Calcium Spiking Patterns and the Role of theCalcium/Calmodulin-Dependent Kinase CCaMK in LateralRoot Base Nodulation of Sesbania rostrata C W

Ward Capoen,a,b,c,1 Jeroen Den Herder,a,b,1 Jongho Sun,c Christa Verplancke,a,b Annick De Keyser,a,b

Riet De Rycke,a,b Sofie Goormachtig,a,b Giles Oldroyd,c,1 and Marcelle Holstersa,b,1,2

a Department of Plant Systems Biology, Flanders Institute for Biotechnology, 9052 Gent, BelgiumbDepartment of Plant Biotechnology and Genetics, Ghent University, 9052 Gent, BelgiumcDepartment of Disease and Stress Biology, John Innes Centre, Norwich NR4 7UH, United Kingdom

Nodulation factor (NF) signal transduction in the legume-rhizobium symbiosis involves calcium oscillations that are

instrumental in eliciting nodulation. To date, Ca2+ spiking has been studied exclusively in the intracellular bacterial invasion

of growing root hairs in zone I. This mechanism is not the only one by which rhizobia gain entry into their hosts; the tropical

legume Sesbania rostrata can be invaded intercellularly by rhizobia at cracks caused by lateral root emergence, and this

process is associated with cell death for formation of infection pockets. We show that epidermal cells at lateral root bases

respond to NFs with Ca2+ oscillations that are faster and more symmetrical than those observed during root hair invasion.

Enhanced jasmonic acid or reduced ethylene levels slowed down the Ca2+ spiking frequency and stimulated intracellular

root hair invasion by rhizobia, but prevented nodule formation. Hence, intracellular invasion in root hairs is linked with a very

specific Ca2+ signature. In parallel experiments, we found that knockdown of the calcium/calmodulin-dependent protein

kinase gene of S. rostrata abolished nodule development but not the formation of infection pockets by intercellular invasion

at lateral root bases, suggesting that the colonization of the outer cortex is independent of Ca2+ spiking decoding.

INTRODUCTION

Leguminous plants have coevolved with nitrogen-fixing rhizobia

to establish a sophisticated root endosymbiosis. In a develop-

mental process guided by reciprocal signal exchange, the plants

form new organs, nodules, to house bacteria that reduce mo-

lecular dinitrogen and feed their host with ammonia. In most

studied interactions, nodulation occurs in a susceptible root

zone with developing root hairs (zone I). This process is activated

by bacterial signals, the lipochitooligosaccharidic nodulation

(Nod) factors (NFs) (D’Haeze and Holsters, 2002; Jones et al.,

2007). In response to NFs, root hairs form a curl that provides a

pocket for a microbial colony. Concomitantly, cortical cell divi-

sion is triggered for organ formation. Purified NFs elicit several

physiological responses in susceptible root hair cells, such as

calcium influx, membrane depolarization, and rhythmic Ca2+

oscillations (spiking) in and around the nucleus (Oldroyd and

Downie, 2004).

Themolecular basis of legume symbiosis has been particularly

well studied in Medicago truncatula and Lotus japonicus, in

which essential nodulation genes have been identified through

forward genetics and map-based cloning. Characterization

of plant mutants that are affected in NF signal perception

or transduction revealed that NFs are perceived by LysM

domain–containing receptor-like kinases, such as lysine motif

receptor-like kinase3 (LYK3)-LYK4/Nod factor perception (NFP)

of M. truncatula and Nod factor receptor1 (NFR1)/NFR5 of L.

japonicus (Ben Amor et al., 2003; Limpens et al., 2003; Madsen

et al., 2003; Radutoiu et al., 2003, 2007; Arrighi et al., 2006). Other

plant nodulation functions are common with the more ancient

symbiosis with arbuscular mycorrhiza and include putative cat-

ion transporters (does not make infection1 [DMI1] of M. trunca-

tula and CASTOR/POLLUX of L. japonicus) (Ane et al., 2004;

Imaizumi-Anraku et al., 2005), a leucine-rich repeat-type recep-

tor-like kinase (DMI2) of M. truncatula, symbiosis receptor-like

kinase (SYMRK) of L. japonicus, and nodulation receptor kinase

of M. sativa (Endre et al., 2002; Stracke et al., 2002), two

nucleoporins (NUP133 and NUP85) of L. japonicus (Kanamori

et al., 2006; Saito et al., 2007), and a calcium/calmodulin-

dependent kinase (CCaMK) of M. truncatula (Levy et al., 2004;

Mitra et al., 2004). Transcription factors of the GRAS (gibberellin-

insensitive, repressor of gal-3, and SCARECROW) family nodu-

lation signaling pathway (NSP1 and NSP2) of M. truncatula and

the ethylene response factor required for nodulation (ERN) family

of M. truncatula are involved in NF-specific gene expression.

Together with the transcriptional regulator nodulation inception

(NIN) identified in L. japonicus and M. truncatula, they regulate

downstream responses, such as cytokinin signaling for primor-

dium initiation (Schauser et al., 1999; Kalo et al., 2005; Smit et al.,

1 These authors contributed equally to this work.2 Address correspondence to marcelle.holsters@psb.vib-ugent.be.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: marcelle.holsters@psb.vib-ugent.be.CSome figures in this article are displayed in color online but in blackand white in the print edition.WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.066233

The Plant Cell, Vol. 21: 1526–1540, May 2009, www.plantcell.org ã 2009 American Society of Plant Biologists

2005; Gonzalez-Rizzo et al., 2006; Heckmann et al., 2006; Marsh

et al., 2007; Middleton et al., 2007; Murray et al., 2007; Tirichine

et al., 2007; Frugier et al., 2008).

Ca2+ oscillations have been shown to direct gene expression in

animal systems (Gu andSpitzer, 1995; Dolmetsch et al., 1997; De

Koninck and Schulman, 1998). In plants, Ca2+ oscillations are

involved in abscisic acid signaling in guard cells, in pollen tube

elongation, and in the NF signal transduction pathway of le-

gumes (Holdaway-Clarke et al., 1997; Li et al., 1998; Allen et al.,

2001; Oldroyd et al., 2001b; Oldroyd and Downie, 2006). NF-

induced Ca2+ spiking is correlated with nodulin gene expression,

is influenced by the hormones ethylene and jasmonate (JA) and

by the developmental context in the root, and is blocked by

inhibitors of Ca2+ pumps and channels (Pingret et al., 1998;

Oldroyd et al., 2001a; Engstrom et al., 2002; Miwa et al., 2006a,

2006b; Sun et al., 2006).

The nonnodulating mutants dmi3 ofM. truncatula and sym9 of

pea (Pisum sativum) show normal Ca2+ spiking and are affected

in a CCaMK-encoding gene (Levy et al., 2004; Mitra et al., 2004),

which is presumably required for the interpretation of the Ca2+

signature (Oldroyd and Downie, 2004). CCaMK has a kinase

domain, a calmodulin (CaM) binding domain with strong homol-

ogy to CaM-dependent kinase II in animals, and a C-terminal

Ca2+ binding domain with three EF hands homologous to the

neuronal Ca2+ binding protein visinin (Patil et al., 1995). Auto-

phosphorylation of CCaMK after EF hand binding of Ca2+ ions

enhances the Ca2+/CaM association that subsequently sup-

presses autoinhibition, thus activating the kinase domain and

allowing substrate phosphorylation (Patil et al., 1995; Gleason

et al., 2006; Tirichine et al., 2006). CCaMK is expressed in roots

and in developing nodules (Levy et al., 2004), and transcripts

have been detected in a few cell layers directly adjacent to the

meristem of indeterminate nodules. CCaMK localizes to the

nucleus of root cells, corresponding to the site where the Ca2+

spiking intensity is the highest and the possible target proteins

NSP1 and NSP2 accumulate (Ehrhardt et al., 1996; Kalo et al.,

2005; Limpens et al., 2005; Smit et al., 2005). Functional analysis

has indicated that the CCaMK proteins are important for nodule

organogenesis and root hair invasion and that gain of function

results in the formation of spontaneous nodules (Gleason et al.,

2006; Tirichine et al., 2006). Nonlegume CCaMK homologs can

interpret the NF-induced Ca2+ signature and restore nodule

formation in the M. truncatula dmi3 mutant, although not neces-

sarily allowing normal bacterial invasion (Gleason et al., 2006;

Godfroy et al., 2006). These data suggest that root hair invasion

has more stringent requirements for correct interpretation of

specific spiking patterns than initiation of organ development.

Nodule initiation mechanisms have mostly been studied in

legumes in which bacteria enter via root hair curling (RHC)

followed by intracellular invasion. However, the legume family is

diverse, and many variations occur on the theme of nodulation.

For instance, the tropical shrub Sesbania rostrata has versatile

nodulation features as an adaptation to temporarily flooded

habitats. Aeroponic roots of S. rostrata are covered with root

hairs, and nodules can form in the susceptible zone I via root hair

invasion according to the classic scheme.S. rostrata can skip the

RHC mode of invasion and nodulate on the stem at bases of

adventitious roots and also at lateral root bases (LRBs) of

submerged roots. During LRB nodulation, the microsymbiont

Azorhizobium caulinodans enters through cracks in the epider-

mis, colonizes the outer cortex, and forms intercellular infection

pockets mediated by NF-induced cell death. Although both RHC

and LRB invasions depend on NFs, the structural NF require-

ments are less stringent for LRB nodulation than for RHC

(D’Haeze and Holsters, 2002; Goormachtig et al., 2004a). Thus,

on the same plant, nodules can be formed at different positions

and, hence, in a different physiological and hormonal context.

Amajor determinant for the switch between nodulation modes

in S. rostrata is the gaseous hormone ethylene that accumulates

upon waterlogging (Goormachtig et al., 2004a). Ethylene is a

potent inhibitor of RHC invasion and of NF-induced Ca2+ spiking

in M. truncatula (Oldroyd et al., 2001a). In S. rostrata, ethylene

inhibits RHC invasion but positively regulates LRB nodulation

and is required, together with H2O2 and gibberellic acid, for

infection pocket formation and nodulation (D’Haeze et al., 2003;

Lievens et al., 2005). Hence, in S. rostrata, LRB invasion allows

nodulation under conditions that inhibit nodulation in zone I

(Goormachtig et al., 2004b). This versatility provides a tool to

investigate requirements that are specific to one invasion mode

or shared between both. At the gene expression level, LRB

nodulation seems simpler than the RHC mechanism (Capoen

et al., 2007).

Ca2+ spiking is a key component in the activation of zone I

nodulation via RHC invasion, but nothing is known about its role

in legume species with alternative modes of nodulation and

infection. We have assessed the importance of Ca2+ spiking

during LRB nodulation in S. rostrata. Under conditions that

promote LRB nodulation, NFs trigger faster Ca2+ oscillations

than during RHC nodulation. Modulation of the ethylene or JA

levels slowed down the Ca2+ spiking frequency and stimulated

RHC invasion but was incompatible with nodule development. In

parallel, we studied the function of CCaMK of S. rostrata during

LRB nodulation using RNA interference (RNAi).

Our data show that, although Ca2+ spiking is a common

component of the signaling pathways for RHC and LRB nodule

formation, the situation is different for each type of bacterial

invasion. Indeed, RHC infection is strongly correlated with Ca2+

oscillations of the appropriate frequency, while intercellular

rhizobial invasion at LRBs probably functions independently of

Ca2+ spiking and CCaMK.

RESULTS

Comparison of NF-InducedCa2+Oscillations at LRBs and in

Root Zone I

As a first approach to investigate the role of Ca2+ spiking during

LRB nodulation, we studiedNF-inducedCa2+ oscillations in zone

I and at LRBs. In aeroponic roots ofS. rostrata, growing root hairs

respond to azorhizobia with curling, infection thread invasion,

and concomitant formation of nodules in zone I (Goormachtig

et al., 2004a). To visualize Ca2+ spiking, we microinjected the

Ca2+-sensitive dye Oregon Green into zone I root hairs after the

root had been briefly submerged in liquidmedium (seeMethods).

The root hairs did not respond to NFs as far as Ca2+ responses

Calcium Spiking in Sesbania rostrata 1527

were concerned, an observation in accordance with the quick

inhibition of RHC nodulation by submergence (Goormachtig

et al., 2004a).

To circumvent this limitation, roots were grown hydroponically

in medium supplemented with 7 mM L-a-(2-aminoethoxyvinyl)-

glycine (AVG), an inhibitor of ethylene synthesis. Such roots have

normal root hairs and RHC nodulation (Goormachtig et al.,

2004a). Growing root hairs in zone I were microinjected with

OregonGreen and subsequently exposed toNFs. Approximately

50%of the cells responded and showedCa2+ oscillationswith an

average period of 179.8 6 77.6 s and an asymmetric spike

shape: the upward and downward phases corresponded to 21

and 79% of the spike duration, respectively (Figures 1A and 1D),

which is similar to the spike shape in M. truncatula zone I root

hairs treatedwithSinorhizobiummelilotiNFs (Figures 1B and 1E).

The M. truncatula spikes showed a rapid increase in Ca2+ that

constituted 16% of the spike duration and a slow reduction in

Ca2+ levels (Figure 1E). In S. rostrata, the average period was

Figure 1. NF-Induced Ca2+ Spiking Patterns in S. rostrata Compared with a M. truncatula Reference.

(A) to (C) Representative Ca2+ spiking traces; the percentage of visualized cells that initiated Ca2+ spiking upon exposure to NFs is shown, and the

number of cells tested is given in parentheses.

(A) Ca2+ spiking traces in zone I root hairs of S. rostrata.

(B) Ca2+ spiking traces in zone I root hairs of M. truncatula.

(C) Ca2+ spiking traces in root hair initials at LRBs of S. rostrata.

(D) to (F) Schematic representation of the average spike shape of traces depicted in (A) to (C), respectively. The numbers in the curve are averages of

the proportion of upward and downward parts of the spike.

(G) Statistical analysis of Ca2+ spiking shapes. Average times taken from at least 30 Ca2+ spikes are presented for several traces under each condition.

The upward phases (dark gray) were identical, but the downward phases (light gray) were significantly different (P < 0.001). Error bars indicate SD.

Statistically significant differences in comparison with S. rostrata LRBs are indicated with an asterisk as well as the P value.

1528 The Plant Cell

longer than inM. truncatula (179.86 77.6 s versus 92.86 24.0 s),

with a greater variation between cells as indicated by the higher

standard deviation (77.6 s compared with 24.0 s inM. truncatula;

Figures 1A, 1B, 1D, and 1E) (see also Miwa et al., 2006a).

In the absence of AVG, hydroponic roots of S. rostrata have no

root hairs, except at LRBs, where several root hair initials are

present (Mergaert et al., 1993). At these positions, NFs trigger

cortical cell division for nodule formation, cell death for invasion,

as well as outgrowth and deformation of the root hair initials.

However, the root hairs are not invaded by infection threads and

the bacteria colonize the cortex intercellularly. Antagonists of

ethylene synthesis or perception as well as inhibitors of reactive

oxygen species production inhibit both NF-dependent root hair

outgrowth and LRB nodulation (D’Haeze et al., 2003). As these

root hair initials located at LRBs were markers for NF responses

during LRB nodulation and were amenable to microinjection, we

screened them for NF-induced Ca2+ spiking. NF application

induced a distinct spiking signature in these root hair initials: 87%

of the cells responded with an average spiking period of 55.5 620.8 s (Figures 1C and 1F). The LRB-associated spikes were

more symmetrical than the zone I–associated spikes in M.

truncatula and S. rostrata, with upward and downward phases

representing 39 and 61% of the spike duration, respectively

(Figures 1F and 1G).

A drawback of Oregon Green as a calcium indicator is that it

cannot be used to measure changes in the amplitude of Ca2+

oscillations. To address this issue, we injected the different

cell types with a ratiometric calcium indicator, dextran-linked

1-[6-amino-2-(5-carboxy-2-oxazolyl)-5-benzofuranyloxy]-2-(2-

amino-5-methylphenoxy) ethane-N,N,N’,N’-tetraacetic acid,

penta-potassium salt (Fura-2). Amplitudes were measured in

zone I cells ofM. truncatula (n = 3) and in LRB cells of S. rostrata

under hydroponic conditions without (n = 6) and with 7 mM AVG

(n = 5). All spikes analyzed confirmed the nuclear localization of

NF-induced Ca2+ spiking and an amplitude (200 to 250 nM)

similar to published data (Figure 2A) (Walker et al., 2000). Thus,

the NF-induced Ca2+ oscillations had similar amplitudes in all

tested tissues (Figure 2B).

Phospholipase D (PLD) and Ca2+-ATPases have been impli-

cated in NF-induced Ca2+ spiking and gene expression in root

hairs of M. truncatula (den Hartog et al., 2001, 2003; Engstrom

et al., 2002). We used the Ca2+-ATPase antagonists cyclopia-

zonic acid (CPA) and 2,5-di-t-1,4-benzohydroquinone (BHQ) and

the PLD antagonist n-butanol to assess whether similar mech-

anisms are involved in the generation of LRB-associated Ca2+

spiking. Both CPA and BHQ inhibited NF-induced LRB Ca2+

spiking in S. rostrata (see Supplemental Figure 1 online). Sup-

pression by BHQ was transient, with spontaneous reversion of

the Ca2+ spiking within ;10 min after application, similarly to

what was observed in M. truncatula (Engstrom et al., 2002). The

primary alcohol n-butanol can accept the phosphatidyl moiety

produced by PLD, resulting in a decreased production of phos-

phatidic acid, in contrast with tert-butanol that provides a valu-

able control (Parmentier et al., 2003). Addition of 0.5% n-butanol

reversibly inhibited LRB-associated Ca2+ spiking, whereas tert-

butanol had no effect (see Supplemental Figure 1 online). These

data suggest that similar mechanisms generate Ca2+ spiking in

zone I root hairs and in root hair initials at LRBs.

Hormonal Modulation of the LRB-Associated Ca2+

Signature Correlates with Root Hair Invasion and Loss

of Nodulation

Ca2+ spiking in M. truncatula root hairs is influenced by hor-

mones. Ethylene has an inhibitory effect because addition of

the ethylene biosynthesis precursor 1-aminocyclopropane-1-

carboxylic acid blocks NF-induced Ca2+ spiking, while the

ethylene biosynthesis inhibitor AVG enhances the number of

cells responsive to both Ca2+ spiking and intracellular infection

(Oldroyd et al., 2001a). JA application also abolishes Ca2+

spiking, or, at lower concentrations, extends the period of the

individual Ca2+ spikes (Sun et al., 2006).

We assessed the effect of these two hormones on the NF-

induced Ca2+ spiking responses at LRBs of S. rostrata. Quanti-

fication of the distribution of spiking signatures showed that the

average spiking period of root hair initials at LRBs tended to be

short; however, it was extended by treatment with 7 mM AVG or

50 mM JA to lengths observed in zone I of S. rostrata or M.

Figure 2. Fura-2 Calibration of Ca2+ Spiking.

(A) Image of aM. truncatula root hair injected with dextran-linked Fura-2,

showing the (peri-)nuclear localization of the calcium responses. Squares

1, 2, and 3 mark the different areas where the measurements were made

to produce the traces on the right: (1) Ca2+ changes in the root hair tip

during a typical experiment, (2) Ca2+ oscillations when only the nucleus is

considered, and (3) an average for the whole root hair.

(B) Representative traces for Ca2+ spiking events in all cell types

discussed, visualized with the Fura-2 ratiometric dye. Traces are given

for M. truncatula root hairs (cells tested n = 3) and S. rostrata cells under

hydroponic conditions without (LRB, n = 6) and with 7 mM AVG (zone I,

n = 5). No significant amplitude differences can be seen.

Calcium Spiking in Sesbania rostrata 1529

truncatula (Figures 3A to 3C). Ca2+ spiking was strongly affected

by 150 mM AVG and totally abolished by 100 mM JA (Figure 3A).

Addition of 1-aminocyclopropane-1-carboxylic acid in concen-

trations ranging from 40 to 1000 mM had no effect.

Hence, Ca2+ spiking at LRBs was suppressed or slowed down

by JA, while increased ethylene levels did not affect the LRB

signature; by contrast, interferencewith ethylene biosynthesis by

the addition of AVG retarded the Ca2+ spiking frequency. Treat-

ment with 7 mM AVG also changed the shape of the Ca2+ spike,

by expanding the second phase, resulting in more asymmetrical

spikes that resembled those inM. truncatula or S. rostrata zone I

root hairs (Figure 3D).

Because application of AVG or JA shifts the Ca2+ spiking

signature at LRBs, we wanted to investigate the effect of the

hormones on nodulation and on rhizobial invasion. For a quan-

titative analysis, hydroponic roots were pretreated for 24 h with

different concentrations of AVG or JA before inoculation with A.

caulinodans strain ORS571 (pRG960SD-32) that expresses the

b-glucuronidase (GUS) reporter gene uidA. Three days after

inoculation (DAI), roots were stained for GUS and LRBs were

excised for stereomicroscopy observation. Ten plants were used

per treatment, and four or five LRBs per root were scored for

numbers of intercellular infection pockets and intracellular infec-

tion threads. The data (Figures 4A and 4B) demonstrate that

pretreatment with low concentrations of AVG or JA stimulated

intracellular invasion of the root hairs. In control roots, infection

threads were observed only very occasionally. Treatment with

1 mM AVG or 12.5 mM JA significantly increased the number of

infection threads in root hairs and decreased the number of

intercellular infection pockets (Figures 4A and 4B). Both infection

thread and infection pocket formation were reduced by higher

concentrations of AVG and JA and completely inhibited by 150 or

100 mM JA.

Previously, 7 mM AVG had been shown to block LRB nodu-

lation completely (D’Haeze et al., 2003). Similar observations

were made upon JA treatment. Treatment with 25 mM JA

reduced the number of LRB nodules by 75% (on average 11.4

nodules on untreated roots versus 2.6 nodules on treated roots).

The concentrations of AVG and JA that affected bacterial

invasion and nodule formation were in general lower than those

required for the Ca2+ spiking switch. Presumably, the 24-h

pretreatment in the nodulation experiments improved penetra-

tion of the compounds, whereas in the Ca2+ spiking analysis, the

effects were measured 10 min after addition of AVG and JA. To

examine this possible explanation, we pretreated hydroponic

roots for 24 h at the low concentrations used in the infection

assays (Figures 4C and 4D) and visualized Ca2+ spiking upon

application of NFs. Indeed, 24 h of pretreatment with 1 mM AVG

(n = 7) and 12.5 mM JA (n = 7) significantly extended the spiking

period compared with untreated cells (n = 10) (Figure 4C) and

shifted the spike shape to one resembling that after 10-min high-

concentration treatment (Figures 3D and 4D). Hence, 24-h pre-

treatments at low concentrations and minute-long treatments at

high concentrations have similar effects on Ca2+ spiking, sug-

gesting that penetration of the compounds is not immediate and

is dose dependent.

A semithin section through a control (Figure 4E) and an AVG-

pretreated (Figure 4F) sample illustrate the differences in the LRB

responses toA. caulinodans inoculation at themicroscopic level.

Whereas in the control a nodule develops opposite infection

pockets (Figure 4E), pretreatment with 7 mM AVG interfered with

nodule development and infection pocket formation, but led to

invaded root hairs (Figure 4F). Limited cell division was occa-

sionally seen (as in Figure 4F), but nodules never developed.

Together, these observations suggest that intracellular root

hair invasion is associated with a particular NF-induced Ca2+

signature. Indeed, shifting the fast spiking pattern at LRBs

toward slower frequencies, resembling the default signature in

susceptible zone I root hairs, correlated with intracellular infec-

tion threads; however, the same conditions were incompatible

with nodule initiation opposite these root hair infection sites.

S. rostrata CCaMK Expression during LRB Nodulation

CCaMK is a critical component of the NF signaling pathway and

presumably functions in decoding NF-induced Ca2+ spiking. To

assess whether Ca2+ spiking decoding is relevant for LRB

nodulation, we investigated the role of CCaMK. To identify S.

rostrata CCaMK, the CCaMK cDNA sequences ofM. truncatula,

pea, rice (Oryza sativa), and tobacco (Nicotiana tabacum) were

aligned. PCR primers were designed to amplify a well-conserved

CCaMK region from a S. rostrata nodule cDNA library. A full-

length cDNA clone was isolated by rapid amplification of cDNA

ends. The corresponding protein of 522 amino acids was most

homologous to L. japonicus CCaMK and M. truncatula DMI3,

with 90.4 and 89.0% amino acid similarity, respectively. The

putative protein had a kinase domain with a conserved Thr (Thr-

270) for autophosphorylation, a CaM binding domain, and three

EF hand domains for Ca2+ binding at the C terminus (see

Supplemental Figure 2A online). DNA gel blot hybridization with

a 652-bp probe containing the CaMbinding domain showed only

one band after genomic DNA digestion with different restriction

enzymes, indicating that S. rostrata CCaMK is a single gene. A

1824-bp promoter region upstream of the start codon was

isolated from genomic DNA of S. rostrata and fused to the

cDNA sequence. The resulting construct was introduced into

roots of the nodulation-defectiveM. truncatula dmi3-1mutant by

Agrobacterium rhizogenes transformation. Transgenic roots

were inoculated with S. meliloti 1021, and functional nodules

were observed (see Supplemental Figure 2B online) in five out of

six transgenic roots. The complementation nodules had a normal

morphology, with an apical meristem, infection zone, and a large

fixation zone with cells fully occupied by symbiosomes (see

Supplemental Figure 2B online). Hence, S. rostrataCCaMK is the

functional ortholog of the M. truncatula CCaMK.

S. rostrata CCaMK transcripts were present in adventitious

rootlets, and the transcript level gradually increased during

stem nodulation, with a maximum at 4 to 5 DAI (Figure 5A). To

localize the expression of CCaMK, the 1824-bp promoter

region was used to drive the transcription of the uidA reporter

gene. In uninoculated transgenic roots, GUS staining was

observed in zones that are potentially responsive to nodula-

tion, i.e., at the LRBs (Figure 5B) and in root zone I (Figure 5C).

At 2 DAI with A. caulinodans, the promoter was active in the

nodule primordia at LRBs (Figure 5D). One day later, when

zonation was initiated, CCaMK was still expressed in the

1530 The Plant Cell

Figure 3. Hormonal Influences on NF-Induced Ca2+ Spiking Signatures.

(A) Representative examples of altered traces observed in root hair cells at LRBs after treatments with AVG and JA. Concentrations are indicated above

the relevant trace; the top trace is a reference, showing NF-induced Ca2+ spiking in untreated roots. The frequency is reduced without inhibition of Ca2+

spiking by 7 mMAVG and 50 mM JA. Spiking is strongly affected by 150 mMAVG and is totally inhibited by 100 mM JA. The percentage of visualized cells

that initiated Ca2+ spiking is shown, and the number of cells tested is given in parentheses.

(B) Effect of treatment with 7 mM AVG and 50 mM JA on the average period of Ca2+ spiking. Error bars indicate SD. Statistically significant differences

between treatment and control are indicated with an asterisk as well as the P value.

(C) Shift in distribution of Ca2+ spiking periods at LRBs toward the distribution found in zone I root hairs after treatment with 7 mMAVG or 50 mM JA. For

each treatment, the percentage of cells with a period in the indicated range was plotted against their spiking periods, and the spiking period was

compared between the different treatments.

(D) Schematic representationof spike shapesderived fromseveral representative traces.Thenumbers in thecurvesareaverageproportionsof upwardand

downwardsegmentsof thespikes. Astatistically significantdifferencewasobserved for thedownwardphaseofAVG-treatedcomparedwithuntreated root

hairs at LRBs (indicated with an asterisk; the P value is also shown).

[See online article for color version of this figure.]

Calcium Spiking in Sesbania rostrata 1531

Figure 4. Effects of AVG on LRB Nodulation and Bacterial Invasion.

(A) Graphs showing the effect of 24-h pretreatment at several AVG concentrations on infection pocket (IP) and infection thread (IT) formation. The dark-

and light-gray bars show the number of IPs and ITs per LRB, respectively, as observed under stereomicroscopy. A significant increase in ITs can be

seen after pretreatment with 1 mMAVGwhen compared with the untreated control plants (indicated with an asterisk; the P value is also given). Error bars

indicate SD.

(B) As for (A) but for JA pretreatment. A significant increase in ITs can be seen after pretreatment with 12.5 mM JA when compared with the untreated

control plants (indicated with an asterisk; the P value is also shown). Error bars indicate SD.

(C) Effect of 24-h pretreatment with 1 mMAVG and 12.5 mM JA on the average period of Ca2+ spiking. Error bars indicate SD. A significant change can be

seen after pretreatment with 1 mMAVG and 12.5 mM JA when compared with the untreated control plants (indicated with an asterisk; the P value is also

shown).

(D) Statistical analysis of the change in Ca2+ spike shapes after 24-h pretreatment with either 1 mMAVG or 12.5 mMJA. The dark and light bars represent

the upward and downward part of the spike, respectively. Error bars indicate SD. A significant change can be seen in the 1 mM AVG and 12.5 mM JA

pretreatments compared with the untreated control plants (indicated with an asterisk; the P value is also shown).

(E) and (F) Semithin sections of a developing LRB nodule at 3 DAI with A. caulinodans (E) and pretreated for 24 h with 7 mMAVG before inoculation with

A. caulinodans (F). Typical features of nodule development and the changes that occur upon hormonal pretreatment are noticeable at the light

microscopic level. Arrowhead and arrow indicate infection pocket and infected root hair, respectively. Bars = 100 mm.

1532 The Plant Cell

nodule primordium (Figure 5E). Sectioning indicated that GUS

was found mainly in the developing nodule and was low in

cortical cells of the infection center (Figures 5F and 5G). In

maturing nodules,CCaMKwas associated with the infection zone

and, to a lesser extent, the fixation zone (Figures 5H to 5J).

A similar pattern of CCaMK expression was observed in

adventitious root nodules on S. rostrata stems by means of

RNA in situ analysis. At the early stages of nodule development,

cells of the nodule primordium accumulated CCaMK transcripts

(Figures 5K and 5L), with the strongest transcript accumulation

Figure 5. Expression Analysis of S. rostrata CCaMK during LRB Nodule Development.

(A) qRT-PCR on developing stem nodules, relative to the constitutive S. rostrata UBI1. Tissues used are peels taken from the stem containing the

dormant adventitious roots that were treated as described. Error bars indicate SD (n = 3).

(B) to (J) CCaMK:GUS expression in S. rostrata hydroponic roots. GUS staining is visible in uninoculated roots ([B] and [C]) and at 2 (D), 3 (E), and 6 (H)

DAI with A. caulinodans ORS571. Microscopic sections are shown of 4-d-old ([F] and [G]) and 6-d-old ([I] and [J]) developing nodules viewed under

bright-field and dark-field optics (signals seen as blue and pink spots, respectively).

(K) to (M) In situ transcript localization of CCaMK during stem nodule development at 3 ([K] and [L]) and 4 (M) DAI. In bright-field and dark-field images,

signals are seen as black and white spots, respectively.

f, fixation zone; i, infection zone; ic, infection center; m, meristem; np, nodule primordium. Bars = 500 mm in (B) to (E) and (H) and 100 mm in (F), (G), and

(I) to (M).

Calcium Spiking in Sesbania rostrata 1533

near the meristem and weakest expression in the infection zone

and in nitrogen-fixing cells (fixation zone). Very low CCaMK

expression was observed around bacterial infection pockets or

progressing intercellular infection threads in the outer cortex

(Figures 5K and 5L) or in the infection center (Figure 5M). This ob-

servation revealed that CCaMK expression correlated strongly

with nodule development and was low in cortical cells during

intercellular rhizobial invasion.

Knockdown of S. rostrata CCaMK Arrests Nodule

Development but Not Intercellular Colonization

To investigate the function ofCCaMK during LRB nodulation, we

reduced CCaMK transcript levels in transgenic S. rostrata roots

by RNAi. Two constructs covering regions of;200 bp (CCaMK-

KO2 and CCaMK-KO3) were introduced into S. rostrata by

transformation with A. rhizogenes 2659 (Van de Velde et al.,

2003). Plants with transgenic roots, identified by green fluores-

cent protein (GFP) cotransformation, were transferred to tubes

with nitrogen-deprived Norris medium and inoculated with A.

caulinodans ORS571 (pBHRdsREDT3) after 9 to 14 d. In control

transgenic roots transformed with the empty vector (n = 34),

developing nodules at LRBs were observed at 4 DAI; mature

determinate nodules formed by 7 DAI (Figures 6A and 6B). In

lines transformed with either of the two knockdown constructs

(n = 99), 59% of the transgenic roots developed normal nodules;

in 25%, the nodulation level was strongly reduced with nodules

smaller than those of the controls. Nevertheless, these small

nodules contained bacteria (Figures 6D and 6E). In the remaining

16% of transgenic roots, only small bumps were observed that

were, nevertheless, colonized by bacteria (Figures 6G and 6H).

CCaMK transcript levels were determined using quantitative

RT-PCR (qRT-PCR) in a subset of the lines with small nodules

(n = 10), mere bumps (n = 8) and in control lines (n = 6) (Figure 6J).

A strong correlation was found between the degree of transcript

reduction and the severity of the phenotype. Lines with the

strongest decrease in expression (lower than 20%; e.g.,

KO3#a3, KO3#a1, KO2#f2, and KO2#b2) formed only bumps

(Figures 6G-I), whereas those with moderately reduced expres-

sion (between 55 and 20%, such as KO2#a7) had small nodule-

like structures (Figures 6D-F). Hence, a decrease in CCaMK

transcript numbers in transgenic RNAi roots had a dosage-

dependent effect on the nodule size, meaning that nodule

development was hampered.

Light and transmission electron microscopy (TEM) showed

that the central tissue of nodules in lines with a moderately

reduced CCaMK expression (such as KO2#a7) had a few

infected cells at 7 DAI, most of which were not completely filled

with symbiosomes (cf. Figures 6F and 6C). Some infection

threads in the infection center were abnormally broad (Figure

6F). TEM analysis indicated that these infection threads had

aberrant shapes with bulged outgrowths and often a rim of low

electron-dense material at the walls (Figures 6L and 6M, in

comparison with Figure 6K). Nodules that appeared on an

incidental GFP-negative root of these lines provided an internal

control: they had a normal size and a central tissue with many

fixing cells that were completely filled with bacteroids, identical

to lines transformed with an empty vector (Figure 6C). In trans-

genic roots with strongly reducedCCaMK expression, only a few

cortical cells divided at 7 DAI, but the cortex at the LRBs was

colonized by bacteria in infection pockets (Figure 6I) and aber-

rant infection threads occurred.

In conclusion, the phenotypes observed in CCaMK RNAi

knockdown roots show that nodule development and infection

thread progression were severely disturbed and nodule-like

structures contained few or almost no bacteroids in the central

tissue, but intercellular invasion of the cortex with infection

pocket formation was hardly affected.

DISCUSSION

Several lines of evidence link Ca2+ spiking in zone I root hairs of

legume roots to NF signaling for nodulation. First, NFs from

incompatible rhizobia or NFs lacking key modifications are

unable to induce proper Ca2+ oscillations (Ehrhardt et al., 1996;

Oldroyd et al., 2001a, 2001b). Second, plant mutants that are

blocked at the earliest stages of the symbiosis are impaired in

NF-induced Ca2+ spiking, including mutations in genes coding

for the putative LysM-type NF receptors, putative ion channels, a

membrane-bound leucine-rich repeat receptor-like kinase, and

components of the nucleoporin complex, functions presumably

involved in generating or regulating spiking downstream of the

NF perception (Catoira et al., 2000; Ben Amor et al., 2003;

Madsen et al., 2003; Radutoiu et al., 2003; Imaizumi-Anraku

et al., 2005; Kanamori et al., 2006; Miwa et al., 2006a; Saito et al.,

2007). Third, one of the essential nodulation genes encodes a

CCaMK protein (Catoira et al., 2000; Miwa et al., 2006a), a strong

candidate for decoding the Ca2+ signal that leads to transcrip-

tional changes. Moreover, gain-of-function mutations of CCaMK

result in spontaneous nodule formation (Gleason et al., 2006;

Tirichine et al., 2006). Besides these genetic data, several

physiological lines of evidence are available. For instance, in-

hibitors of Ca2+ spiking suppress the induction of nodulin genes,

such as early nodulin 11 (ENOD11) (Pingret et al., 1998;

Engstrom et al., 2002; Miwa et al., 2006b), and, on average,

;36 uninterrupted spikes are required to allow ENOD11 ex-

pression (Miwa et al., 2006a).

Until now, the role of Ca2+ spiking in symbiosis had been

studied exclusively in legumes with zone I nodulation and

intracellular invasion in growing root hairs. S. rostrata presents

an alternative nodulation that differs in position, physiological

environment, invasion mode, and the need for ethylene and has

less stringent NF structural requirements (Goormachtig et al.,

2004a). During LRB nodulation in S. rostrata, bacterial invasion

skips the epidermis, allowing the study of the role of NF

signaling components in the intercellular colonization of the

cortex.

Here, we examined Ca2+ spiking patterns at LRBs and in root

zone I of S. rostrata, and, in parallel, we studied the role of S.

rostrataCCaMK in LRB nodulation. The outer cortical cells, where

invasion takes place, were not amenable for microinjection with a

Ca2+-sensitive dye. The only accessible targets were root hair

initials present at LRBs of the otherwise hairless hydroponic

roots. These epidermal cells respond to NFs and to bacterial

inoculation by outgrowth and deformation, but they do not

1534 The Plant Cell

become invaded. Just like LRB nodulation, this response de-

pends on NFs, H2O2, and ethylene (D’Haeze et al., 2003). As

these root hair initial responses have the same signaling require-

ments as nodulation at LRBs, we used these cells to analyze

Ca2+ spiking. We found that spike shape and frequency were

different in LRB cells and zone I root hairs. The Ca2+ spiking at

LRBs was faster than in zone I, and the shape of the spikes was

more symmetrical (Figure 1). Despite these differences, a phar-

macological analysis suggests a similar underlying mechanism

for the generation of RHC- and LRB-associated Ca2+ spiking

Figure 6. CCaMK RNAi Knockdown Phenotypes in S. rostrata Hairy Roots.

(A) to (I) Root nodule at 7 DAI grown on a control line ([A] to [C]) and a moderate KO2 a7 ([D] to [F]) and strong KO2 b2 ([G] to [I]) CCaMK knockdown

line. A bright-field image (left column), a dsRED fluorescence image (depicting bacteria; middle column), and a microscopic section (right column) are

shown in each row. Arrows in (F) indicate broad infection threads.

(J) Relative CCaMK expression level of knockdown root lines (KO2 and KO3) compared with controls (empty PZP vector), as obtained from qRT-PCR

with constitutive Sr UBI1. Error bars indicate SD (n = 3).

(K) to (M) TEM images of infection thread structures in a control (K) and in CCaMK knockdown nodules ([L] and [M]). Arrowheads and asterisks indicate

low electron-dense rims and bulges in infection threads, respectively.

ct, central tissue; f, fixation zone; ip, infection pocket; np, nodule primordium. Bars = 500 mm in (A), (B), (D), (E), (G), and (H), 100 mm in (C), (F), and (I),

and 2 mm in (K) to (M).

Calcium Spiking in Sesbania rostrata 1535

patterns, involving phospholipid signaling and Ca2+-ATPases

(see Supplemental Figure 1 online).

The LRB Ca2+ spiking pattern could be modulated by altering

hormone levels. As observed in M. truncatula (Sun et al., 2006),

JA extended the period of Ca2+ spikes. In contrast with the

situation inM. truncatula (Oldroyd et al., 2001a), excess ethylene

had no effect on LRB Ca2+ spiking; on the contrary, inhibition of

ethylene production either blocked it, or, at low concentrations,

extended its period. Increased JA levels or reduced ethylene

levels also affected the spike shape, rendering it more asym-

metric, thus shifting the signature toward the pattern observed in

zone I root hairs. Modulating the levels of these hormones

prominently influenced the downward phase of the Ca2+ spike,

which is associated with the resequestration of Ca2+ into the

internal store (Figure 3). Therefore, the target for ethylene and JA

might be the Ca2+-ATPase that functions in the reuptake of

calcium.

Interestingly, the hormonal modulations that conferred zone I

identity to the Ca2+ spiking pattern at LRBs stimulated infection

thread formation. In the absence of AVG or JA, intracellular

infection threads were rarely observed. At low concentrations,

AVG or JA promoted intracellular invasion in root hairs, whereas

at high concentrations they negatively affected LRB nodulation

and reduced the number of both infection pockets and infected

root hairs. With Fura-2 as a ratiometric dye, no significant

amplitude differences were found between the cell types, indi-

cating that shape and period are the determining factors for the

shift in infection strategy (Figures 2 and 4). These findings

correlate a defined Ca2+ spiking profile with the capacity for

intracellular root hair invasion, be it in a zone I or in a LRB

developmental context. The hormonal changes negatively af-

fected LRB nodule development and infection pocket formation

(Figures 4E and 4F), which could be caused either via the altered

spiking signature or directly by the modified hormone levels.

Additional data were obtained from the study of the role of

CCaMK in LRB nodulation. CCaMK proteins are plausible can-

didates to interpret the NF-triggered Ca2+ signature and to

transmit information for gene expression, nodule formation,

and infection thread progression. A S. rostrata CCaMK cDNA

clone, corresponding to a unique gene, complemented the

dmi3-1 mutation of the M. truncatula CCaMK gene. qRT-PCR

demonstrated that expression of CCaMK is upregulated during

nodulation at adventitious root bases. A promoter-GUS reporter

construct and in situ hybridization revealed expression in nodule

primordia and in the proximal cells of the meristematic zone of

developing nodules (Figure 5). InM. truncatula nodules,CCaMK,

DMI1, and DMI2 transcripts are localized in the apical preinfec-

tion zone (Bersoult et al., 2005; Limpens et al., 2005; Riely et al.,

2007), andDMI2 andNFPpromoter activity has been observed in

the nodule primordium prior to penetration by infection threads

(Bersoult et al., 2005; Arrighi et al., 2006).

Interestingly, downregulation of CCaMK expression by RNAi

severely interfered with nodule formation but hardly affected the

primary intercellular cortical invasion. The degree of transcript

reduction and the impairment of nodule development correlated

well. Lines with <20% residual transcripts merely developed

small bumps at the LRBs, implying that CCaMK is important for

nodulation. All suppressed lines had large infection pockets and

intercellular infection threads, suggesting that signaling via

CCaMK is not essential to initiate these structures (Figure 6).

However, intracellular cortical infection threads were abnormal,

resembling the lumpy structures observed in S. rostrata SYMRK

knockdown lines and wild-type plants upon invasion with NF-

deficient bacteria (Figure 6) (Capoen et al., 2005; Den Herder

et al., 2007). Thus, proper infection thread progression in S.

rostrata requires cell-autonomous NF perception with signal

transduction that involves both CCaMK and, as shown previ-

ously, SYMRK (Capoen et al., 2005).

Our data show that initiation and development of nodule

primordia at LRBs require CCaMK and that CCaMK plays a

role in the progression of infection threads (Figure 6). The similar

requirements for CCaMK in RHC nodulation in M. truncatula

(Levy et al., 2004) allow us to conclude that Ca2+ oscillations are a

basic component in nodule primordia initiation and infection

thread progression both in an RHC and LRB context. Different

Ca2+ signatures are associated with RHC and LRB nodulation,

but, at present, we cannot conclude whether the Ca2+ spiking

pattern observed in root hair initials is representative of the

pattern in cortical cells during LRB nodulation.

Rhizobial invasion via RHC depends on CCaMK (Catoira et al.,

2000; Tirichine et al 2006). Here, we report that this type of

invasion correlates with a specific Ca2+ spiking pattern, suggest-

ing that a frequency-dependent phosphorylation of CCaMK

targets might be an important step in this process. By contrast,

infection pocket formation seems independent of CCaMK (Fig-

ure 6), implying a mechanism for the initiation of cortical cell

death that does not depend on Ca2+ oscillations.

Based on these observations, we propose a model with a dual

pathway downstream of the primary NF perception at LRBs. NF

perception by specific receptors would cause Ca2+ spiking, of

which the decoding by CCaMK is essential for nodule formation.

In parallel, NF perception generates secondary signals that,

independently of Ca2+ spiking, are essential for the outer cortex

colonization with infection pocket formation. Plausiblemediators

of these primary intercellular invasion events are H2O2 and

ethylene (D’Haeze et al., 2003).

Our data also confirm the key role for ethylene in submer-

gence-adapted nodulation in S. rostrata: under waterlogged

conditions, when ethylene accumulates, RHC nodulation in zone

I is suppressed (D’Haeze et al., 2003), while LRB nodulation that

requires ethylene is prevalent.Wedemonstrate that the root hairs

present on hydroponic roots can perceive NFs but cannot

mount a calcium response with the appropriate period for root

hair infection, presumably because of inhibiting hormones that

accumulate in hydroponic roots. This problem is circumvented

via a Ca2+-independent intercellular infection pathway with in-

fection pocket formation allowing the accumulation of sufficient

numbers of rhizobia to generate a greatly amplified signal for

further nodule invasion.

In conclusion, we have shown that ethylene and JA modulate

the Ca2+ oscillations that are activated by rhizobial NFs at LRBs

of S. rostrata and that a specific Ca2+ signature correlates well

with the intracellular invasion mode. The physiological context

and, in particular, the ethylene concentration, influences Ca2+

spiking and the choice of the developmental pathway that is

activated by NF signaling in S. rostrata, thus contributing to the

1536 The Plant Cell

phenotypic plasticity that is characteristic of nodulation of this

tropical legume.

METHODS

Biological Material

Sesbania rostrata Brem seedlings were germinated and grown in tubes

with liquid medium (hydroponic roots) or in Leonard jars (aeroponic roots)

according to the procedures described (Fernandez-Lopez et al., 1998).

Medicago truncatula A17 seedlings were germinated and grown as

described (Sun et al., 2006). Azorhizobium caulinodans ORS571 labeled

with the red fluorescent protein (DsRED; Clontech) was obtained by

introducing the plasmid pBHRdsREDT3 (Smit et al., 2005) into A.

caulinodans ORS571 by electroporation. The A. caulinodans ORS571

(pBHRdsREDT3), ORS571 (pBBR5-hem-gfp5-S65T) (D’Haeze et al.,

2004), and ORS571 (pRG960SD-32) (D’Haeze et al., 1998) strains were

grown on yeast extract broth medium with appropriate antibiotics (Van

den Eede et al., 1987). NFs were purified from A. caulinodans cultures as

previously described (Mergaert et al., 1993).

Ca2+ Spiking Analysis

For Ca2+ spiking analysis (Ehrhardt et al., 1996; Wais et al., 2000),

micropipettes were made from borosilicate capillaries with an electrode

puller (model 773; Campden Instruments). Needles were preloaded with

either dextran-linked Fura-2 or Oregon Green and Texas Red (10,000

molecular weight; Molecular Probes). Cells were injected via iontopho-

resis with a cell amplifier (model Intra 767; World Precision Instruments)

and a stimulus generator (World Precision Instruments).

Plants were mounted on slides containing buffered nodulation medium

(Ehrhardt et al., 1992) for microinjection of suitable root hairs and

subsequent imaging. After microinjection, cells were visualized for 10

min to verify viability; only cells that still showed cytoplasmic streaming

were challenged with 10 pM purified A. caulinodans or Sinorhizobium

meliloti NFs. For visualization, an inverted epifluorescence microscope

(TE2000; Nikon) equipped with a monochromator (Optoscan; Cairn

Research) was used. Cells were imaged with a CCD camera (ORCA-

ER; Hamamatsu) and an image splitter (Optosplit; Cairn Research).

Subsequently, data were analyzed with MetaFluor software (Universal

Imaging). All traces shown are the fluorescence intensity ratio of Oregon

Green to Texas Red; although not considered a ratiometric method, all

intensity shifts caused by cytoplasmic streaming in the cells being

visualized were canceled out.

For Fura-2 calibrations, measured ratios were calibrated in vitro with a

series of standards using the Fura-2 Ca2+ calibration kit according to the

manufacturer’s instructions (Invitrogen). The emission ratio was linear for

calcium concentrations within a biologically relevant range (0 to 650 nM).

For the pharmacological tests on Ca2+ spiking at LRBs, concentrations

of compounds were 1, 3, 7, or 150 mMAVG (Sigma-Aldrich); 12.5, 25, 50,

or 100 mM JA (Sigma-Aldrich), 0.5% (v/v) n-butanol (Sigma-Aldrich), and

10 mM CPA (Sigma-Aldrich). Spiking cells were visualized for at least 30

min before addition of the compounds, and changes were measured 10

min afterward to avoid transition effects.

To obtain zone I root hairs on hydroponic roots, S. rostrata seedlings

were germinated overnight and directly transferred to 7 mMAVG. Spiking

was tested 7 to 10 d later.

Excel software (Microsoft) was used for statistical analysis of the Ca2+

spiking experiments. To measure the frequencies and periodicity of the

spikes, the time between Ca2+ spike maxima was measured and aver-

aged, and the standard deviations were subsequently calculated. For the

spike shape analysis, individual spikes were assessed for the time to

reach amaximum frombaseline (the upward slope) to the time to return to

baseline (the downward slope). Typically, for all statistical analyses, at

least 30 spikes were measured from a minimum of five traces. When

necessary, we ascertained statistical significance with t tests assuming

unequal variance.

Some traces were detrended with a moving average. The number of

points used in the moving average is particular to each trace and was

chosen to be as low as possible while retaining features of interest.

Outliers in the traces were removed and replaced with linear interpolation

(Brockwell and Davis, 2002). The number of points used to calculate the

moving average for every trace are 150 forM. truncatula, 19 forS. rostrata,

25 for 7 mM AVG, 25 for LRB (Figure 1), and 25 and 19 for BHQ and CPA,

respectively (see Supplemental Figure 1 online). It should be noted that

this detrending differs from the derivation often used in the literature on

NF-inducedCa2+ spiking literature: Y =X(n+1)-Xn, where Y is the point-to-

point change in fluorescence, and X(n+1) and Xn are the intensity

measurements at time points (n + 1) and (n) (Wais et al., 2000). This

formula was not used in this study.

Pharmacological Assays on LRB Nodulation

All experiments were repeated at least twice per time point. Stock

solutions (10003) of AVG (Sigma-Aldrich) and JA (Sigma-Aldrich) were

made according to themanufacturer’s instructions, and equal amounts of

the solvent were tested as controls. Compounds were added 6 d after

germination, and 24 h after their addition, S. rostrata seeds were inoc-

ulated with A. caulinodans ORS571 (pRG960SD-32). At the concentra-

tions used, neither the solvents nor the inhibitors influenced growth of

plants and bacteria. For the statistical analysis of the infection assays

(Figure 4), frequencies of infection thread and infection pocket formation

were analyzed by generalized linear modeling, using a logarithmic link

function and a Poisson distribution of frequencies in the Genstat software

suite (Payne and Lane, 2005).

Microscopy

Roots were stained for GUS activity as previously described (D’Haeze

et al., 1998) before overnight clearing in a chloral hydrate solution (Sabatini

et al., 1999). Subsequently, infection threads and infection pockets were

counted under a stereomicroscope. For semithin sectioning and light

microscopy, LRBs were excised and fixed with 2.5% glutaraldehyde and

embedded in Technovit 7100 (Kulzer Histo-Technik). Sections of 4 mm

were cut, stained with toluidine blue or Ruthenium Red, mounted with

Depex (Sigma-Aldrich) (Den Herder et al., 2007), and analyzed with a

Diaplan bright-field microscope (Leitz). GFP and dsRED analyses (Van de

Velde et al., 2003) andTEM (D’Haeze et al., 1998)were doneasdescribed.

Identification of the S. rostrata CCaMK Promoter and Open

Reading Frame

Nested primers were chosen in conserved regions of CCaMK proteins

from other plant species. RT-PCR on S. rostrata nodule cDNA revealed a

652-bp fragment. The full-length sequence was obtained by 59 and 39

rapid amplification of cDNA ends (Smart RACE cDNA amplification kit;

Clontech), and the complete open reading frame was amplified with

primers SrDmi3FLS (59-GGGAATTCAAAGACTTGAT-39) and SrDmi3FLAS

(59-GATGCAATAAAAACTAAATTTGGAAG-39) and anchored into the

pCRII-TOPO vector (Invitrogen). The full-length open reading frame was

transferred to the pDONR221 entry vector (Invitrogen).

The S. rostrata CCaMK promoter was identified with the Universal

GenomeWalker kit (Clontech) applied to genomic DNA, and the 1824-bp

region upstream of the start codon was amplified with primers attB4-

SrDMI3promFullS (59-GGGGACAACTTTGTATAGAAAAGTTGTGATG-

GACCACTTTG-39) and attB1-SrDMI3promFullAS (59-GGGGACTGCTT-

TTTTGTACAAACTTGGTGGTGCACAAAACA-39) and recombined in the

pDONR P4-P1 (Invitrogen).

Calcium Spiking in Sesbania rostrata 1537

Expression Analysis of S. rostrata CCaMK

qRT-PCR was performed with primers Dmi3-Q2S (59-TTTCAT-

TGCTCCGTCTAATCGC-39) and Dmi3-Q2AS (59-GCTTTGCTGATTGG-

GAAATGCC-39) or Dmi3-Q3S (59-AACAAAAGGTGGAGAGAAAAGC-39)

and Dmi3-Q3AS (59-ACAAGGCATCTGAGACTGAAAC-39) on the Light-

cycler 480 (Roche Diagnostics) with the SYBRGreen I master mix (Roche

Diagnostics) according to the manufacturer’s instructions. The constitu-

tively active S. rostrata UBI1 gene was amplified with primers SrUBI1Q-S

(59-GGGAAGCAGTTGGAGGATGG-39) and SrUBI1Q-AS (59-AGACGCA-

GAACAAGGTGAAGG-39) (Capoen et al., 2007) to determine relative

values with the qBase software (Hellemans et al., 2007). One represen-

tative result from three biological repeats is shown.

To analyze the promoter-GUS activity, the Multisite Gateway three-

fragment vector construction kit (Invitrogen) was used to fuse the pro-

moter with the uidA gene (in pDONR207-GUS) and the T35S terminator (in

pENTR-R2-T35S-L3) into vector pKm43GW-rolD for coexpression with

GFP. To obtain transgenic roots, S. rostrata embryonic axes were

transformed (Van de Velde et al., 2003). A 652-bp region was used as

template to produce a 35S-labeled antisense probe for the in situ hybrid-

ization that was performed according to Goormachtig et al. (1997).

RNAi of S. rostrata CCaMK

Knockdown constructs (SrCCaMK-KO2 and SrCCaMK-KO3) were pro-

duced by recombining two regions of CCaMK in the pK7GWIWG2-D

binary GATEWAY vector (Invitrogen) (Karimi et al., 2002). For the

SrCCaMK-KO2 and SrCCaMK-KO3 constructs, the primers Dmi3KO2-

Sense (59-CAAAAAAGCAGGCTTCACACTACAAGGAAAAG-39) and

Dmi3KO2-Asense (59-AGAAAGCTGGGTAAATTTGGAAGAATTTG-39);

and Dmi3KO3-Sense (59-CAAAAAAGCAGGCTTGGATGCAAATAGT-

GATG-39) and Dmi3KO3-Asense (59-AGAAAGCTGGGTCAGAAGTTA-

GATGGCACAG -39) were used, respectively. To obtain transgenic

roots, S. rostrata embryonic axes were transformed as described, and

composite plants carrying transgenic roots were selected byGFP (Van de

Velde et al., 2003). The empty vector pPZP200-egfp was used as control.

The knockdown level was determined from transgenic roots cultured in

vitro on a plate by qRT-PCR as described above.

Dmi3-1 Complementation

For complementation of the M. truncatula dmi3-1 mutant, the S. rostrata

CCaMK promoter and open reading frame were recombined with Mul-

tisite Gateway recombination into the vector pKm43GW-rolD (Invitrogen).

The generation of transgenic roots onM. truncatula dmi3-1 seedlings and

infection with S. meliloti was performed according to the previously

described procedure (Boisson-Dernier et al., 2001).

Accession Numbers

Sequence data from this article can be found in the GenBank/EMBL data

libraries under accession numbers EU622875 (S. rostrata CCaMK, com-

plete coding sequence) and EU622876 (S. rostrata CCaMK, promoter

region and 59 untranslated region).

Supplemental Data

The following materials are available in the online version of this article.

Supplemental Figure 1. Mechanisms of Ca2+ Spiking in S. rostrata

LRB-Associated Root Hair Cells.

Supplemental Figure 2. CCaMK Sequence Alignment and Functional

Complementation.

ACKNOWLEDGMENTS

We thank Grant Calder, Hiroki Miwa, and Allan Downie for useful

discussions, Saul Hazledine and James Brown for statistical analyses,

Patrick Smit and Sharon Long for providing the dsRED plasmid and M.

truncatula NFs, respectively, and Martine De Cock for help in preparing

the manuscript. This work was supported by the European Union Grain

Legume Integrated Project (Food-CT-2004-506223). G.O. is funded by a

David Philips Fellowship and a grant-in-aid of the Biotechnology and

Biological Science Research Council, a Wolfson Research Merit award

of the Royal Society, and the European Molecular Biology Organization

Young Investigator Program. W.C. is grateful to the Institute for the

Promotion of Innovation by Science and Technology in Flanders for a

predoctoral fellowship and the European Molecular Biology Organiza-

tion for both short- and long-term fellowships, respectively. J.D.H. was a

Research Fellow of the Research Foundation-Flanders.

Received February 12, 2009; revised April 30, 2009; accepted May 8,

2009; published May 26, 2009.

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1540 The Plant Cell

DOI 10.1105/tpc.109.066233; originally published online May 26, 2009; 2009;21;1526-1540Plant Cell

Sofie Goormachtig, Giles Oldroyd and Marcelle HolstersWard Capoen, Jeroen Den Herder, Jongho Sun, Christa Verplancke, Annick De Keyser, Riet De Rycke,

Sesbania rostrataLateral Root Base Nodulation of Calcium Spiking Patterns and the Role of the Calcium/Calmodulin-Dependent Kinase CCaMK in

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