arabidopsis snc2-1d activates receptor-like protein ... · arabidopsis snc2-1d activates...

Arabidopsis snc2-1D Activates Receptor-LikeProtein-Mediated Immunity Transduced through WRKY70 C W

Yaxi Zhang,a,b Yuanai Yang,a Bin Fang,a Patrick Gannon,c Pingtao Ding,a Xin Li,c and Yuelin Zhanga,1

a National Institute of Biological Sciences, Beijing 102206, People’s Republic of Chinab Beijing Normal University, Beijing 100875, People’s Republic of ChinacMichael Smith Laboratories, University of British Columbia, Vancouver V6T 1Z4, Canada

Plant immune receptors belonging to the receptor-like protein (RLP) family contain extracellular leucine-rich repeats (LRRs)

and a short cytoplasmic tail linked by a single transmembrane motif. Here, we report the identification of snc2-1D (for

suppressor of npr1-1, constitutive 2), a semidominant Arabidopsis thaliana mutant with constitutively activated defense

responses. Map-based cloning of snc2-1D showed that it encodes an RLP. The point mutation in snc2-1D leads to

substitution of the second Gly for Arg in the conserved GXXXG motif of the transmembrane helix, suggesting that this

residue is important for negative regulation of the protein. Epistasis analysis revealed that the snc2-1D mutant phenotype is

not affected by mutations in genes known to be required for the nucleotide binding (NB)-LRR Resistance (R) protein

signaling. A suppressor screen of snc2-1D was performed, and map-based cloning of one suppressor revealed that

mutations in WRKY70 suppress the constitutive defense responses in snc2-1D, suggesting that WRKY70 functions

downstream of snc2-1D. The identification of snc2-1D provides us with a unique system for genetic analysis of resistance

pathways downstream of RLPs, which may be distinct from those downstream of NB-LRR type R proteins.

INTRODUCTION

Plantshaveevolvedsophisticateddefensemechanismsto fendoff

a vast variety of microbial pathogens in nature. The plant immune

system consists of pathogen-associated molecular pattern-trig-

gered immunity (PTI) and effector-triggered immunity (Jones and

Dangl, 2006). Recognition of pathogen-associated molecular pat-

terns is facilitated by surface receptors such as FLS2, EFR, and

CERK1 (Gomez-Gomez and Boller, 2000; Zipfel et al., 2006; Miya

et al., 2007; Wan et al., 2008). By contrast, detection of pathogen

effectors involves both intracellular and transmembrane receptors

collectively called Resistance (R) proteins. Effector-triggered im-

munity is usually a stronger immune response comparedwith PTI,

and it is often associated with the hypersensitive response (HR), a

form of localized cell death at the site of infection that may help

restrict the growth and spread of pathogens.

There are three major classes of R genes in plants (Dangl and

Jones, 2001). The largest class encodes nucleotide binding-

leucine-rich repeat (NB-LRR)–type R proteins with anNB domain

and LRRs. The NB-LRR class of R proteins is predicted to be

intracellular. The two remaining classes of R genes encode

transmembrane receptor-like kinases (RLKs) and receptor-like

proteins (RLPs). Unlike RLKs, RLPs do not have an intracellular

kinase domain. The functional R proteins known in RLPs include

tomato (Solanum lycopersicum) Cf and Ve proteins and the apple

(Malus domestica) HcrVf2 protein, which confer resistance

against the fungal pathogens Cladosporium fulvum, Verticillium

species, and Venturia inaequalis, respectively (Jones et al., 1994;

Kawchuk et al., 2001; Belfanti et al., 2004; Fradin et al., 2009).

Interestingly, two RLPs in tomato, Eix1 and Eix2, also function as

receptors of the fungal ethylene-inducing xylanase, which is a

potent elicitor of plant defense responses (Ron and Avni, 2004).

Reverse genetic analysis of Avr9/Cf-9 Rapidly Elicited genes

identified multiple components, including the protein kinase

ACIK1, the U-box protein CMPG1, and F-box protein ACIF1

that are required for Cf-mediated HR or resistance against C.

fulvum (Rowland et al., 2005; Gonzalez-Lamothe et al., 2006; van

den Burg et al., 2008). Another interesting protein found to

be required for Cf-mediated resistance responses is NRC1, an

NB-LRR–type R protein. Silencing NRC1 in Nicotiana benthami-

ana compromises HR induced by Eix and multiple R proteins,

including Cf-9, Pto, Rx, and Mi (Gabriels et al., 2007).

InArabidopsis thaliana, there are 57 putative RLP genes (Wang

et al., 2008). The functions of these proteins are largely unknown,

with the exception of CLAVATA2 (CLV2) and TOO MANY

MOUTHS (TMM). CLV2 regulates meristem and organ develop-

ment (Jeong et al., 1999), whereas TMM regulates proper sto-

mata distribution (Nadeau and Sack, 2002). Arabidopsis RLP52

was implicated in resistance against the powdery mildew path-

ogen Erysiphe cichoracearum (Ramonell et al., 2005), whereas

Arabidopsis RLP30 was suggested to influence nonhost resis-

tance toward Pseudomonas syringae pv phaseolicola (Wang

et al., 2008). None of the Arabidopsis RLP proteins have been

shown to function as R proteins.

In this study, we report the characterization of snc2-1D (for

suppressor of npr1-1, constitutive 2), which was identified in a

1Address correspondence to [email protected] 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: Yuelin Zhang([email protected]) .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.110.074120

This article is a Plant Cell Advance Online Publication. The date of its first appearance online is the official date of publication. The article has been

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The Plant Cell Preview, www.aspb.org ã 2010 American Society of Plant Biologists 1 of 11

forward genetic screen to search for components functioning in

the NONEXPRESSOR OF PATHOGENESIS-RELATED GENES1

(NPR1)-independent resistance pathways. snc2-1D mutant plants

accumulate high levels of endogenous salicylic acid (SA) and exhibit

enhanced disease resistance against the virulent oomycete Hyalo-

peronosporaarabidopsidis (H.a.)Noco2.Positionalcloning revealed

that snc2-1D encodes an RLP with a gain-of-function mutation in

the conservedGXXXGmotif in the transmembrane domain. A sup-

pressor screen of snc2-1D further revealed that WRKY70 is re-

quired forsnc2-1D–mediatedresistance,suggesting thatWRKY70

is an important component of RLP-mediated innate immunity.

RESULTS

Identification and Characterization of snc2-1D

To search for defense signaling components independent of

NPR1, we screened for suppressor mutations that caused the

activation of defense gene expression in the absence of NPR1.

To that end, seeds of npr1-1 were mutagenized with ethane

methylsulfonate (EMS), and the M2 seedlings were screened for

constitutive expression of a BGL2 (PR2)-GUS reporter gene,

which is not expressed in the npr1-1 single mutants. One mutant

with strong GUS staining displayed dwarf morphology similar to

the previously identified snc1 (Li et al., 2001); therefore, we

named the new mutation suppressor of npr1-1, constitutive 2

(snc2) (Figure 1A). When backcrossed with the wild type, the F1

plants exhibited an intermediate morphology, indicating that the

mutation is semidominant. Thus, we named thismutant snc2-1D.

Macroscopically, like snc1, snc2-1D plants do not exhibit spon-

taneous lesions. The dwarf morphology of snc2-1D can be

partially suppressed by growing the plants at high temperature

(see Supplemental Figure 1 online).

Real-time RT-PCR analysis confirmed that the endogenous

defense marker gene PR-2 (BGL2) was constitutively expressed

in snc2-1D plants (Figure 1B). The expression level of PR-1 was

also elevated, but not to the same level as PR-2 (Figure 1C).

Salicylic acid (SA), a key signal molecule of plant defense, was

found to accumulate more in snc2-1D npr1-1 plants (Figure 1D).

Figure 1. Characterization of the snc2-1D Mutant.

(A) Phenotypes of the wild type, npr1-1, and snc2-1D npr1-1. Plants

were grown on soil and photographed 4 weeks after planting.

(B) and (C) PR-2 (B) and PR-1 (C) expression in the wild type, npr1-1, and

snc2-1D npr1-1 determined by quantitative RT-PCR. Values were nor-

malized to the expression of ACTIN1. Statistical differences among the

samples are labeled with different letters (P < 0.001).

(D) Free SA and SAG in wild-type, npr1-1, and snc2-1D npr1-1 plants.

Statistical differences among the samples are labeled with different

letters (P < 0.01).

(E) Growth of H. a. Noco2 on the wild type, npr1-1, and snc2-1D npr1-1.

Three-week-old seedlings were sprayed with H. a. Noco2 spores (50,000

spores/mL). Infection was scored 7 d after inoculation by counting the

number of spores per gram of leaf samples. Error bars represent SD from

three measurements. Statistical differences among the samples are

labeled with different letters (P < 0.01).

(F) Growth of P. s. t. DC3000 on the wild type, npr1-1, and snc2-1D

npr1-1. Leaves of 5-week old plants were infiltrated with a bacterial

suspension at OD600 = 0.0002. The values presented are averages of four

replicates 6 SD. Statistical differences among the samples are labeled

with different letters (P < 0.01). c.f.u., colony-forming units.

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

2 of 11 The Plant Cell

To determine whether pathogen resistance was constitutively

activated in snc2-1D npr1-1, the mutant plants were challenged

with the virulent oomycete pathogen H. a. Noco2 and the

bacterial pathogen Pseudomonas syringae pv tomato (P. s. t.)

DC3000. As shown in Figure 1E, H. a. Noco2 growth on the

snc2-1D npr1-1 plants is much lower than that on the wild-type

plants. Growth of P. s. t. DC3000 on the snc2-1D npr1-1 plants

was also lower than on npr1-1 plants (Figure 1F), indicating that

snc2-1D confers enhanced pathogen resistance.

Epistasis Analysis

To determine whether snc2-1D activates resistance pathways

shared by NB-LRR–type R proteins, snc2-1D npr1-1 was

crossed with known defense signaling mutants, and triple

mutants were obtained in the F2. These known mutants carry

mutations in ENHANCED DISEASE SUSCEPTIBILITY1 (EDS1),

PHYTOALEXIN DEFICIENT4 (PAD4), and NON-RACE-SPE-

CIFIC DISEASE RESISTANCE1 (NDR1). EDS1 and PAD4 are

required generally for resistance mediated by NB-LRR–type R

proteins with an N-terminal Toll Interleukin I Receptor (TIR)

domain (Glazebrook et al., 1996; Parker et al., 1996; Aarts et al.,

1998), whereas NDR1 is required for NB-LRR–type R proteins

with an N-terminal coiled coil domain (Century et al., 1995). As

shown in Supplemental Figure 2A online, mutations in EDS1,

PAD4, andNDR1 had little effect on themorphology of snc2-1D

npr1-1. In addition, the mutations in EDS1, PAD4, and NDR1

had no obvious effects on the expression of PR-2 (see Sup-

plemental Figure 2B online) or resistance to H. a. Noco2 (see

Supplemental Figure 2C online), suggesting that snc2-1D ac-

tivates resistance pathways distinct from those downstream of

NB-LRR–type R proteins.

Since the snc2-1D mutation lead to the accumulation of high

levels of SA, the requirement of SA for the phenotypes of snc2-1D

npr1-1 was tested by making the snc2-1D eds5-3 npr1-1 triple

mutant. EDS5 encodes a member of the MATE transporter family.

Mutations in EDS5 abolish pathogen-induced SA accumulation in

Arabidopsis (Nawrath et al., 2002). In snc2-1D eds5-3 npr1-1, SA

accumulation was blocked (see Supplemental Figure 2D online).

The eds5-3 mutation had little effect on the morphology (see

Supplemental Figure 2A online) and resistance to H. a. Noco2 in

snc2-1D npr1-1 (see Supplemental Figure 2C online), but the

expression of PR-2 was modestly reduced in snc2-1D eds5-3

npr1-1 (seeSupplemental Figure 2Bonline). This suggests that the

contributionof theheightenedSA levels to the constitutivedefense

phenotypes in snc2-1D is relatively small.

Map-Based Cloning of snc2-1D

To map the snc2-1D mutation, snc2-1D (in Columbia [Col]

background) was crossed with Landsberg erecta (Ler), and F2

Figure 2. Map-Based Cloning of snc2-1D.

(A) Map position and the mutation in snc2-1D.

(B) Predicted protein structure of SNC2. TM, transmembrane motif.

(C) Morphological phenotypes of 4-week-old wild-type and transgenic

plants expressing the snc2-1D mutant gene.

(D) and (E) PR-1 (D) and PR-2 (E) expression in wild-type and transgenic

plants expressing the snc2-1D mutant gene under its own promoter

determined by quantitative RT-PCR. Values were normalized to the

expression of ACTIN1. Statistical differences among the samples are

labeled with different letters (P < 0.001).

(F) Growth of H. a. Noco2 on the wild type and transgenic plants

expressing the snc2-1Dmutant gene. The experiment was performed as

in Figure 1E. Statistical differences among the samples are labeled with

different letters (P < 0.01).


RLP-Mediated Immunity in Arabidopsis 3 of 11

plants with snc2-1D morphology were selected for linkage

analysis. The snc2-1D mutation was initially flanked between

FCA8 and F9F13 on chromosome 4. Further analysis of 1100 F2

plants flanked the mutation between F28A21 and F13C5, a

region of ;47 kb (Figure 2A). The coding sequences in this

region were amplified from snc2-1D genomic DNA by PCR and

sequenced. Comparison of the sequences from snc2-1D with

wild-type Arabidopsis genomic sequence revealed a single

G-to-A mutation in At4g18760.

At4g18760 encodes a receptor-like protein, also named

Receptor-like protein 51 (RLP 51). It contains a predicted

N-terminal signal peptide, four extracellular LRRs, a single

transmembrane domain, and a short cytoplasmic tail of four

amino acids (Figure 2B). Compared with the Cf-9 class of R

proteins previously characterized, the number of LRRs in

RLP51 is rather small, and the domain between the signal

peptide and the LRRs is atypically long. The transmembrane

domain contains a GXXXG motif that is highly conserved in

RLPs and many RLKs (Fritz-Laylin et al., 2005). The mutation in

snc2-1D resulted in the substitution of the second Gly (Gly-412)

in the GXXXG motif for Arg (Arg-412). To confirm that the

mutation identified in snc2-1D is responsible for the constitutive

defense phenotypes of the mutant, genomic clones containing

the wild type or the mutant At4g18760 gene were constructed

and transformed into wild-type plants. While transgenic plants

with the wild-type transgene all displayed wild-type morphol-

ogy, about half of the transgenic plants with the mutant trans-

gene exhibited snc2-1D–like morphology (Figure 2C). Two

representative lines with snc2-1D morphology were analyzed

further. As shown in Figures 2D and 2E, PR-1 and PR-2 were

constitutively expressed in both lines. When challenged with H.

a. Noco2, the transgenic plants displayed enhanced resistance

to H. a. Noco2 (Figure 2F). These results indicate that

At4g18760 is SNC2, and the point mutation in snc1-2D is

responsible for the autoimmunity phenotypes of the mutant.

SNC2 Is Localized to the PlasmaMembrane

To determine the subcellular localization of SNC2, transgenic

plants expressing SNC2 with N- or C-terminal green fluores-

cent protein (GFP) tags under its native promoter were gener-

ated. GFP fluorescence was not detected in these transgenic

plants, suggesting that the SNC2 protein is expressed at very

low levels. As shown in Supplemental Figure 3 online, wild-type

plants expressing the snc2-1D-GFP fusion protein displayed

snc2-1D–like morphology, suggesting that the fusion protein is

functional. Subsequently, we transformed Arabidopsis meso-

phyll protoplasts with a construct expressing the SNC2-GFP

fusion protein under the cauliflower mosaic virus 35S promoter.

As shown in Supplemental Figure 4A online, the SNC2-GFP

fusion protein was localized to the plasma membrane, sug-

gesting that SNC2 is a plasma membrane protein as predicted.

To examine whether themutation in snc2-1D affects the protein

localization, snc2-1D-GFP fusion protein was also expressed in

mesophyll protoplasts under 35S promoter. As shown in Sup-

plemental Figure 4A online, snc2-1D-GFP was localized to the

plasma membrane, suggesting that the mutation in snc2-1D

does not affect the localization of the protein. Membrane

Figure 3. Characterization of Transgenic Plants Expressing the

snc3G404R Mutant Gene in the Wild-Type Background under Its Own

Promoter.

(A) Morphological phenotypes of 4-week-old wild-type and transgenic

plants expressing the snc3G404R mutant gene.

(B) and (C) PR-1 (B) and PR-2 (C) expression in wild-type and transgenic

plants expressing the snc3G404R mutant gene determined by quantita-

tive RT-PCR. Values were normalized to the expression of ACTIN1. *P <

0.001, significant difference from the wild type.

(D) Growth of H. a. Noco2 on the wild-type and transgenic plants

expressing the snc3G404R mutant gene. Error bars represent SD from

three measurements. *P < 0.01, significant difference from the wild type.



localization of SNC2 was further confirmed by fractionation

analysis using transgenic plants expressing the SNC2-HA

protein (see Supplemental Figure 4B online).

Mutations in the GXXXGMotif of SNC3 Constitutively

Activate Defense Responses

Unlike other R genes, SNC2 and its close homolog At5g45770 in

Arabidopsis, which we named SNC3, are quite conserved

among different plant species (Fritz-Laylin et al., 2005). SNC3

is 63% identical to SNC2 at amino acid level. Gly-404 in SNC3 is

the second Gly in the GXXXG motif that corresponds to Gly-412

in SNC2. To test whether substituting the Gly-404 in SNC3 for

Arg results in activation of defense responses, constructs

expressing the wild-type SNC3-HA and snc3G404R-HA, both

under its native promoter, were made. Upon transformation into

wild-type plants, all transgenic plants carrying the SNC3 trans-

gene displayed wild-type morphology. By contrast, about half of

the transgenic plants with the snc3G404R mutant transgene

exhibited snc2-1D–like morphology (Figure 3A). Analysis of two

representative snc3G404R transgenic lines showed that expres-

sion of PR-1 and PR-2 is constitutively activated (Figures 3B and

3C). Higher PR-1 expression in the transgenic lines than that

in snc2-1D npr1-1 is probably due to the presence of npr1-1

mutation in snc2-1D npr1-1. In addition, these transgenic lines

displayed enhanced resistance to H. a. Noco2 (Figure 3D),

suggesting that substituting Gly-404 for Arg in SNC3 constitu-

tively activates defense responses.

We also tested whether similar mutations in TMM and CLV2

lead to snc2-1D–like phenotypes. We made transgenic plants

expressing mutant TMM or CLV2 with the second G or S in the

conserved (G/S/T)XXX(G/S/T) motif changed to R and found that

all transgenic lines displayed wild-type morphology (see Sup-

plemental Figure 5A online). Real-time RT-PCR analysis showed

that TMM and CLV2 are overexpressed in the transgenic lines

(see Supplemental Figures 5B and 5C online). These data sug-

gest that the G/S to R mutations do not lead to gain of defense–

related functions in these proteins that function in the regulation

of plant development.

Identification of Intragenic Suppressors of snc2-1D

To identify intragenic suppressor mutations of snc2-1D and

components functioning downstream of snc2-1D, snc2-1D

npr1-1 seeds were mutagenized with EMS, and the M2 popula-

tion was screened for mutants with wild-type morphology. Six

intragenic suppressors with mutations in SNC2 were obtained

from the screen (Figure 4A). Five of the mutations are missense

mutations of amino acids conserved in SNC2, SNC3, and their

homologs in other plants. Among the SNC2 mutations, three

were located in the region between the signal peptide and the

Figure 4. Analysis of Intragenic Suppressors of snc2-1D.

(A) Morphological phenotypes of the intragenic suppressors of snc2-1D

npr1-1. Plants were grown on soil and photographed 4 weeks after

planting. All mutants are in the npr1-1 background.

(B) Map of six intragenic SNC2 mutations.

(C) PR-2 expression in the intragenic suppressors of snc2-1D deter-

mined by quantitative RT-PCR. Values were normalized to the expres-

sion of ACTIN1. *P < 0.001, significant difference from all other

genotypes.

(D) Growth of H. a. Noco2 on the intragenic suppressors of snc2-1D.

Statistical differences among the samples are labeled with different

letters (P < 0.05).



LRRs, one in the LRRs, and another in the region between the

LRRs and the transmembrane helix (Figure 4B). The only non-

sense mutation identified was located in the cytoplasmic exten-

sion and results in the loss of the last three amino acids of the

protein, suggesting that the C-terminal cytoplasmic extension is

important for the functions of SNC2. Real-time RT-PCR indi-

cated that PR-2 expression was also suppressed in the snc2-1D

revertants (Figure 4C). Further analysis showed that resistance

to H. a. Noco2 was suppressed to different extent in the rever-

tants except snc2-2 (Figure 4D).

SNC2 Is Required for Basal Resistance against

P. s. t. DC3000

To test whether knocking out SNC2 affects resistance to path-

ogens, we obtained two T-DNA insertion lines, SALK_143038

andSAIL_740_C06, from theABRC.Wedidnot find thepredicted

T-DNA insertion in the SALK_143038 line. The T-DNA insertion in

SAIL_740_C06 is located upstream of the 59-untranslated region

(UTR) of SNC2, and we found that the expression of SNC2 was

not reduced in the homozygous line for SAIL_740_C06. To

identify a knockout mutant for SNC2, we screened for additional

intragenic suppressors of snc2-1D. One mutant, snc2-8, was

found to revert snc2-1D to wild-type morphology and contain a

mutation in SNC2 that changes Gln-52 to a stop codon. Since

snc2-8 results in early termination of the protein, it is most likely a

null allele of snc2. When spray inoculated with P. s. t. DC3000,

snc2-8 npr1-1 supported ;10-fold more bacterial growth than

npr1-1 plants (Figure 5). Analysis of another intragenic suppres-

sor of snc2-1D, snc2-5, showed that bacterial growth in snc2-5

npr1-1 is also higher than in npr1-1. These data suggest that

SNC2 is required for basal resistance against P. s. t. DC3000. As

bacterial growth in snc2-5 npr1-1 is lower than that in snc2-8

npr1-1, the point mutation in snc2-5 probably results in only

partial loss of the function of SNC2.

WRKY70 Functions Downstream of SNC2

To map second-site suppressor mutants of snc2-1D npr1-1 that

do not contain mutations in SNC2, we crossed each mutant with

Ler to generate amapping population. F2 plants homozygous for

the snc2-1D mutation with wild-type morphology were selected

for linkage analysis. Crude mapping showed that two allelic

recessive mutants, bda2-1 (bian da 2-1; bian da means becom-

ing big in Chinese) and bda2-2 (Figure 6A), were located on the

lower arm of chromosome 3. Fine mapping of bda2-1 using

roughly 2400 F2 plants positioned the mutation in a 220-kb

region between marker F27K19 and T5P19. Sequence analysis

of genes within this region identified a G-to-A mutation in

WRKY70 (Figure 6B). This mutation changes Gly-101 in the

protein to Glu-101. Sequence analysis of the WRKY70 locus in

the bda2-2 identified a C-to-Tmutation that creates an early stop

codon in the cDNA, which probably results in complete loss of

the function of the protein. These data suggest that mutations in

WRKY70 were responsible for the suppression of snc2-1D

mutant morphology. Since suppression of snc2-1D morphology

by bda2-1 is not as complete as that by bda2-2, the point

mutation in bda2-1 probably results in only partial loss of the

function of WRKY70. As shown in Supplemental Figure 6 online,

bda2-1 and bda2-2 did not affect the expression of SNC2.

Further analysis of the bda2-1 snc2-1D npr1-1 and bda2-2

snc2-1D npr1-1 mutant plants showed that constitutive

Figure 5. Growth of P. s. t. DC3000 on npr1-1, snc2-5 npr1-1, and

snc2-8 npr1-1.

Leaves were collected 2 d after spray inoculation with a bacterial

suspension at OD600 = 0.2. The values presented are averages of five

replicates 6 SD. Statistical differences among the samples are labeled

with different letters (P < 0.01). cfu, colony-forming units.

Figure 6. bda2-1 and bda2-2 Contain Mutations in WRKY70.

(A) Morphological phenotypes of 4-week-old bda2-1 snc2-1D npr1-1

and bda2-2 snc2-1D npr1-1.

(B) Map-based cloning of BDA2 (WRKY70).



expression of PR-1 and PR-2 was reduced in bda2-1 snc2-1D

npr1-1 and completely suppressed in bda2-2 snc2-1D npr1-1

(Figures 7A and 7B). In addition, enhanced resistance to H. a.

Noco2was attenuated inbda2-1 snc2-1D npr1-1 and completely

suppressed in bda2-2 snc2-1D npr1-1 (Figure 7C), suggesting

that snc2-1D activatesWRKY70-mediated resistance pathways.

To test whether SA accumulation in snc2-1D npr1-1 is affected

by thewrky70mutations, SA levels in the bda2-1 snc2-1D npr1-1

and bda2-2 snc2-1D npr1-1 mutant plants were measured. As

shown in Figure 7D, free SA levels in bda2-1 snc2-1D npr1-1 and

bda2-2 snc2-1D npr1-1 are comparable to those in snc2-1D

npr1-1, suggesting thatWRKY70 functions in an SA-independent

pathway downstream of snc2-1D. Interestingly, accumulation of

salicylic acid glucoside (SAG) was modestly reduced in bda2-1

snc2-1D npr1-1 and bda2-2 snc2-1D npr1-1 compared to that in

snc2-1D npr1-1, suggesting that WRKY70 may have a role in the

regulation of conversion of SA to SAG.

DISCUSSION

Cf-like R genes have been scarcely studied in Arabidopsis. Here,

we report the identification and characterization of snc2-1D, a

semidominant mutant that exhibits constitutive activation of PR

genes and pathogen resistance. The mutant phenotypes of

snc2-1D are very similar to those previously reported in the

snc1mutant (Li et al., 2001). Unlike snc1, which is a TIR-NB-LRR

R gene mutant (Zhang et al., 2003a), snc2-1D carries a mutation

in a receptor-like protein. Since similar mutations in CLV2 and

TMM do not result in the same phenotypes as snc2-1D, snc2-1D

Figure 7. WRKY70 Is Required for snc2-1D–Mediated Defense Responses.

(A) and (B) PR-1 (A) and PR-2 (B) expression in the wild type, snc2-1D npr1-1, bda2-1 snc2-1D npr1-1, and bda2-2 snc2-1D npr1-1 determined by

quantitative RT-PCR. Statistical differences among the samples are labeled with different letters (P < 0.01).

(C) Growth of H. a. Noco2 on the wild type, snc2-1D npr1-1, bda2-1 snc2-1D npr1-1, and bda2-2 snc2-1D npr1-1. Error bars represent SD from three

measurements. Statistical differences among the samples are labeled with different letters (P < 0.05).

(D) Free SA and SAG in the wild type, snc2-1D npr1-1, bda2-1 snc2-1D npr1-1, and bda2-2 snc2-1D npr1-1. Statistical differences among the samples

are labeled with different letters (P < 0.01).


is probably not a neomorphic mutation. The identification of

snc2-1D provides us with a unique system to perform genetic

analysis of RLP-mediated immunity in Arabidopsis. It will be

interesting to test whether SNC2 and Cf-9-like R proteins iden-

tified in other plant species use similar downstream components

to activate defense responses.

Unlike the previously characterized RLP-type R proteins,

which are generally not well conserved, phylogenetic analysis

indicates that SNC2 is highly conserved in plants (Fritz-Laylin

et al., 2005). What activates SNC2 remains to be determined.

Since loss-of-function mutations in SNC2 cause enhanced sus-

ceptibility to P. s. t. DC3000, it is likely that SNC2 functions as a

receptor for a conserved molecule from microbial pathogens.

Detection of this molecule by SNC2 could activate downstream

defense responses. Alternatively, SNC2 could be involved in

guarding a conserved component of plant innate immunity.

Alteration of this component by microbial pathogens would

trigger activation of SNC2.

The conserved GXXXG motif is found in the transmembrane

domains of many proteins where it facilitates protein–protein

interactions in the plasma membrane (Senes et al., 2004). The

mutation in snc2-1D changes the second Gly in the GXXXGmotif

to Arg. Site-directedmutagenesis ofSNC3 showed that changing

the corresponding Gly to Arg in SNC3 also results in activation of

defense responses, suggesting that this residue may play impor-

tant roles in preventing autoactivation of theseRLPs. Activation of

the SNC2 and SNC3 through the Gly to Arg mutations might be

the result of disrupting their interaction with an unidentified

negative regulator that associates with SNC2 or SNC3 through

the GXXXGmotif. Interestingly, a similar mutation in Cf-9 (G825R)

abolished the function of Cf-9 (Wulff et al., 2004). Whether this

mutation causes alteration of the structure or stability of Cf-9 is

unknown. It is possible that the GXXXG motif in different RLPs

may function through interacting with different proteins.

Epistasis analysis showed that snc2-1D–mediated defense

responses do not require NDR1, EDS1, or PAD4, suggesting that

it activates resistance pathways distinct from the NB-LRR–type

R proteins. Like snc1, snc2-1D also accumulates high levels of

SA. Blocking the SA synthesis through the loss of EDS5 function

partially blocks the expression of the defense marker gene PR-2

but has limited effects on the snc2-1D mutant morphology,

suggesting that snc2-1D activates both SA-dependent and SA-

independent resistance pathways.

In a suppressor screen of snc2-1D, we found that the snc2-1D

mutant phenotypes were largely suppressed by mutations in

WRKY70, indicating that this WRKY transcription factor plays a

crucial role in RLP-mediated immunity. In Arabidopsis, there are

more than 70WRKY transcription factors, andmany are induced

during plant defense (Eulgem and Somssich, 2007). It has been

suggested that WRKY transcription factors play important roles

in the regulation of plant immune responses (Maleck et al., 2000).

Using reverse genetics, a number of WRKY transcription factors

have been identified as critical regulators of plant defense

responses (Journot-Catalino et al., 2006; Wang et al., 2006; Xu

et al., 2006; Zheng et al., 2006; Shen et al., 2007; Kim et al., 2008).

However, WRKY transcription factors have rarely been identified

in forward genetic screens to search for regulators of plant

immunity. One possibility to explain this rarity is that individual

WRKY transcription factors may have specialized functions in

regulating expression of a subset of defense genes and the

mutant phenotypes of theseWRKY transcription factors can only

be identified in sensitized mutant backgrounds.

Previous studies have shown that WRKY70 plays complex

roles in modulating defense responses and senescence (Li et al.,

2004; Knoth et al., 2007; Ulker et al., 2007). Based on epistasis

analysis, WRKY70 was suggested to function downstream of

NPR1 in an SA-dependent signal pathway where it activates

SA-inducible PR gene expression and represses jasmonate-

inducible PDF1.2 expression. WRKY70 was proposed to be a

determinant of the balance between SA and jasmonate signal-

ing (Li et al., 2004). In addition, knocking out WRKY70 causes

modest reduction of resistance to H. a. Emoy2 mediated by

the TIR-NB-LRR–type R protein, RPP4 (Knoth et al., 2007). Our

data suggest that WRKY70 functions independent of SA

and NPR1 in the regulation of snc2-1D–mediated defense

responses.

A working model is proposed in Figure 8. Accumulation of

high levels of SA in snc2-1D suggests that the SA-dependent

resistance pathway is activated in the mutant. Since blocking

SA synthesis in snc2-1D does not completely block PR gene

expression and pathogen resistance, snc2-1D also activates

an SA-independent defense pathway.WRKY70 probably func-

tions as a critical regulator of the SA-independent pathway

because it blocks the defense responses but not SA synthesis

in snc2-1D npr1-1 plants. It remains to be determined how the

signal is transduced from the plasma membrane–localized

SNC2 to nucleus to activate WRKY70. Previous studies have

shown that recognition of Avr4 by Cf-4 or Avr9 by Cf-9 leads

to rapid activation of downstream mitogen-activated pro-

tein kinases in tomato and tobacco (Romeis et al., 1999;

Stulemeijer et al., 2007). Studies of RLP TMM in stomata devel-

opment also suggest that a mitogen-activated protein kinase

cascade links the surface receptors to its downstream target

SPEECHLESS, a transcription factor required for stomata for-

mation. Future research will reveal whether there is also a

mitogen-activated protein kinase cascade functioning down-

stream of snc2-1D to regulate WRKY70-mediated activation of

defense responses.

Figure 8. A Working Model for snc2-1D–Induced Resistance.

The snc2-1D mutation constitutively activates both SA-dependent and

SA-independent resistance pathways downstream. SA-dependent re-

sistance pathway requires NPR1. The SA-independent resistance re-

sponses are mediated by WRKY70.


METHODS

Mutant Screening and Characterization

The snc2-1D npr1-1 mutant was identified from an EMS-mutagenized

npr1-1 mutant population by GUS staining (Gao et al., 2008). To identify

the suppressor mutants of snc2-1D, the snc2-1D npr1-1 mutant seeds

were treated with EMS. About 40,000 M2 plants representing;2000 M1

families were grown on soil and screened for loss of the snc2-1D

morphological phenotype. All plants were grown under 16 h light at

238C and 8 h dark at 208C.

SA was extracted from 0.1 g of leaf tissue using a previously described

protocol andmeasured by HPLC (Li et al., 1999). To analyze resistance to

H. a.Noco2, 2-week-old seedlings were sprayed withH. a.Noco2 spores

at a concentration of 50,000 spores per mL of water. Plants were grown

for another 7 d at 188C in 12-h-light/12-h-dark cycles with 95% humidity

before infections were scored by counting the number of spores with a

hemocytometer. P. s. t. DC3000 infections were performed by infiltrating

or spraying leaves of 5-week-old plants grown at 228C under 12-h-light/

12-h-dark cycles with bacterial suspensions at different concentrations.

Quantitative Real-Time RT-PCR

For gene expression analysis, RNA was extracted from the 2-week-old

seedlings grown on half Murashige and Skoog medium using the RNAiso

reagent (Takara). Reverse transcription was performed using the M-MLV

reverse transcriptase (Takara). Quantitative real-time RT-PCR was per-

formed using the SYBR Premix Ex Taq II kit (Takara). PCR reactions were

performed in triplicate with three independent RNA samples. Statistical

analyses were performed with one-way analysis of variance by Stats-

Direct statistical software (StatsDirect). Primers used for amplification of

PR-1, PR-2, and Actin1 were described previously (Zhang et al., 2003b).

Epistasis Analysis

Thepad4-1,ndr1-1, andeds5-3mutantsused forepistasisweredescribed

previously (Century et al., 1995; Glazebrook et al., 1996; Nawrath and

Metraux, 1999). The eds1-2mutant (Parker et al., 1996) backcrossed into

Col background was kindly provided by Jane Parker. To generate vari-

ous mutant combinations, the snc2-1D npr1-1 plants were crossed with

pad4-1, eds1-2, ndr1-1, and eds5-3. Triplemutant plantswere identified in

the F2 progeny by PCR (Li et al., 2001). To check for npr1-1 homozygosity,

mutant lines were plated on Murashige and Skoog plates with 0.2 mM SA

because npr1-1mutants bleach to death on high concentrations of SA.

Map-Based Cloning

The markers used for mapping were designed based on the Monsanto

Arabidopsis thaliana polymorphism and Ler sequence collections (Jander

et al., 2002). For mapping the snc2-1D and bda2-1 mutations, the

phenotypes of the recombinants were determined by assessing the

segregation of snc2-1D morphological phenotype in their progeny.

Primer sequences of markers used for mapping are shown in Supple-

mental Table 1 online.

To test whether themutation inAt4g18760 is responsible for themutant

phenotypes in the snc2-1D mutant, a 3.3-kb genomic fragment of

At4g18760 (SNC2) containing its own promoter but without the stop

codon and 39-UTR was amplified from snc2-1D or wild-type genomic

DNA using primers 59-CGGGGTACCGATCATCATGTTCATCGACC-39

and 59-CGCGGATCCACCACACCATTTGGCGAGAAG-39 and cloned

into modified pCAMBIA1305 vectors to obtain pCAMBIA1305-snc2-1D

(or SNC2)-HA andpCAMBIA1305-snc2-1D (or SNC2)-GFP to express the

wild type or mutant snc2-1D fusion proteins under its own promoter. The

resulting plasmids were used to transform wild-type plants by the floral

dip method (Clough and Bent, 1998).

Mutation Analysis of SNC3

Agenomic fragment ofAt5g45770 (SNC3) without the stopcodonand39-UTR

was amplified from wild-type genomic DNA using primers 59-CCGGAA-

TTCTAGAGGTGATGGAATTGTC-39 and 59-CGCGGATCCTCAAATCA-

AACGACACCTTTTAG-39and cloned into a modified pCAMBIA1305

vector to obtain pCAMBIA1305-SNC3-HA to express the wild-type

SNC3 fusion protein under its own promoter. The snc3G404R mutation

was introduced into pCAMBIA1305-SNC3-HA by site-directed mutagen-

esis using a PCR-based linker scanning method (Li and Shapiro, 1993).

The resulting plasmids were transformed into wild-type plants using the

floral dip method (Clough and Bent, 1998).

Accession Numbers

Sequence data from this article can be found in the Arabidopsis Genome

Initiative or GenBank/EMBL databases under the following accession

numbers: At4g18760 (SNC2), At5g45770 (SNC3), AT3G56400 (WRKY70),

At2g14610 (PR1), At3g57260 (PR2), and At2g37620 (Actin1).

Supplemental Data

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

Supplemental Figure 1. Morphological Phenotypes of Wild Type,

npr1-1, and snc2-1D npr1-1 Plants Grown on Soil at 23 or 288C.

Supplemental Figure 2. Analysis of snc2-1D pad4-1 npr1-1, snc2-1D

eds1-2 npr1-1, snc2-1D ndr1-1 npr1-1, and snc2-1D eds5-3 npr1-1

Mutant Plants.

Supplemental Figure 3. Morphological Phenotypes of Wild-Type,

npr1-1, snc2-1D npr1-1, and Transgenic Plants Expressing the snc2-

1D-GFP Fusion Protein under Its Own Promoter in the Wild-Type

Background.

Supplemental Figure 4. SNC2-GFP and snc2-1D-GFP Are Localized

to the Plasma Membrane.

Supplemental Figure 5. Analysis of Transgenic Plants Expressing

clv2S697R or tmmG475R under the 35S Promoter in the Wild-Type

Background.

Supplemental Figure 6. SNC2 Expression in npr1-1, snc2-1D

npr1-1, bda2-1 snc2-1D npr1-1, and bda2-2 snc2-1D npr1-1 Deter-

mined by Quantitative RT-PCR.

Supplemental Table 1. Markers Used for Map-Based Cloning.

ACKNOWLEDGMENTS

We thank Dongling Bi, Yan Huang, and Yi-Ti Cheng for their help with

confocal microscopy and SA measurement. We also thank Youjun

Zhang, Shaoning Chen, and Pei Wen for their help on the mutant

screening and mapping. Jane Parker is thanked for seeds of eds1-2 in

Col. We are grateful for financial support from the Chinese Ministry of

Science and Technology to Y.Z.

Received January 18, 2010; revised August 23, 2010; accepted August

30, 2010; published September 14, 2010.

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DOI 10.1105/tpc.110.074120; originally published online September 14, 2010;Plant Cell

Yaxi Zhang, Yuanai Yang, Bin Fang, Patrick Gannon, Pingtao Ding, Xin Li and Yuelin ZhangWRKY70

Activates Receptor-Like Protein-Mediated Immunity Transduced throughArabidopsis snc2-1D

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Supplemental Data /content/suppl/2010/09/08/tpc.110.074120.DC1.html

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