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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
edited and the authors have corrected proofs, but minor changes could be made before the final version is published. Posting this version online
reduces the time to publication by several weeks.
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).
[See online article for color version of this figure.]
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.
[See online article for color version of this figure.]
4 of 11 The Plant Cell
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).
[See online article for color version of this figure.]
RLP-Mediated Immunity in Arabidopsis 5 of 11
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).
[See online article for color version of this figure.]
6 of 11 The Plant Cell
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).
RLP-Mediated Immunity in Arabidopsis 7 of 11
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.
8 of 11 The Plant Cell
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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RLP-Mediated Immunity in Arabidopsis 11 of 11
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
This information is current as of February 1, 2019
Supplemental Data /content/suppl/2010/09/08/tpc.110.074120.DC1.html
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