses the emerging role of the nucleus J Exp Bot 65 1689-1697 1066

Sorek N Poraty L Sternberg H Bar E Lewinsohn E Yalovsky S (2007) Activation 1067

status-coupled transient S acylation determines membrane partitioning of a plant Rho-related 1068

GTPase Mol Cell Biol 27 2144-54 1069

Taliansky ME Brown JW Rajamaumlki ML Valkonen JP Kalinina NO (2010) 1070

Involvement of the plant nucleolus in virus and viroid infections parallels with animal 1071

pathosystems Adv Virus Res 77 119-158 1072

Tehseen M Cairns N Sherson S Cobbett CS (2010) Metallochaperone-like genes in 1073

Arabidopsis thaliana Metallomics 2 556ndash564 1074

Tilsner J Taliansky ME Torrance L (2014) Plant Virus Movement eLS published 1075

online 16 JUN 2014 | DOI 1010029780470015902a0020711pub2 Wiley 1076

Tran LS Nakashima K Sakuma Y Simpson SD Fujita Y Maruyama K Fujita M 1077

Seki M Shinozaki K Yamaguchi-Shinozaki K (2004) Isolation and functional analysis of 1078

arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-1079

element in the early responsive to dehydration stress 1 Promoter Plant Cell 16 2481-2498 1080

Tran LS Nakashima K Sakuma Y Osakabe Y Qin F Simpson SD Maruyama K 1081

Fujita Y Shinozaki K Yamaguchi-Shinozaki K (2007) Co-expression of the stress-1082

inducible zinc finger homeodomain ZFHD1 and NAC transcription factors enhances 1083

expression of the ERD1 gene in Arabidopsis Plant J 49 46-63 1084

1085

Torrance L Cowan GH Gillespie T Ziegler A Lacomme C (2006) Barley stripe mosaic 1086

virus-encoded proteins triple-gene block 2 and gamma-b localize to chloroplasts in virus-1087


36

infected monocot and dicot plants revealing hitherto-unknown roles in virus replication J 1088

Gen Virol 87 2403-11 1089

Torrance L Lukhovitskaya NI Schepetilnikov MV Cowan GH Ziegler A Savenkov 1090

EI (2009) Unusual Long-Distance Movement Strategies of Potato mop-top virus RNAs in 1091

Nicotiana benthamiana MPMI 22 381ndash390 1092

Torrance L Wright KM Crutzen F Cowan GH Lukhovitskaya NI Bragard C 1093

Savenkov EI (2011) Unusual features of pomoviral RNA movement Frontiers in 1094

Microbiology 2 article 259 1-7 1095

Turgeon R Wolf S (2009) Phloem transport cellular pathways and molecular trafficking 1096

Annu Rev Plant Biol 60 207-221 1097

Verchot-Lubicz J Torrance L Solovyev AG Morozov SY Jackson AO Gilmer D 1098

(2010) Viral movement strategies employed by triple gene block encoding viruses MPMI 1099

23 1231-1247 1100

Voinnet O Rivas S Mestre P Baulcombe D (2003) An enhanced transient expression 1101

system in plants based on suppression of gene silencing by the p19 protein of tomato bushy 1102

stunt virus Plant J 33 949-956 1103

Vojtek A Hollenberg SM (1995) Ras-Raf interaction two-hybrid analysis Method 1104

Enzymol 255 331-42 1105

1106

Waterworth HE Hadidi A (1998) Economic Losses Due to Plant Viruses In A Hadidi RK 1107

Khetarpal and N Koganezawa N eds Plant Virus Disease Control APS Press Minnesota 1108

pp 1-13 1109

1110

Wessel D Flugge UI (1984) A method for the quantitative recovery of protein in dilute 1111

solution in the presence of detergents and lipids Anal Biochem 138 141-3 1112

1113

Wojcik J Boneca IG Legrain P (2002) Prediction assessment and validation of protein 1114

interaction maps in bacteria J Mol Biol 323 763-70 1115


37

Wright K Cowan GH Lukhovitskaya N Tilsner J Roberts A Savenkov E Torrance L 1116

(2010) The N-terminal domain of PMTV TGB1 movement protein is required for nucleolar 1117

localization microtubule association and long-distance movement MPMI 23 1486-1497 1118

Xu P Chen F Mannas JP Feldman T Sumner LW Roossinck MJ (2008) Virus 1119

infection improves drought tolerance New Phytologist 180 911-921 1120

1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion

Duration of drought stress (min)

0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B

Figure 7 Relative expression of NbHIPP26 in leaves of N benthamiana (A) after 30-240

min drought stress (representative experiment showing means plusmnSDs for each time point n =

3) and (B) in PMTV-infected leaves 12 days post inoculation (representative experiment

showing means plusmnSDs for each time point n = 8) showing that both drought stress and

PMTV infection up-regulate NbHIPP26 expression





Parsed CitationsAltschul SF Gish W Miller W Myers EW Lipma D (1990) Basic local alignment search tool J Mol Biol 215 403-410

Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Ashery U Yizhar O Rotblat B Elad-Sfadia G Barkan B Haklai R Kloog Y (2006) Spatio temporal organization of Ras signalingrasosomes and the galectin switch Cell Mol Neurobiol 26 471-95


Bartel PL Chien CT Sternglanz R Fields S (1993) Using the two-hybrid system to detect protein-protein interactions In CellularInteractions in Development A Practical Approach D A Hartley (ed) Oxford University Press Oxford pp 153-179


Barth O Vogt S Uhlemann R Zschiesche W Humbeck K (2009) Stress induced and nuclear localized HIPP26 from Arabidopsis thalianainteracts via its heavy metal associated domain with the drought stress related zinc finger transcription factor ATHB29 Plant Mol Biol69 213ndash226


Baulcombe DC Chapman S Santa Cruz S (1995) Jellyfish green fluorescent protein as a reporter for virus infections Plant J Jun 71045-1053


Benson DA Cavanaugh M Clark K Karsch-Mizrachi I Lipman DJ Ostell J Sayers EW (2013) GenBank Nucleic Acids Res Jan41(Database issue) D36-42


Boulon S Westman BJ Hutten S Boisvert F-M Lamond AI (2010) The Nucleolus under Stress Molecular Cell 40 216-227Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Buhtz A Springer F Chappell L Baulcombe DC Kehr J (2008) Identification and characterization of small RNAs from the phloem ofBrassica napus Plant J 53 739ndash749


Buhtz A Pieritz Janin Springer F Kehr J (2010) Phloem small RNAs nutrient stress responses and systemic mobility BMC PlantBiology 10 64


Choy E Chui VK Silletti J Feoktistov M Morimoto T Michaelson D Ivanov IE Philips MR (1999) Endomembrane trafficking of RasThe CAAX motif targets proteins to the ER and Golgi Cell 9869-80


Clemente T (2006) Nicotiana (Nicotiana tobacum Nicotiana benthamiana) pp 143ndash154 In Wang K (ed) Methods in Molecular Biologyvol 343 Agrobacterium Protocols 2e volume 1 Humana Press Inc Totowa New Jersey


Collum TD Padmanabhan MS Hsieh Y-C Culver JN (2016) Tobacco mosaic virus-directed reprogramming of auxinindole acetic acidprotein transcriptional responses enhances virus phloem loading Proc Natl Acad Sci USA 10 113 (19)E2740-9


Cowan GH Torrance L Reavy B (1997) Detection of potato mop-top virus capsid readthrough protein in virus particles J Gen Virol 78 wwwplantphysiolorgon September 4 2020 - Published by Downloaded from

Copyright copy 2018 American Society of Plant Biologists All rights reserved

1779-1783Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Coutu C Brandle J Brown D Brown K Miki B Simmonds J Hegedus DD (2007) pORE A modular binary vector series suited for bothmonocot and dicot plant transformation Transgenic Res 16 771ndash781


Crowell DN (2000) Functional implications of protein isoprenylation in plants Progress in Lipid Research 39 393-408Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

de Abreu-Neto JB Turchetto-Zolet AC de Oliveira LF Zanettini MH Margis-Pinheiro M (2013) Heavy metal-associated isoprenylatedplant protein (HIPP) characterization of a family of proteins exclusive to plants FEBS J 280 1604-16


Dykema PE Sipes PR Marie A Biermann BJ Crowell DN Randall SK (1999) A new class of proteins capable of binding transitionmetals Plant Mol Biol 41 139ndash150


Edgar RC (2004) MUSCLE multiple sequence alignment with high accuracy and high throughput Nucleic Acids Res 32(5) 1792ndash1797Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Fernandez-Calvino L Faulkner C Walshaw J Saalbach G Bayer E Benitez-Alfonso Y Maule A (2011) Arabidopsis PlasmodesmalProteome PLoS ONE 6(4) e18880


Fernandez-Pozo N Menda N Edwards JD Saha S Tecle IY Strickler SR Bombarely A Fisher-York T Pujar A Foerster H Yan AMueller LA The Sol Genomics Network (SGN)mdashfrom genotype to phenotype to breeding Nucleic Acids Res (28 January 2015) 43 (D1)D1036-D1041


Forrester MT Hess DT Thompson JW Hultman R Moseley MA Stamler JSCasey PJ (2011) Site-specific analysis of protein S-acylation by resin-assisted capture M J Lipid Res 52393-8


Formstecher E Aresta S Collura V Hamburger A Meil A Trehin A Reverdy C Betin V Maire S Brun C Jacq B Arpin M Bellaiche YBellusci S Benaroch P Bornens M Chanet R Chavrier P Delattre O Doye V Fehon R Faye G Galli T Girault JA Goud B deGunzburg J Johannes L Junier MP Mirouse V Mukherjee A Papadopoulo D Perez F Plessis A Rosseacute C Saule S Stoppa-LyonnetD Vincent A White M Legrain P Wojcik J Camonis J Daviet L (2005) Protein interaction mapping a Drosophila case study GenomeRes 15 376-84


Fromont-Racine M Rain JC Legrain P (1997) Toward a functional analysis of the yeast genome through exhaustive two-hybridscreens Nat Genet 16 277-82


Fukuoka S Saka N Koga H Ono K Shimizu T Ebana K Hayashi N Takahashi A Hirochiko H Okuno K Yano M (2009) Loss of functionof a proline-containing protein confers durable disease resistance in rice Science 325 998ndash1001


Gao W Xiao S Li H-Y Tsao S-W Chye M-L (2009) Arabidopsis thaliana acyl-CoA-binding protein ACBP2 interacts with heavy-metal-binding farnesylated protein AtFP6 New Phytologist 181 89ndash102



Giavalisco P Kapitza K Kolasa A Buhtz A Kehr J (2006) Towards the proteome of Brassica napus phloem sap Proteomics 6 896ndash909Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Goodin MM Chakrabarty R Banerjee R Yelton S DeBolt S (2007) New gateways to discovery Update on live-cell imaging in plantsPlant Physiol 145 1100-1109


Hemsley PA Taylor L Grierson CS (2008) Assaying protein palmitoylation in plants Plant Methods 11 42Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Hemsley PA (2015) The importance of lipid modified proteins in plants New Phytologist 205 476-89Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Karimi M Inzeacute D Depicker A (2002) Gateway vectors for agrobacterium-mediated plant transformation Trends Plant Sci 7 193-195Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Kehr J Buhtz A (2008) Long distance transport and movement of RNA through the phloem J Exp Bot 59 85ndash92Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Kim SH Ryabov EV Kalinina NO Rakitina DV Gillespie T MacFarlane S Taliansky M (2007) Cajal bodies and the nucleolus arerequired for a plant virus systemic infection EMBO J 26 2169-2179


Livak KJ (1997) Relative quantitation of gene expression Foster City CA USA PE Applied Biosystems 11ndash15Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Lough TJ Lucas WJ (2006) Integrative Plant Biology role of the phloem in long-distance macromolecular trafficking Annu Rev PlantBiol 57 203-232


Lukhovitskaya NI Cowan GH Vetukuri RR Tilsner J Torrance L Savenkov EI (2015) Importin-α-guided accumulation of TGB1 in thenucleolus in virus systemic movement in Nicotiana benthamiana Plant Physiol 167 738-752


Martin K Kopperud K Chakrabarty R Banerjee R Brooks R Goodin MM (2009) Transient expression in Nicotiana benthamianafluorescent marker lines provides enhanced definition of protein localization movement and interactions in planta Plant J 59 150ndash162


Marmagne A Ferro M Meinnel T Christophe Bruley C Kuhn L Garin J Barbier-Brygoo H Ephritikhine G (2007) A high content inlipid-modified peripheral proteins and integral receptor kinases features in the Arabidopsis plasma membrane proteome Mol CellProteomics 6 1980-96


Maurer-Stroh S Eisenhaber F (2005) Refinement and prediction of protein prenylation motifs Genome Biol 26 (6)R55Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title


Padmanabhan MS Shiferaw H Culver JN (2006) The Tobacco mosaic virus replicase protein disrupts the localization and function ofinteracting AuxIAA proteins Mol Plant Microbe Interact 19864-73


Pennazio S Roggero P Conti M (1996) Yield losses in virus-infected crops Arch Phytopathol Plant Prot 30 283-296Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Rambault A (2012) Figtree 142 httptreebioedacuksoftwarefigtreePubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Rain JC Selig L De Reuse H Battaglia V Reverdy C Simon S Lenzen G Petel F Wojcik J Schaumlchter V Chemama Y Labigne ALegrain P (2001) The protein-protein interaction map of Helicobacter pylori Nature 409 211-215


Reavy B Arif M Cowan GH Torrance L (1998) Association of sequences in the coat proteinreadthrough domain of potato mop-topvirus with transmission by Spongospora subterranean J Gen Virol 79 2343-2347


Shivprasad S Pogue GP Lewandowski DJ Hidalgo J Donson J Grill LK Dawson WO (1999) Heterologous sequences greatly affectforeign gene expression in tobacco mosaic virus-based vectors Virology 255 312ndash323


Solovyev AG Savenkov EI (2014) Factors involved in systemic transport of plant RNA viruses the emerging role of the nucleus J ExpBot 65 1689-1697


Sorek N Poraty L Sternberg H Bar E Lewinsohn E Yalovsky S (2007) Activation status-coupled transient S acylation determinesmembrane partitioning of a plant Rho-related GTPase Mol Cell Biol 27 2144-54


Taliansky ME Brown JW Rajamaumlki ML Valkonen JP Kalinina NO (2010) Involvement of the plant nucleolus in virus and viroidinfections parallels with animal pathosystems Adv Virus Res 77 119-158


Tehseen M Cairns N Sherson S Cobbett CS (2010) Metallochaperone-like genes in Arabidopsis thaliana Metallomics 2 556ndash564Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Tilsner J Taliansky ME Torrance L (2014) Plant Virus Movement eLS published online 16 JUN 2014 | DOI1010029780470015902a0020711pub2 Wiley


Tran LS Nakashima K Sakuma Y Simpson SD Fujita Y Maruyama K Fujita M Seki M Shinozaki K Yamaguchi-Shinozaki K (2004)Isolation and functional analysis of arabidopsis stress-inducible NAC transcription factors that bind to a drought-responsive cis-element in the early responsive to dehydration stress 1 Promoter Plant Cell 16 2481-2498


Tran LS Nakashima K Sakuma Y Osakabe Y Qin F Simpson SD Maruyama K Fujita Y Shinozaki K Yamaguchi-Shinozaki K (2007)Co-expression of the stress-inducible zinc finger homeodomain ZFHD1 and NAC transcription factors enhances expression of theERD1 gene in Arabidopsis Plant J 49 46-63

Pubmed Author and TitleCrossRef Author and Title


Google Scholar Author Only Title Only Author and Title

Torrance L Cowan GH Gillespie T Ziegler A Lacomme C (2006) Barley stripe mosaic virus-encoded proteins triple-gene block 2 andgamma-b localize to chloroplasts in virus-infected monocot and dicot plants revealing hitherto-unknown roles in virus replication JGen Virol 87 2403-11


Torrance L Lukhovitskaya NI Schepetilnikov MV Cowan GH Ziegler A Savenkov EI (2009) Unusual Long-Distance MovementStrategies of Potato mop-top virus RNAs in Nicotiana benthamiana MPMI 22 381ndash390


Torrance L Wright KM Crutzen F Cowan GH Lukhovitskaya NI Bragard C Savenkov EI (2011) Unusual features of pomoviral RNAmovement Frontiers in Microbiology 2 article 259 1-7


Turgeon R Wolf S (2009) Phloem transport cellular pathways and molecular trafficking Annu Rev Plant Biol 60 207-221Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Verchot-Lubicz J Torrance L Solovyev AG Morozov SY Jackson AO Gilmer D (2010) Viral movement strategies employed by triplegene block encoding viruses MPMI 23 1231-1247


Voinnet O Rivas S Mestre P Baulcombe D (2003) An enhanced transient expression system in plants based on suppression of genesilencing by the p19 protein of tomato bushy stunt virus Plant J 33 949-956


Vojtek A Hollenberg SM (1995) Ras-Raf interaction two-hybrid analysis Method Enzymol 255 331-42Pubmed Author and TitleCrossRef Author and TitleGoogle Scholar Author Only Title Only Author and Title

Waterworth HE Hadidi A (1998) Economic Losses Due to Plant Viruses In A Hadidi RK Khetarpal and N Koganezawa N eds PlantVirus Disease Control APS Press Minnesota pp 1-13


Wessel D Flugge UI (1984) A method for the quantitative recovery of protein in dilute solution in the presence of detergents andlipids Anal Biochem 138 141-3


Wojcik J Boneca IG Legrain P (2002) Prediction assessment and validation of protein interaction maps in bacteria J Mol Biol 323763-70


Wright K Cowan GH Lukhovitskaya N Tilsner J Roberts A Savenkov E Torrance L (2010) The N-terminal domain of PMTV TGB1movement protein is required for nucleolar localization microtubule association and long-distance movement MPMI 23 1486-1497


Xu P Chen F Mannas JP Feldman T Sumner LW Roossinck MJ (2008) Virus infection improves drought tolerance New Phytologist180 911-921



Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

2

The authors responsible for distribution of materials integral to the findings presented in this 29

article are Lesley Torrance (LesleyTorrancehuttonacuk) and Eugene Savenkov 30

(eugenesavenkovsluse) 31

Abstract 32

Virus movement proteins facilitate virus entry into the vascular system to initiate systemic 33

infection The potato mop-top virus (PMTV) movement protein TGB1 is involved in long-34

distance movement of both viral ribonucleoprotein complexes and virions Here our analysis 35

of TGB1 interactions with host Nicotiana benthamiana proteins revealed an interaction with 36

a member of the heavy metal-associated isoprenylated plant protein family HIPP26 which 37

acts as a plasma membrane-to-nucleus signal during abiotic stress We found that knockdown 38

of NbHIPP26 expression inhibited virus long-distance movement but did not affect cell-to-39

cell movement Drought and PMTV infection upregulated NbHIPP26 gene expression and 40

PMTV infection protected plants from drought In addition NbHIPP26 promoter-reporter 41

fusions revealed vascular tissue-specific expression Mutational and biochemical analysis 42

indicated that NbHIPP26 sub-cellular localisation at the plasma membrane and 43

plasmodesmata was mediated by lipidation (S-acylation and prenylation) as non-lipidated 44

NbHIPP26 was predominantly in the nucleus Notably co-expression of NbHIPP26 with 45

TGB1 resulted in a similar nuclear accumulation of NbHIPP26 TGB1 interacted with the C-46

terminal CVVM (prenyl) domain of NbHIPP26 and bimolecular fluorescence 47

complementation revealed that the TGB1-HIPP26 complex localized to microtubules and 48

accumulated in the nucleolus with little signal at the plasma membrane or plasmodesmata 49

These data support a mechanism where interaction with TGB1 negates or reverses NbHIPP26 50

lipidation thus releasing membrane-associated NbHIPP26 and re-directing it via 51

microtubules to the nucleus thereby activating the drought stress response and facilitating 52

virus long-distance movement 53

54

INTRODUCTION 55

The systemic infection of plants by viruses results in major economic losses in yield andor 56

quality of harvested tissues (Pennazio et al 1996 Waterworth and Hadidi 1998) To 57

achieve a systemic infection viruses must move from the initial point of entry to 58

neighbouring cells and then long distance in the vasculature to other leaves and organs 59


3





disease control 64














et al 2016) 78















4













104

RESULTS 105



















5
















1B and C) 138


















6













yeast 167





















7


































8


































9





255





























10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

3





disease control 64














et al 2016) 78















4













104

RESULTS 105



















5
















1B and C) 138


















6













yeast 167





















7


































8


































9





255





























10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

4













104

RESULTS 105



















5
















1B and C) 138


















6













yeast 167





















7


































8


































9





255





























10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

5
















1B and C) 138


















6













yeast 167





















7


































8


































9





255





























10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

6













yeast 167





















7


































8


































9





255





























10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

7


































8


































9





255





























10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

8


































9





255





























10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

9





255





























10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

10


































11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

11


























with PMTV 67 340

341






12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

12






























375

376

DISCUSSION 377


13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

13



































14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

14





















(Kim et al 2007) 431












15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

15


































16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

16

474

475
































17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

17


et al 2002) 507






























18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

18






Plant material 541






















563




19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

19




569










579



















598


20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

20























621











21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

21





635

















652











22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

22





















682









691



23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

23
















708

709






715




719


24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

24

Figure legends 720












732












744










25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

25







arrowheads) 759

760
















776











26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

26





plusmnSD 790






























27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

27






















840







847





28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

28

























875









29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

29



















systemic spread 901
















30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

30









924


926


tool J Mol Biol 215 403-410 928



Neurobiol 26 471-95 931







Biol 69 213ndash226 938

939



942



945


31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

31



948



951












963



966









975


32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

32



978








(D1) D1036-D1041 986



52393-8 989








997



1000




1004


33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

33




1008



1011



1014



1017


205 476-89 1019





1024



EMBO J 26 2169-2179 1027

1028



1031




34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

34







1040
















1056




1060


35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

35




1064





















1085




36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

36


Gen Virol 87 2403-11 1089











23 1231-1247 1100





Enzymol 255 331-42 1105

1106



pp 1-13 1109

1110



1113




37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

37






1121

1122








Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations







Control 30 60 90 120 240

Rel

ativ

e G

ene

Exp

ress

ion


0

05

1

15

2

Rel

ativ

e G

ene

Exp

ress

ion

Mock PMTV0

05

1

15

2A B































































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

























































































































Article File

Figure 1

Figure 2

Figure 3

Figure 4

Figure 5

Figure 6

Figure 7

Figure 8

Figure 9

Figure 10

Parsed Citations

Potato mop-top virus co-opts the stress sensor HIPP26 for long … · 3 60 (reviewed in Tilsner et...

Documents

Transcript of Potato mop-top virus co-opts the stress sensor HIPP26 for long … · 3 60 (reviewed in Tilsner et...