Lotus japonicus symRK-14 uncouples the corticaland epidermal symbiotic program

Sonja Kosuta1,†, Mark Held1,2, Md Shakhawat Hossain1, Giulia Morieri3, Amanda MacGillivary1, Christopher Johansen1,2,

Meritxell Antolın-Llovera4, Martin Parniske4, Giles E. D. Oldroyd3, Allan J. Downie3, Bogumil Karas1,2,‡ and Krzysztof

Szczyglowski1,2,*

1Agriculture and Agri-Food Canada, Southern Crop Protection and Food Research Centre, London, ON N5V 4T3 Canada,2Department of Biology, University of Western Ontario, London, ON N6A 5BF Canada,3John Innes Centre, Colney, Norwich NR4 7UH, UK, and4Biocenter University of Munich (LMU), Genetics, 82152 Martinsried, Germany

Received 15 April 2011; revised 13 May 2011; accepted 18 May 2011.*For correspondence (fax +519 457 3997; e-mail krzysztof.szczyglowski@agr.gc.ca).†Present address: Patent Branch, Canadian Intellectual Property Office, Industry Canada, Gatineau, QC K1A OC9, Canada.‡Present address: J. Craig Venter Institute, San Diego, CA 9212, USA.

SUMMARY

SYMRK is a leucine-rich-repeat (LRR)-receptor kinase that mediates intracellular symbioses of legumes with

rhizobia and arbuscular mycorrhizal fungi. It participates in signalling events that lead to epidermal calcium

spiking, an early cellular response that is typically considered as central for intracellular accommodation and

nodule organogenesis. Here, we describe the Lotus japonicus symRK-14 mutation that alters a conserved

GDPC amino-acid sequence in the SYMRK extracellular domain. Normal infection of the epidermis by fungal

or bacterial symbionts was aborted in symRK-14. Likewise, epidermal responses of symRK-14 to bacterial

signalling, including calcium spiking, NIN gene expression and infection thread formation, were significantly

reduced. In contrast, no major negative effects on the formation of nodule primordia and cortical infection

were detected. Cumulatively, our data show that the symRK-14 mutation uncouples the epidermal and cortical

symbiotic program, while indicating that the SYMRK extracellular domain participates in transduction of non-

equivalent signalling events. The GDPC sequence was found to be highly conserved in LRR-receptor kinases

in legumes and non-legumes, including the evolutionarily distant bryophytes. Conservation of the GDPC

sequence in nearly one-fourth of LRR-receptor-like kinases in the genome of Arabidopsis thaliana suggests,

however, that this sequence might also play an important non-symbiotic function in this plant.

Keywords: legumes, root, symbiosis, SYMRK, signalling, calcium.

INTRODUCTION

Arbuscular mycorrhiza (AM) root endosymbiosis with

Glomeromycota fungi appears in the fossil record with early

land plants about 450 million years ago and has been

retained by approximately 80% of extant plant species,

including bryophytes and other primitive plants (Parniske,

2008; and references therein). The colonization of roots by

AM fungi is mediated by a signalling process, which in

legumes involves a receptor-like kinase (RLK) called SYMRK/

NORK/DMI2 (Endre et al., 2002; Stracke et al., 2002), here-

after referred to as SYMRK. SYMRK is also indispensable for

the root nodule symbiosis (RNS) of legumes with nitrogen-

fixing rhizobia, which accounts for its classification as an

element of the so-called common symbiosis signalling

pathway. This signalling pathway mediates early root

responses to both AM fungi and nitrogen-fixing bacteria

(Kistner et al., 2005; Parniske, 2008).

During nodulation, SYMRK acts downstream of nodula-

tion factor (NF) perception (Madsen et al., 2003; Radutoiu

et al., 2003) and together with CASTOR and POLLUX ion

channels (Ane et al., 2004; Charpentier et al., 2008) and three

nuclear pore proteins NUP85, NUP133 and SEH1 (Kanamori

et al., 2006; Saito et al., 2007; Groth et al., 2010), is required

in Lotus japonicus to generate epidermal calcium spiking

(Miwa et al., 2006), one of the earliest plant cellular

responses to both fungal and bacterial symbionts (Oldroyd

and Downie, 2006; Kosuta et al., 2008). Arbuscular mycor-

rhizal fungal and rhizobial induced epidermal calcium spik-

ing are believed to be decoded by another protein complex,

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The Plant Journal (2011) doi: 10.1111/j.1365-313X.2011.04645.x

which includes the calcium and calmodulin-dependent

receptor kinase (CCaMK) and CYCLOPS (Levy et al., 2004;

Mitra et al., 2004; Messinese et al., 2007; Yano et al., 2008).

This leads to the symbiont-specific transcriptional repro-

gramming that is required to support root colonization by

both AM fungi and bacterial microsymbionts and also to

initiate nodule organogenesis (Kistner et al., 2005; Høgslund

et al., 2009).

Mutations in SYMRK abort, or significantly restrict, root

infection by both AM fungi and rhizobia (Catoira et al., 2000;

Endre et al., 2002; Stracke et al., 2002). The initiation of

nodule organogenesis is also prevented in symRK mutants.

These observations highlight a critical role for SYMRK in

both early events at the root epidermis, where the initial

bacterial entry occurs, and also in a signalling pathway that

is usually required for the initiation of nodule primordia (NP)

in the root cortex. Nodule organogenesis is assumed to

operate by linking the NF-dependent early root responses,

which require SYMRK, with intrinsic plant developmental

mechanisms, such as the induction of cytokinin signalling,

which is required for and also sufficient to initiate nodule

development (Gonzalez-Rizzo et al., 2006; Murray et al.,

2007; Tirichine et al., 2007). However, how SYMRK acts

during signal transduction to initiate the symbiosis-relevant

process remains unclear.

We describe here an allele of SYMRK, symRK-14, which

carries a mutation that alters a highly conserved GDPC

amino-acid sequence in the SYMRK extracellular domain.

Unlike previously described symRK mutations, symRK-14

allows rhizobial-induced nodule morphogenesis even

though epidermal calcium spiking and infection thread

(IT)-dependent root colonization by bacteria are significantly

attenuated. We show that the symRK-14 mutation also

greatly attenuates AM symbiosis in L. japonicus. The GDPC

motif is highly conserved in leucine-rich repeat (LRR)-RLKs

of many non-legume species including Arabidopsis thali-

ana, rice and also two bryophytes, Physcomitrella patens

and Marchantia polymorpha.

RESULTS

Two SYMRK alleles

Based on a genetic screen for suppressors of the L. japoni-

cus har1-1 hypernodulation phenotype (Murray et al., 2006),

two symRK mutant alleles, symRK-13 and symRK-14, were

identified (Figure 1a). Both were recessive but conferred

rather different nodulation phenotypes (Murray et al., 2006).

Nodulation and mycorrhization were completely blocked

in the symRK-13 har1-1 double mutant (Nod), Myc) pheno-

type), which had a bushy root phenotype (Figure S1a) that

Figure 1. Genetic lesions in Lotus japonicus symRK-13 and symRK-14 and the conservation of the GDPC sequence in the SYMRK extracellular domain.

(a) A scheme of the wild-type SYMRK protein; main domains and the locations of amino-acid substitutions in mutant alleles are indicated. EC, extracellular domain;

SP, signal peptide; NEC, N-terminal region of unknown function; GDPC, short amino-acid sequence residing within the conserved extracellular region (CEC domain)

of SYMRK EC; LRR, leucine-reach repeat; TM, transmembrane domain, PK, protein kinase domain. Domain nomenclature was adopted from Markmann and

Parniske (2009).

(b) Protein alignment of a GDPC-containing portion of the L. japonicus SYMRK EC domain with the corresponding regions of SYMRK and SYMRK-like proteins from

different legumes and non-legumes, including a non-vascular bryophyte, Physcomitrella patens. Highly conserved amino-acid residues are indicated. Asterisks (*)

indicate the pair of conserved cysteine residues; the arrow marks the position of the first leucine in the LRR motif. The following accession numbers correspond to

proteins used in the alignment: L. japonius (LjSYMRK, AAM67481.1); Sesbania rostrata (SrSYMRK, AAV88632.1); Medicago truncatula (MtDMI2, CAD10810.1);

Medicago sativa (MsNORK, CAD10815.1); Melilotus alba (CAD22013.1); Pisum sativum (SYMRK19, CAD10812.1); Lathyrus sativus (ABD64156.1); Vicia hirsuta

(CAD22012.1); Astragalus sinicus (AAX53605.1); Lupinus albus (AAY22390.1); Tropaeolum majus (AAY22388.1); Alnus glutinosa (AAY22387.1); Lycopersicon

esculentum (AAY22055.1); Arabidopsis thaliana (CAA66376.1); Oryza sativa (BAF21940.1); Physcomitrella patens (EDQ64798.1); and Zea mays (ABD67490.1).

2 Sonja Kosuta et al.

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

was indistinguishable from un-inoculated roots of the

har1-1 parent (Wopereis et al., 2000; Krusell et al., 2002).

The symRK-13 mutation resulted in a change in amino acid

A620 to T in the protein kinase domain and was predicted

to affect the presumed ATP-binding region of the SYMRK

protein (Figure 1a). As symRK-13 har1-1 and the corre-

sponding symRK-13 single mutant (see below) had no novel

phenotypic features beyond what has been already

described for other SYMRK mutant alleles, it was only used

in a limited set of comparative analyses (see below).

symRK-14 har1-1 showed an interesting symbiotic phe-

notype because it formed a few wild-type nodules (Fig-

ure S1a), many NP and/or uncolonized small nodules and

was also significantly reduced for AM formation (see below).

The symRK-14 mutation changed amino acid P386 to L in

the extracellular domain upstream of the first LRR in the

predicted SYMRK protein (Figure 1a). A protein alignment

(Figure 1b) revealed that the proline residue affected in

symRK-14 is part of a short amino-acid sequence, GDPC,

which showed complete conservation in legume SYMRK

sequences.

The symRK-13 and symRK-14 mutants in a HAR1 back-

ground (see Experimental Procedures) had wild-type root

and shoot morphology and symRK-13 was Nod) and AM)

(data not shown), whereas symRK-14 formed uncolonized

and some colonized nodules when inoculated with Meso-

rhizobium loti (Figure S1b). A strong defect in the interaction

with AM fungi was invariably observed in the symRK-14

mutant (see below).

In order to verify that the different phenotypes of symRK-

13 and symRK-14 were indeed caused by the symRK-13 and

symRK-14 alleles, the mutations were complemented. Hairy

roots formed on symRK-13 or symRK-14 shoots retained the

symbiotic phenotypes of the mutants (Figure S2). Transfor-

mation with the wild-type L. japonicus SYMRK gene

restored normal nodulation and AM infection in both

symRK-13 and symRK-14 (Figure S2), confirming the caus-

ative nature of the symRK mutations.

The symRK-14 mutation affects an evolutionarily conserved

sequence in the extracellular domain

The extracellular portion of the domain that is located

upstream of the LRRs in L. japonicus SYMRK shares

approximately 80% similarity with proteins in legumes such

as Medicago truncatula and Pisum sativum, and 50–60%

similarity with homologous proteins in non-legumes such as

rice, tomato and maize (Endre et al., 2002; Markmann et al.,

2008). The GDPC motif is conserved in the same location,

immediately upstream of the first LRR and encompassing

the first cysteine of the cysteine pair motif, in LRR-RLKs of

non-legume species, such as tomato, maize and rice (Fig-

ure 1b), from which SYMRK homologues capable of sup-

porting AM formation have been identified (Markmann

et al., 2008). A similarly positioned GDPC sequence is also

present in a subset of A. thaliana LRR-RLKs, as well as in a

limited number of LRR-RLKs in non-vascular plants such

as moss (Physcomitrella patens; Figure 1b) suggesting, an

ancient origin of the GDPC-containing LRR-RLKs.

Infection thread formation is blocked in the symRK-14

mutant

symRK-14 har1-1 formed a similar number of uncolonized

NP as the har1-1 mutant (Figure 2a–c) but nodule infection

was clearly delayed. At early time points the number of

infected nodules (referred to hereafter as ‘nodules’), was

reduced by about 90% 7 and 14 days after inoculation (dai)

and by about 50% 21 dai (Figure 2a–c). These results

indicated that the progression from uncolonized NP to

nodules was impaired by the symRK-14 mutation.

The symRK-14 mutant (in the homozygous HAR1 back-

ground) initiated a similar number of nodulation events as

the wild-type control, with the main differences being a

higher proportion of uncolonized NP and delayed nodule

colonization in the case of already well-formed nodules

(Figure 2d–f). This confirmed a slow transition from NP to

nodules as observed in symRK-14 har1-1.

Microscopic examination of roots inoculated with M. loti

carrying a constitutively expressed hemA:lacZ reporter

allowed more detailed characterization of root colonization

events (Figure 3). Unlike har1-1 and wild-type Gifu, in which

M. loti microcolonies were observed within tightly formed

shepherd’s crook-curled root hairs (Figure 3a), root hairs of

both symRK-14 har1-1 and symRK-14 showed exaggerated

curling (Figure 3b–e). At 7 dai, symRK-14 and symRK-14

har1-1 formed abnormally enlarged, diffuse agglomerations

of M. loti (Figure 3b,d) within the apical portion of root hairs

or abnormal ITs whose progress was blocked within the root

hair (Figure 3e,f). Only a very few ITs could be found by 7

and 14 dai on roots of symRK-14 and the vast majority of

them terminated before reaching the bottom of the epider-

mal cell (Figure 3e–h). In contrast, more than 100 ITs per root

were observed in the wild type at the corresponding time-

points after infection (Figure 3a,h). Wild-type plants also

display many arrested infections, but unlike symRK-14,

where blockage of infection was often observed within the

root hair shaft (Figure 3e,f), arrested infection in the wild

type typically terminates at the epidermal–cortical interface.

In both symRK-14 and symRK-14 har1-1 roots, NP often

formed below the entrapped clumps of bacteria that resided

on the surface of the epidermis or within entangled super-

curled root hairs (Figure 4a). This suggested that in the

symRK-14 mutant, NF signalling could induce root cortical

divisions, despite the apparent abnormality in micro-colony

formation and defects in IT development and bacterial entry

into the roots.

Over time, an increasing number of fully colonized

nodules were observed in symRK-14 (Figures 2 and 4b),

but, in most cases no root-hair ITs leading to these nodules

GDPC sequence and the evolution of SYMRK function 3

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

were detected. Instead, M. loti bacteria were present on

the surface of growing NP and empty nodules, where they

formed extensive extracellular infection patches (Fig-

ure 4c,d). These were often associated with bacteria present

within the interior of root hairs, where they remained

dispersed (Figure 4d) and/or heavily accumulated at the

tip (Figure 3d) or the base (Figure 4e) of epidermal cells. The

subsequent colonization of the nodule interior was fre-

quently observed to be preceded by the formation of cortical

infection regions (Figure 4d,f), reminiscent of the infection

pockets formed by M. loti in the L. japonicus root hairless

mutant (Karas et al., 2005). Such infection pockets were

typically found in the periphery of fully infected nodules

(Figure 4g,h), suggesting that they constitute an intermedi-

ate step in successful intracellular infection of nodule

cortical cells. Infrequent epidermal ITs that managed to

progress inside the nodule cortex were also observed.

However, in the majority of cases they appeared misshapen

and terminated prematurely, resulting in the accumulation

of bacteria in the sub-epidermal, cortical region of growing

NP (Figure 4i), from which colonization of the nodule interior

was initiated, as described above. Although somewhat

delayed, mature nodules that formed in symRK-14 har1-1

and symRK-14 were fully colonized by bacteria (Fig-

ure 4b,g,h), similar to wild-type nodules (Figure 4j).

symRK-14 is defective in AM symbiosis

symRK-14 har1-1 was initially categorized as defective for

AM symbiosis (Myc); Murray et al., 2006). In wild-type

plants, after attaching to the root surface, Glomus sp.

hyphae cross the epidermis to reach the inner cortex where

they form characteristic arbuscule and vesicle structures

(Figure 5a). Arbuscular mycorrhizal infection of both sym-

RK-14 har1-1 and symRK-14 was generally blocked at the

epidermal cell layer. On or within symRK-14 epidermal cells,

the fungus formed balloon-shaped hyphal swellings char-

acteristic of the ‘common symbiosis’ mutants (Figure 5b,c;

see also Kistner et al., 2005). In symRK-14 har1-1, and to a

lesser extent in symRK-14, hyphal growth occasionally

continued, leading to apparently normal infections of seg-

ments of symRK-14 and symRK-14 har1-1 roots, including

the formation of vesicles and arbuscules (Figure 5d).

The har1-1 mutant is hypermycorrhized (Solaiman et al.,

2000) but the har1-1 symRK-14 double mutant had slightly

less than half the colonized root length of the har1-1 parent

(Figure 5e). In the symRK-14 mutant (HAR1 background),

AM colonization was only one-tenth that of the wild type

(Figure 5f).

symRK-14 is defective for Nod factor-induced epidermal

calcium spiking

The lack of normal root infection but the presence of nodule

morphogenesis in the symRK-14 mutant could indicate that

this allele does not fully block Nod-factor signalling.

To determine if this is the case, we tested whether calcium

spiking was induced by the Nod factor from M. loti using

microinjection of root hair cells with the calcium-sensitive

dye Oregon Green. To increase the probability of observing

calcium spiking, the experiments were done with the sym-

RK-14 har1-1 double mutant in which many NP are induced

in response to M. loti (Figure S3). None of the cells tested

showed calcium spiking, and so in this respect the symRK-14

Figure 2. Kinetics of nodule formation.

(a)–(c) Lotus japonicus har1-1 parental line and the corresponding symRK-14 har1-1 double mutant.

(d)–(f) Lotus japonicus Gifu (wild-type) and the corresponding symRK-14 single mutant. Note that all nodulation events that were not accompanied by

Mesorhizobium loti colonization are defined as nodule primordia. Ten to 16 individuals were scored for each time point (averages � 95% confidence intervals are

given).

4 Sonja Kosuta et al.

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

har1-1 mutant behaved similarly to the symRK13 har-1-1

mutant (Figure S3b,c). As expected, the har1-1 mutant had

normal calcium spiking (Figure S3a).

Microinjection of root hairs has the disadvantage that

only a limited number of cells can be analysed. To analyse a

larger number of epidermal cells, the nuclear-targeted

chameleon (NupYC2.1; Sieberer et al., 2009) was introduced

into wild-type and symRK-14 roots via Agrobacterium

rhizogenes-mediated transformation and epidermal cells of

the susceptible zone were analysed for calcium spiking.

In the wild type, regular calcium spiking was observed in 63

out of 285 epidermal cells examined (Figure 6a) and a few

irregular calcium spikes (Figure 6b) were detected in another

16 cells. When transgenic roots of the symRK-14 mutant

were analysed, only two cells (out of 376) showed clear

calcium spiking (Figure 6c), and a few calcium spikes

(Figure 6d) were observed in another 10 cells. Occasional

cells showing calcium spiking was previously reported with

one of the weak alleles (dmi2-2) of the SYMRK orthologue in

M. truncatula (Wais et al., 2000). These observations indi-

cate that although the symRK-14 mutation greatly reduces

Nod factor-induced calcium spiking, a few cells retain the

ability to mount this cellular response.

symRK-14 is defective for early induction of the nodulation

gene NIN

Given the large reduction in the number of calcium spiking

cells in the symRK-14 mutant, we evaluated the signalling

that occurs in wild-type L. japonicus downstream of SYMRK

and CCaMK using the promoter proNIN:GUS reporter gene

fusion. We assessed early symbiotic responses and cellular

differentiation in the root epidermis in response to M. loti

inoculation in F2 progeny derived from the cross between

homozygous symRK-14 and proNIN:GUS transgene-con-

taining wild-type L. japonicus (see Experimental Proce-

dures). As expected, in wild-type proNIN:GUS plants and

(a)

(d)

(g) (h)

(e) (f)

(b) (c)

Figure 3. symRK-14 is defective in the infection thread-dependent root hair colonization by Mesorhizobium loti.

Mesorhizobium loti strain NZP2235 tagged with a constitutive hemA:lacZ reporter gene fusion was used for inoculation and roots were stained for b-galactosidase

activity to reveal the location of bacteria (blue). (a) Root colonization by M. loti in the wild-type Lotus japonicus Gifu. MC denotes a microcolony within the curled

root hair; IT denotes an infection thread that contains bacteria. (b)–(d) Unsuccessful colonization attempts at the symRK-14 root epidermis 10 days after inoculation

(dai). Grossly enlarged colonies of M. loti formed within exaggeratedly coiled root hairs (b, d); accumulation of bacteria at the base of tangled root hairs (c). (e), (f)

Unsuccessful root hair colonization/IT formation events in symRK-14 har1-1 14 dai. (g) Rare example of successful IT formation in symRK-14 har1-1. (h) Combined

numbers (averages � 95% confidence intervals) of intact and aborted ITs per root in the wild-type and symRK-14 single mutant are shown 7 and 14 dai (n = 10).

GDPC sequence and the evolution of SYMRK function 5

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

those heterozygous for symRK-14, proNIN:GUS induction

was observed in the root epidermis, including root hairs, 24–

72 h after inoculation with M. loti (Figure 7a,c). This induc-

tion also appeared to be cell autonomous (Figure 7d). In

contrast, all the proNIN:GUS progeny homozygous for

symRK-14 lacked epidermal b-glucuronidase (Figure 7e,g),

although later during development (7–21 dai), b-glucuroni-

dase was detected in these plants (Figure 7f). It was localized

to cell division foci of developing NP within the root cortex

and to nodules, similarly to wild-type proNIN:GUS trans-

genic plants (Figure 7b). These observations were con-

firmed in F3 progeny from homozygous symRK-14

segregants (data not shown).

Quantitative PCR was used to validate the reporter gene

data by evaluating the steady-state levels of NIN mRNA in

roots of proNIN:GUS wild-type and symRK-14 plants (Fig-

ure 7h). An early time point, 48 h after inoculation, was

chosen for this analysis to avoid potential interference from

the NIN mRNA that is normally associated with nodule

organogenesis in the root cortex. A similar, background

level of NIN mRNA was detected in un-inoculated roots

regardless of the genotype tested. Upon inoculation, this

level was increased approximately 25 times in the wild type

and only twice in symRK-14 mutant roots (Figure 7h).

DISCUSSION

symRK-14 uncouples epidermal and cortical programs

The symRK-14 mutation specifically altered the SYMRK-

dependent signalling for epidermal colonization by bacterial

and fungal partners, without having a significant negative

impact on the cortical programs. The symRK-14 mutant was

defective in all epidermal responses to M. loti inoculation/

NF application analysed, including root hair curling, micro-

colony formation, calcium spiking, NIN-gene expression and

IT initiation/persistence. The symRK-14 mutant was also

significantly impaired in entry of the AM fungus through

the outermost cell layers, which is consistent with previous

(a)

(d)

(g) (h) (i) (j)

(e) (f)

(b) (c)

Figure 4. Colonization of nodule primordia by Mesorhizobium loti.

(a) symRK-14 forms numerous empty nodule primordia (arrows). (b) Fully colonized, wild-type looking nodules formed on roots of symRK-14 21 days after

inoculation (dai). (c) Close-up to the surface of an uncolonized nodule primordium (NP) showing M. loti (blue) occupying its surface. (d) Uncolonized nodule

primordium, where bacteria are present within the interior of the root hair; an extensive accumulation of M. loti occurred at the surface (*) and also within a sub-

epidermal cortical region of the NP (arrow), from which further colonization of the NP interior has just begun (arrowhead). (e) Advancing colonization of the nodule

interior by M. loti. Note the presence of numerous cortical ITs (arrow) and a root hair IT that failed to progress beyond root epidermis (arrowhead); as a result,

bacteria have accumulated at the base of the root hair cell. (f) A cross-section of an uncolonized NP showing accumulation of M. loti (dark blue color) within the sub-

epidermal, cortical region of the NP. (g) A small symRK-14 nodule colonized by M. loti (blue), showing the presence of a sub-epidermal infection pocket (arrow). (h) A

cross-section through the nodule shown in panel (g). (i) A longitudinal section of a growing NP, showing accumulation of M. loti (blue) within grossly deformed and

entangled root hairs. An IT (arrowhead) was formed but failed to ramify within the nodule interior, leading to the accumulation of M. loti within a sub-epidermal

cortical region of the NP. (j) An example of fully colonized nodules in wild-type L. japonicus Gifu; note ITs on the top of the nodule. The images shown in panels (d),

(f), (h) and (i) reflect 30-lm thick sections.

6 Sonja Kosuta et al.

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

observations (Demchenko et al., 2004). Taken together,

these data clearly demonstrate loss of early symbiotic signal

transduction in the epidermis caused by the symRK-14

mutation. However, the cortical responses including, NP

organogenesis, NIN induction in nodules, cortical IT and

arbuscule/vesicle formation were unaffected.

symRK-14 restricts epidermal calcium spiking and NIN

expression

As observed previously with mutations in NFR1 and NFR5

receptor genes and also in the common symbiosis loci

SYMRK, NUP85, NUP133, CASTOR and POLLUX (Miwa

et al., 2006), no calcium spiking was detected in symRK-14

when assayed in a limited number of root hair cells injected

with a calcium-sensitive dye. This was supported by the

observation of greatly reduced expression of NIN in the

symRK-14 epidermis. It was therefore puzzling that a lack of

these cellular responses, which would normally be expected

to result in a non-nodulating phenotype, coincided with

almost normal nodule organogenesis in symRK-14.

Analysis of a large number of epidermal cells in symRK-14

roots using a nuclear-targeted chameleon showed that a few

of these cells maintained the ability to respond to Nod factor

by inducing calcium spiking. The simplest explanation of

this is that symRK-14 is slightly leaky, therefore allowing rare

calcium spiking events in the root epidermis. This could

explain the limited initiation of epidermal ITs in symRK-14.

SYMRK might handle several non-equivalent inputs

In contrast to AM infection and IT-dependent bacterial entry,

the presence of the intact GDPC sequence was apparently

dispensable for NP organogenesis. The basis for this dif-

ferential effect of the symRK-14 mutation remains unclear;

however, as the proline residue was replaced by leucine as

the result of symRK-14 mutation, this might have simply

limited a signalling capacity of the receptor due to a struc-

tural change to its extracellular domain.

The structural requirements for NF were reported to be

more stringent for IT-dependent bacterial entry than for the

induction of cortical cell divisions for NP organogenesis

(Ardouel et al., 1994; Walker and Downie, 2000). Further-

more, more rigorous constraints in terms of NF structure

were observed for root hair-dependent invasion in compar-

ison with intercellular, cortical entry in the semi-aquatic

legume Sesbania rostrata (D’Haeze et al., 2000; Goormach-

tig et al., 2004; for recent review see Capoen et al., 2010).

(a) (b)

(e) (f)

(c) (d)

Figure 5. symRK-14 is defective in arbuscular

mycorrhizal (AM) symbiosis.

(a) A representative fragment of Lotus japonicus

wild-type roots that was successfully colonized

by an AM fungus, Glomus intraradices. (b), (c)

Unsuccessful colonization attempts associated

with characteristically swollen hyphae (arrows)

on symRK-14 and symRK-14 har1-1 roots,

respectively. (d) A section of a symRK-14 har1-1

root successfully colonized by the fungus as

evidenced by wild-type like arbuscules and ves-

icles. EH, extraradical hypae; V, vesicle; Ar,

arbuscule, as in panel (a). Panels (a)–(d) repre-

sent samples collected 8 weeks post-inoculation.

(e), (f) Scores of root colonizations (aver-

ages � 95% confidence intervals) by G. intrara-

dices, as reflected by the presence of intraradical

hyphae, vasicles and arbuscules, in the wild-type

and har1-1 parental line and the corresponding

single and double mutants.

GDPC sequence and the evolution of SYMRK function 7

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

Thus, depending on upstream events, different inputs

probably converge at and are processed by SYMRK, leading

to non-equivalent outputs.

symRK-14 does not affect the formation of nodule primordia

Very few epidermal cells showed calcium spiking in symRK-

14, but nevertheless the activation of these cells could pos-

sibly explain nodule formation in symRK-14, since the much

longer time frames involved (several days) might, even at

low signalling intensity, allow sufficient cytokinin produc-

tion to trigger organogenesis in the cortex. It is possible that

the low nodulation phenotype of some nup85, nup133 and

pollux mutants may also be caused by the presence of a

limited number of calcium spiking cells (Kanamori et al.,

2006; Saito et al., 2007; Martin Parniske, unpublished data).

This interpretation is further supported by the phenotype of

the L. japonicus nena-1 mutant (Groth et al., 2010). Like

symRK-14, nena-1 significantly reduced epidermal calcium

spiking, and this prevented epidermal IT formation while

leaving the cortical program for nodule organogenesis

almost intact (Groth et al., 2010). However, it remains

possible that epidermal calcium is not as essential in sig-

nalling for NP organogenesis as previously suggested.

It has recently been shown that the activation of

CCaMK is sufficient to explain SYMRK action (Hayashi

et al., 2010; Madsen et al., 2010) and from this we may

infer that induction of CCaMK may be sufficient to

activate nodule organogenesis in the cortex. Importantly,

nodule organogenesis in symRK-14 requires functional

CCaMK as the symRK-14 ccamk-5 double mutant (Murray

et al., 2006) was unable to form nodules (see Appen-

dix S1). Thus, either a limited number of responsive

epidermal cells in symRK-14 and nena-1 are still sufficient

to activate CCaMK-dependent NP organogenesis, or this is

achieved in a manner independent of epidermal calcium

spiking.

The cortical infection program

snf1, a mutant version of CCaMK, can induce nodule devel-

opment in the absence of rhizobia or Nod factors (Gleason

et al., 2006; Tirichine et al., 2006). Importantly, the infection

of spontaneously induced nodules by M. loti in the symrk-3

snf1 double mutant (Madsen et al., 2010) appears to mimic

at least a subset of infection events observed in symRK-14

(Figure 4i). Thus, in both symRK-14 and symrk-3 snf1

mutants, a limited number of root hair ITs that appeared

misguided and were characterized by very distinctive local-

ized swellings were observed (Figure 4i; Madsen et al.,

2010), suggesting aberrant epidermal signalling in symrk-3

snf1. In symRK-14, formation of intermediate infection

pockets preceded colonization of the nodule interior, which

was reminiscent of the nena-1 phenotype (Groth et al.,

2010). The nodule-associated infection pockets were not

reported for symrk3 snf1; however, like symRK-14 (Fig-

ure 4e) and nena-1 (Groth et al., 2010), numerous cortical ITs

were found in the interior of colonized symrk3 snf1 nodules

(Madsen et al., 2010).

Madsen et al. (2010) provided evidence for a NFR1-,

NFR5- and SYMRK-independent Nod factor perception

system in the cortex, since nfr1 nfr5 symrk-3 snf1 quadruple

mutants were still able to form nodules containing trans-

cellular cortical ITs. The presence of these ITs was depen-

dent on the production of Nod factor by the rhizobia. Taken

together with our observation of wild-type-like cortical

symbiotic development in the symRK-14 mutant, these

observations are congruent with the idea that the main role

of SYMRK during the infection process is in the epidermis.

This also adds to the growing body of evidence in support

of alternative signalling components/pathways leading to

the activation of the cortical infection program. This would

allow the cortical infection process, which in symRK-14 and

nena-1 originates mainly from nodule-associated infection

pockets, to proceed normally in spite of the defective

epidermal program. It is worth noting, however, that this is

unlike the aberrant cortical infection phenotypes of RNA

interference (RNAi) lines in Sesbania or Medicago, where

down-regulation of the SYMRK expression lead to aberrant

Figure 6. Nuclear calcium oscillation in response to Mesorhizobium loti Nod

factor monitored in Lotus japonicus wild-type and symRK-14 roots expressing

cameleon NupYC2.1.

Changes in nuclear calcium were recorded following the addition of 100 nM

Nod factor (vertical line) in L. japonicus wild-type Gifu (a, b) and symRK-14

epidermal cells (c, d). The yellow/cyan fluorescent protein ratio (YFP/CFP) was

plotted against time. Images were taken at 5-sec intervals for more than

30 min. The traces show representative cells positive for calcium spiking (a, c)

and representative cells showing a few spikes (b, d). Cells showing traces a

and b are from seven and six independent plants, respectively. Cells showing

traces c and d are from two and three independent plants, respectively. The

number of epidermal cells positive for calcium oscillation divided by the total

number of cells tested is indicated next to each trace.

8 Sonja Kosuta et al.

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

symbiosome formation (Capoen et al., 2005; Limpens et al.,

2005).

Presence of the GDPC motif in RLKs ranging from mosses

to angiosperms suggests an ancient origin

The symRK-14 mutation occurred in a highly conserved

GDPC motif found immediately upstream of the first LRR

region, a location conserved in LRR-RLKs in legumes and

many non-legume species, including the moss P. patens

and liverwort M. polymorpha (Figure S4). Strong conserva-

tion of GDPC in terms of both its amino-acid sequence and

topological position in nearly one-third of LRR-RLKs in the

non-AM plant Arabidopsis suggests additional functional

significance of this motif outside of symbiosis. Nevertheless,

the fact that we have found only seven and six LRR-RLKs

in P. patens and M. polymorpha, respectively, that contain

GDPC (Figure S4), opens the possibility of identifying an

orthologue of the L. japonicus SYMRK among a limited

number of GDPC-bearing LRR-RLKs in non-vascular plants.

This should further aid in understanding the evolution of this

interesting gene and the origin of its complex function in

symbiosis (Markmann et al., 2008).

EXPERIMENTAL PROCEDURES

Plant material and growth conditions

The L. japonicus symRK mutants were isolated as suppressors ofhar1-1 hypernodulation and identified by map-based cloning asdescribed previously (Murray et al., 2006). The double symRK-13har1-1 and symRK-14 har1-1 mutant lines were crossed with wild-type L. japonicus Gifu and the F2 progenies homozygous for bothHAR1 and symRK-13 or symRK-14 were selected.

Seeds were scarified with sandpaper before surface-sterilizationfor 3 min with concentrated bleach. Seeds were then rinsed fourtimes with sterile water and imbibed overnight before placing themin Petri plates on filter paper wetted with sterile water. Seven-day-old seedlings were transplanted to pots for the evaluation ofsymbiotic phenotypes as described below. For all phenotypicassays, plants were maintained in a growth room under a 16-h/8-hday/night regime, with 200–250 lE sec)1 m)2 light intensity at 22�C,and watered with tap water unless otherwise indicated.

Evaluation of symbiotic phenotypes

For nodulation assays, L. japonicus seedlings were transferredfrom germination plates to pots containing an autoclaved 6:1mixture of vermiculite and sand soaked to field capacity with B&Dnutrient solution (Broughton and Dilworth, 1971) containing a low

(a)

(e) (f) (h)

(b) (c)

(g)

(d)

Figure 7. Mesorhizobium loti-induced proNIN:GUS reporter gene activity in roots of Lotus japonicus wild-type (a–d) and symRK-14 mutant (e–g).

Transgenic wild-type Gifu and symRK-14 mutant plants carrying the proNIN:GUS reporter gene construct were inoculated and the GUS activity (blue) was visualized

by histochemical staining 24 h (a, e) and 21 days (b, f) after inoculation. Dark-field images of 30-lm-thick sections of proNIN:GUS containing wild-type (c) and

homozygous symRK-14 (g) transgenic hairy roots 48 h after inoculation (hai) are shown, where GUS activity is indicated by the red colour. Note that the reporter

gene activity is present in root hairs and epidermis of the proNIN:GUS wild-type roots but is absent from proNIN:GUS symRK-14 roots. (d) Close up to the

proNIN:GUS wild-type root epidermis showing GUS activity in a single root hair epidermis cell; note that surrounding epidermal cells do not express the reporter

gene activity. (h) Quantitative RT-PCR of the NIN mRNA in un-inoculated (U) and inoculated (I) proNIN:GUS wild-type and symRK-14 roots 48 hai; average

values � SD are given).

GDPC sequence and the evolution of SYMRK function 9

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

concentration (0.5 mM) of KNO3. Plants were inoculated after7 days with M. loti strain NZP2235 carrying a hemA:lacZ reportergene fusion. At 7, 14 and 21 days after inoculation, roots were fixed,stained for b-galactosidase activity, cleared as previously described(Wopereis et al., 2000) and evaluated using brightfield micros-copy (Nikon SMZ1500; http://www.nikon.com/). Nodule countswere performed on at least 10–16 independent individuals fromeach line and were expressed as mean nodule number perplant � 95% confidence interval. Infection threads were scored 7and 14 dai on at least 10 individuals. Root and nodule sectionswere obtained and processed as previously described (Karaset al., 2005).

For evaluation of mycorrhizal phenotypes, L. japonicus seedlingswere transferred from germination plates to pots containing a 1:1mixture of Turface and sand soaked to field capacity with 1/2Hoagland’s solution (Hoagland and Arnon, 1950). The AM inoculumconsisted of the Turface:sand (1:1) substrate and roots (choppedinto 1-cm pieces) of a 6-month-old Glomus intraradices (Schenckand Smith)–Porrus alum L. culture. At 4 and 8 weeks after inocu-lation, roots were ink-stained as previously described (Kosuta et al.,2005) and observed microscopically as above. The percentage ofroot length colonization was determined for a minimum of 10individuals per line and expressed as mean percentage of colonizedroot length � 95% confidence interval.

Agrobacterium rhizogenes-mediated root transformation

The symRK-13 and symRK-14 complementation experiments wereperformed as in Murray et al. (2006). The A. rhizogenes AR10 straincarrying either a Gateway pK7RWG2,0 empty destination vector orthe same vector containing the entire SYMRK genomic locus wasused for these experiments. The nodulation phenotypes wereevaluated at 10 and 12 dai, while AM symbiosis was scored 8 weeksafter inoculation.

The nucleoplasmin chameleon YC2.1 (NupYC2.1; kindly providedby D. Barker, INRA-CNRS, France) was introduced into L. japonicuswild-type plants and in symRK14 plants by hairy root transforma-tion as described (Diaz et al., 2005) using the A. rhizogenes strainAR1193. Plants were grown in plates containing B5 medium(Gamborg et al., 1968) for at least 21 dai before calcium imagingexperiments.

Calcium analysis in L. japonicus epidermal cells

For analysis of calcium spiking using microinjected dyes, seedlingswere sterilized, germinated and grown as described previously(Miwa et al., 2006), except that the agar growth medium was thatdescribed by Fahraeus (1957). Seedlings were microinjected asdescribed previously, except that the liquid medium used in thebath was as described by Fahraeus (1957) and the volume of theincubation bath was 100 ll. Oregon Green 488 BAPTA-1-dextran10 000 MW and Texas Red-dextran 10 000 MW (Molecular Probes;http://www.invitrogen.com/site/us/en/home/brands/Molecular-Pro-bes.html) were microinjected and their fluorescence imaged asdescribed previously. After taking a series of images, the ratiometrictraces were calculated by dividing Oregon Green fluorescence bythat of Texas Red at each time point. Traces were generated usingMicrosoft Excel.

For calcium imaging experiments using NupYC2.1 (Siebereret al., 2009), plants transformed by hairy root transformation weretransferred to plates containing Fahraeus agar medium 5–10 daysbefore calcium analysis. Filter paper (grade 0860; Schleicherand Schull, http://www.gogenlab.com/products/manufacturers/schleicher-schuell) was placed between the agar and the roots toprevent the roots growing into the agar. The roots were then

covered by another filter paper to keep them moist. The Petri disheswere incubated in a vertical position in a controlled environment(20�C/15�C, day/night cycles of 18 h/6 h) and the region of the Petridish containing the roots was covered with black plastic.

For microscopic observation on an inverted epifluorescentmicroscope, plants were placed into a chamber’ made on a largecover glass using high-vacuum grease (Dow Corning; http://www.dowcorning.com/) and filled with liquid Fahraeus medium.Roots with a high fluorescence level in root cell nuclei wereselected, excised and transferred to a smaller chamber containing100 ll of liquid Fahraeus medium. After the addition of Nod factor ata final concentration of 100 nM changes in nuclear calciumconcentrations were analysed by calculating fluorescence reso-nance energy transfer using measurements of fluorescence of cyanand yellow fluorescent protein (CFP and YFP, respectively) asdescribed by Miwa et al. (2006). All cells tested for calcium spikingwere in the area of growing root hair cells. About half of the roothair cells were imaged with their axes horizontal to the longitu-dinal root plane. Epidermal cells were also imaged with their axesperpendicular to the root; with this latter approach it was possibleto image a larger number of cells. There was no significantdifference (based on a chi-square test) in the number of cells thatwere positive for calcium spiking regardless of the techniqueused.

Images were collected every 5 sec and analysed using MetaFluorsoftware. Values were exported into EXCEL (Microsoft; http://www.microsoft.com/), converted to a ratio between YFP and CFPfluorescence, and plotted against time.

proNIN:GUS expression

Transgenic plants carrying the homozygous proNIN:GUS reportergene fusion (kindly provided by J. Stougaard, Aarhus University,Denmark; see also Radutoiu et al., 2003) were crossed with homo-zygous symRK-14. The segregating F2 population was analyzed forearly root responses to M. loti inoculation by histochemical detec-tion of b-glucuronidase activity. The same population was simul-taneously genotyped for the presence of the symRK-14 mutationand the proNIN:GUS transgene by sequencing and PCR, respec-tively.

For expression assays, approximately 100 F2 seeds were germi-nated for 2 days on inverted 0.8% agar plates. The 69 selectedhealthy seedlings were placed on sterile filter paper laid over B&Dwith reduced nitrate as above, solidified with 0.8% agar. The rootportion of each seedling was entirely covered with an additionalpiece of B&D-wetted Whatman filter paper grade 597 (Schleicher &Schull). Plates were propped in a vertical position and incubated for2 days at 22�C with 16-h daylight. The bottom half of the Petri dishwas covered to obscure the light. After 2 days, each seedling wasinoculated with 20 ll of wild-type M. loti suspension, which wasdiluted 1:100 in distilled water. Two days later, the bottom 5 cm ofeach root was removed and placed into a Farhaeus slide.To visualize GUS activity, roots were incubated overnight at 37�Cwith the substrate 5-bromo-4-chloro-3-indolyl glucuronide cyclo-hexylammonium salt (X-gluc; Inalco, http://www.inalco.it/), asdescribed previously (Journet et al., 1994). Seedlings were imme-diately planted into a 4:4:1 mixture of Pro-mix (Premier Horticulture;http://www.premierhort.com/), medium size vermiculite (Therm-O-Rock East Inc.; http://www.therm-o-rock.com/), and perlite (Therm-O-Rock East Inc.) and watered with ½ Hoagland’s nutrient solution(Hoagland and Arnon, 1950) or water as needed. At planting,seedlings were re-inoculated with wild-type M. loti and wateredwhen necessary with tap water. After 2 weeks, a portion ofnodulated roots was harvested, washed thoroughly and stainedfor GUS activity as above.

10 Sonja Kosuta et al.

The Plant Journal ª 2011 Blackwell Publishing Ltd, The Plant Journal, (2011), doi: 10.1111/j.1365-313X.2011.04645.xNo claim to original US government works

The same analyses were performed on 180 F3 progenies of theselected F2 individual that was homozygous for the symRK-14 alleleand was carrying the proNIN:GUS transgene, except that rootssegments from three time points at 24, 48 and 72 h after inoculationwith M. loti, were harvested and analyzed for the reporter geneactivity. An independent batch of the same F3 seeds were planted tosoil and analysed for the reporter gene activity 21 dai.

For genotyping, 69 F2 progeny of the F1 parental line derived fromthe cross between homozygous symRK14 and the proNIN:GUStransgenic Gifu plant were analysed. The SYMRK extracellularregion encompassing the site of the symRK-14 mutation wasPCR-amplified from all individuals using 5¢-GTATTGGCTG-GAGGGTCAAA-3¢ and 5¢-GAAATGAGGGGACAGAACCA-3¢ as thegene-specific forward and reverse primers and the correspondingsequences were determined. The presence of proNIN:GUS wasanalysed using the forward (5¢-CAAGCACTGCTTATTAATTAC-3¢)and reverse (5¢-CGCGATCCAGACTGAATGCCC-3¢) primers. Theratio of 48:10:8:3 of wild-type plants with (48) and without (10) theproNIN:GUS transgene and symRK-14 with (8) and withoutthe transgene (3) was obtained. Forty-seven out of 48 wild-type plants carrying the transgene were GUS positive, while allsymRK-14 plants were GUS negative.

The analysis of NIN gene expression

Total RNA from un-inoculated and inoculated proNIN:GUS wild-typeand symRK-14 mutant roots was extracted and processed asdescribed earlier (Murray et al., 2007). Three biological replicas wereused and quantitative RT-PCR reactions were performed in triplicateon 2 ll cDNA using the SYBR-GREEN PCR Master Kit (Perkin-ElmerApplied Biosystems; http://www.appliedbiosystems.com/) as previ-ously described (Murray et al., 2007). The NIN mRNA-specificprimers used were as described in Tirichine et al. (2007).

Computer analysis of sequence data

Databases were searched with BLAST (http://www.ncbi.nlm.nih.gov/) using the L. japonicus SYMRK as a query to identify all serine/threonine kinases with the GDPC in the organisms of interest.Additional LRR-RLKs were identified in rice by the Putative FunctionSearch on the Rice Genome Annotation site (http://rice.plantbiolo-gy.msu.edu/cgi-bin/putative_function_search.pl) and in P. patensby a keyword search on the Joint Genome Institute website (http://genome.jgi-psf.org/annotator/servlet/jgi.annotation.Annotation?pDb=Phypa1_1). GDPC domains were identified by string searchand/or by alignments by CLUSTALW2 (http://www.ebi.ac.uk/Tools/msa/clustalw2/). Secondary protein structure was predicted andcompared using PredictProtein (http://www.embl-heidelberg.de/)and comparisons were done using Pfam (http://pfam.janelia.org/).Results were then compiled and plotted in Excel.

ACKNOWLEDGEMENTS

We thank A. Molnar for his expert help in preparation of the figures.The plasmid expressing the nuclear-targeted chameleonNupYC2.1 was a generous gift from D. Barker. This work wassupported by grants from Agriculture and Agri-Food Canada CropGenomics Initiative and National Science and Engineering ResearchCouncil of Canada (NSERC grant no. 3277A01) to KS and by theBiotechnology and Biological Sciences Research Council withgrants to JAD and GEDO. MH and BK were supported by NSERCPGS-D while CJ received Ontario Genomic Institute (OGI) summerfellowship. GM was supported by a European Union grant (MRTN-CT-2006-035546) awarded to JAD and GEOD in the ‘Nodperception’network. MAL and MP were supported by the Priority ProgramSPP1212 ‘Plant-Micro’ of the German Research Council (DFG).

SUPPORTING INFORMATION

Additional Supporting Information may be found in the onlineversion of this article:Figure S1. Symbiotic phenotypes of symRK-13 and symRK-14.Figure S2. Complementation of the symRK-13 and symRK-14mutant symbiotic phenotypes.Figure S3. Mesorhizobium loti Nod factor-induced epidermalcalcium spiking in the Lotus japonicus har1-1 parental line andsymRK-13 har1-1 and symRK-14 har1-1 double mutants.Figure S4. The presence of the GDPC sequence coincides with a lownumber of leucine-rich repeats.Appendix S1. Supporting data: nodule formation in symRK-14requires the intact calcium and calmodulin-dependent receptorkinase (CCaMK) function.Please note: As a service to our authors and readers, this journalprovides supporting information supplied by the authors. Suchmaterials are peer-reviewed and may be re-organized for onlinedelivery, but are not copy-edited or typeset. Technical supportissues arising from supporting information (other than missingfiles) should be addressed to the authors.

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