PEPR2 Is a Second Receptor for the Pep1 and Pep2 ...PEPR2 Is a Second Receptor for the Pep1 and Pep2 Peptides and Contributes to Defense Responses in Arabidopsis W Yube Yamaguchi,a,1,2

PEPR2 Is a Second Receptor for the Pep1 and Pep2 Peptidesand Contributes to Defense Responses in Arabidopsis W

Yube Yamaguchi,a,1,2 Alisa Huffaker,a,3 Anthony C. Bryan,b Frans E. Tax,b and Clarence A. Ryana,4

a Institute of Biological Chemistry, Washington State University, Pullman, Washington 99164b Department of Molecular and Cellular Biology, University of Arizona, Tucson, Arizona 85721

Pep1 is a 23–amino acid peptide that enhances resistance to a root pathogen, Pythium irregulare. Pep1 and its homologs

(Pep2 to Pep7) are endogenous amplifiers of innate immunity of Arabidopsis thaliana that induce the transcription of

defense-related genes and bind to PEPR1, a plasma membrane leucine-rich repeat (LRR) receptor kinase. Here, we identify

a plasma membrane LRR receptor kinase, designated PEPR2, that has 76% amino acid similarity to PEPR1, and we

characterize its role in the perception of Pep peptides and defense responses. Both PEPR1 and PEPR2 were transcrip-

tionally induced by wounding, treatment with methyl jasmonate, Pep peptides, and pathogen-associated molecular

patterns. The effects of Pep1 application on defense-related gene induction and enhancement of resistance to Pseudo-

monas syringae pv tomato DC3000 were partially reduced in single mutants of PEPR1 and PEPR2 and abolished completely

in double mutants. Photoaffinity labeling and binding assays using transgenic tobacco (Nicotiana tabacum) cells expressing

PEPR1 and PEPR2 clearly demonstrated that PEPR1 is a receptor for Pep1-6 and that PEPR2 is a receptor for Pep1 and

Pep2. Our analysis demonstrates differential binding affinities of two receptors with a family of peptide ligands and the

corresponding physiological effects of the specific receptor–ligand interactions. Therefore, we demonstrate that, through

perception of Peps, PEPR1 and PEPR2 contribute to defense responses in Arabidopsis.

INTRODUCTION

Plants are sessile organisms surrounded by a wide range of

microorganisms, including plant pathogens. However, infection

is established between a limited combination of plants and

pathogens because plants can detect the presence of many

potential pathogens through common pathogen-associated

molecular patterns (PAMPs) or microbe-associated molecular

patterns and subsequently mount a defense response called

PAMP-triggered immunity (PTI) (He et al., 2007; Schwessinger

and Zipfel, 2008). PAMPs include lipopolysaccharides, flagellin,

and translation elongation factor Tu; all of these are released

from bacterial pathogens (Nurnberger et al., 2004; Boller and

Felix, 2009). Calcium-dependent cell wall transglutaminase and

lipid-transfer proteins (elicitins) were identified as PAMPs from

oomycetes (Nurnberger et al., 2004; Boller and Felix, 2009).

Fungal cell wall components, such as b-glucan, ergosterol, and

chitin, are also recognized as PAMPs (Nurnberger et al., 2004;

Boller and Felix, 2009). In addition to sensing invading pathogens

by PAMP (infectious non-self) recognition, plants can also sense

infectious-self ormodified-self patterns called damage-associated

molecular patterns (Matzinger, 2007; Boller and Felix, 2009), such

as oligogalacturonides that are released from plant cell wall pectin

by fungal polygalacturonase (Ridley et al., 2001).

PAMPs are perceived by pattern recognition receptors (PRRs)

in the plasma membrane, and the resulting signals are trans-

duced into the cytosol, inducing PTI (Schwessinger and Zipfel,

2008; Zipfel, 2008). Bacterial flagellin and translation elongation

factor Tu are perceived by leucine-rich repeat receptor-like

kinases (LRR-RLKs) FLS2 and EFR, respectively (Chinchilla

et al., 2006; Zipfel et al., 2006). An Arabidopsis thaliana lysine

motif (LysM)–containing receptor-like kinase, CERK1/LysM-

RLK1, and a rice (Oryza sativa) LysM-containing transmembrane

protein, CEBiP, are required for chitin responses (Kaku et al.,

2006; Miya et al., 2007; Wan et al., 2008). A heptaglucoside from

Phytophtora sojae and a xylanase from bacteria bind to extra-

cellular glucan binding protein and tomato (Solanum lycopersi-

cum) receptor-like proteins (Le EiX1 and Le EiX2), respectively

(Schwessinger and Zipfel, 2008). Although the PAMPs are per-

ceived by specific PRRs, comparative analysis of microarray

data from Arabidopsis treated with different PAMPs indicated

that PAMP signals converge and share a downstream defense

response, including induction of WRKY transcription factors and

mitogen-activated protein kinases (MAPKs; Wan et al., 2008).

Recently, a 23–amino acid peptide, Pep1, was isolated from

Arabidopsis and shown to activate defense genes associated

with the innate immune response (Huffaker et al., 2006). Pep1

and its homologs, Pep2-7, are derived from the C-terminal

portion of their precursor proteins PROPEP1-7, respectively

(Huffaker et al., 2006). Although PROPEP7 was recently anno-

tated as At5g09978, we were unable to detect a transcript of this

1Current address: Laboratory of Crop Physiology, Graduate School ofAgriculture, Hokkaido University, Sapporo, 060-8589, Japan.2 Address correspondence to [email protected] Current address: Center of Medical, Agricultural, and VeterinaryEntomology, USDA, Gainesville, FL 32608.4 Deceased.The author responsible for distribution of materials integral to thefindings presented in this article in accordance with the policy describedin the Instructions for Authors (www.plantcell.org) is: Yube Yamaguchi([email protected]).WOnline version contains Web-only data.www.plantcell.org/cgi/doi/10.1105/tpc.109.068874

The Plant Cell, Vol. 22: 508–522, February 2010, www.plantcell.org ã 2010 American Society of Plant Biologists

gene in seedlings or leaf tissue from Arabidopsis plants. Tran-

scripts of the PROPEP1-3 genes were differentially induced by

the defense-related hormones methyl salicylate (MeSA) and

methyl jasmonate (MeJA) by pathogen infection, application of

PAMPs, and by treatment with synthetic Pep peptides (Huffaker

et al., 2006; Huffaker and Ryan, 2007). Transcription of a

pathogenesis-related protein-1 (PR-1) gene was dramatically

induced by Pep1-3 and Pep5-6, and transcription of an antimi-

crobial peptide gene, defensin (PDF1.2), was induced by Pep1

and Pep2 (Huffaker and Ryan, 2007). Furthermore, transgenic

Arabidopsis overexpressing the PROPEP1 and PROPEP2 genes

exhibited higher PDF1.2 and PR-1 expression and increased

resistance to the oomycete pathogen Pythium irregulare

(Huffaker et al., 2006; Huffaker and Ryan, 2007). Because Pep

peptides induced the transcription of their own precursor genes

in addition to defense genes, it is likely that Pep peptides, which

are initially induced by PAMPs, feed back into the signaling

pathways to generate additional processed peptides to further

upregulate downstream defense responses (Ryan et al., 2007).

The Pep1 receptor, PEPR1, was isolated from Arabidopsis

suspension cultured cells using a photoaffinity labeling tech-

nique (Yamaguchi et al., 2006). PEPR1 is a typical LRR receptor

kinase, having an extracellular LRR domain and an intracellular

protein kinase domain, and belongs to the LRR XI subfamily of

the 15 LRR-RLK subfamilies (Shiu et al., 2004). Based on the

effects of Pep peptides on defense responses, and on the

similarity of the receptors between PEPR1 and PRRs, some

researchers classify Pep peptides as damage-associated mo-

lecular patterns (Boller and Felix, 2009). However, the specific

mechanisms through which PEP peptides and PEPR1 influence

defense response are largely unknown.

Since T-DNA insertional mutants of PEPR1 did not show any

obvious difference from wild-type plants upon pathogen infec-

tion, it was speculated that there is another receptor for Pep

peptides. In this study, we selected At1g17750 (PEPR2) as a

candidate for a second receptor for Pep peptides based on its

phylogenetic relationship with PEPR1. We show that both

PEPR1 and PEPR2 are transcriptionally induced by wounding

of plants and by treatment withMeJA, Pep peptides, and specific

PAMPs. Functional analysis of PEPR1 and PEPR2 using SALK

T-DNA insertional mutants demonstrate that the double mutants

do not activate transcription of defense-related genes when

plants were treated with Pep1. Pretreatment of double mutant

plants with Pep1was not able to inhibit bacterial growth asmuch

as it did in wild-type controls. Binding assays with Pep peptides

and PEPR1 andPEPR2 demonstrated that PEPR1 can recognize

Pep1-6 and that PEPR2 only recognizes Pep1 and Pep2. These

and other results provide evidence that PEPR1 and PEPR2 have

differential responses to the Pep peptides and play a role in

defense response signaling.

RESULTS

Phylogenetic Analysis of LRR XI Subfamily of LRR

Receptor Kinases

Phylogenetic analysis among the LRR RLK XI subfamily of

Arabidopsis (Shiu et al., 2004) was conducted (Figure 1A) to

identify candidates that share recent common ancestry with

PEPR1. The result showed that At1g17750 was the most closely

related gene to PEPR1. Other known receptors in this subfamily

are involved in development and differentiation (Figure 1A).

CLAVATA1 (CLV1), BAM1 (for barely any meristem 1), BAM2,

and BAM3 are required for meristem function (Clark, et al., 1997;

DeYoung, et al., 2006). HAESA (Jinn et al., 2000) and HSL2 (for

HAESA like 2) regulate floral organ abscission (Cho et al., 2008;

Stenvik et al., 2008). PXY/TDR is a receptor of TDIF (for tracheary

element differentiation inhibitory factor) (Fisher and Turner, 2007;

Hirakawa et al., 2008). IKU2 regulates seed size (Luo et al., 2005).

GSO1 and GSO2 are required for formation of a normal epider-

mal surface during embryogenesis (Tsuwamoto et al., 2008).

The At1g17750 gene encodes a predicted protein with 1088

amino acid residues (119 kD) and all the characteristic domains

of an LRR-RLK (Figure 1B). The N terminus contains a hydro-

phobic secretion signal followed by an extracellular domain with

25 tandem copies of a 24-residue LRR (residues 101 to 699). The

LRR domain is flanked by two pairs of Cys residues. A single

transmembrane domain (residues 741 to 761) is predicted to

separate the extracellular domain from an intracellular Ser-Thr

kinase domain (residues 794 to 1080) in which all important

subdomains, including guanilyl cyclase catalytic domain (Kwezi

et al., 2007) and residues for catalysis, are conserved (see

Supplemental Figure 1 online). At1g17750 and PEPR1 are 64%

identical and 76% similar at the amino acid level over their entire

lengths with one LRR domain fewer in At1g17750 (Figure 1B). In

this study, we designated At1g17750 as PEPR2 and further

analyzed PEPR2 in pathogen response pathways and Pep

perception and response.

Induction of PEPR1 and PEPR2 Gene Expression by

Wounding and MeJA

Since the Pep1 precursor gene PROPEP1 was induced by

wounding (Huffaker et al., 2006), quantitative RT-PCR (qRT-

PCR) was conducted using total RNA extracted from wounded

and unwounded upper leaves of 4-week-old soil-grown Arabi-

dopsis plants to analyze PEPR1 and PEPR2 gene expression

(Figures 2A and 2B). Both PEPR1 and PEPR2 mRNA accumu-

lated within 15 min in wounded leaves. The maximum mRNA

accumulation occurred 0.5 to 1 h after wounding and then

decreased to basal levels at 4 h. In the unwounded upper leaves

from the wounded plant, the induction of PEPR1 and PEPR2was

not observed (Figures 2A and 2B).

PROPEP genes were shown to be differentially induced by

several plant hormones involved in defense responses, including

MeJA and MeSA, and ethephon, an ethylene releaser (Huffaker

et al., 2006; Huffaker and Ryan, 2007). To determine whether

any of these compounds also induce the PEPR genes, we

sprayed 4-week-old Arabidopsis plants with MeJA, MeSA, or

1-aminocyclopropan-1-carboxylic acid, an ethylene precursor,

and total RNA was subjected to qRT-PCR to detect PEPR1 and

PEPR2 expression. MeJA induced transcription of both PEPR1

andPEPR2within 30min (Figures 2C and 2D), whereasMeSAand

1-aminocyclopropan-1-carboxylic acid did not induce either

PEPR1 or PEPR2 (see Supplemental Figure 2 online). These

results were consistent with the data from the AtGenExpress

PEPR2, a Receptor for Pep Peptides 509

Figure 1. Phylogenetic Analysis of the LRR XI Subfamily of Arabidopsis LRR Receptor Protein Kinases.

(A) The phylogenetic relationships of the LRR XI subfamily of Arabidopsis LRR receptor protein kinases. The phylogenetic relationships (unrooted) were

510 The Plant Cell

microarray database (http://www.uni-tuebingen.de/plantphys/

AFGN/atgenex.htm).

Pep Peptide- and PAMP-Induced Transcription of PEPR1

and PEPR2

Treatment with Pep1 has been shown to induce transcription of its

precursor gene, PROPEP1, when Pep1 is supplied through the

petiole (Huffaker et al., 2006). In order to test the effects of Pep1

on PEPR1 and PEPR2 expression in the absence of wounding,

2-week-old Arabidopsis seedlings grown in liquid medium were

incubated with Pep1 (10 nM) and then subjected to qRT-PCR

analysis in comparison with PROPEP1 gene expression (Figures

3A to 3C). The levels of induction ofPROPEP1,PEPR1, andPEPR2

gene expression by Pep1 were very similar, with rapid induction

within 30 min and maximal accumulation (10-fold) observed 0.5 to

1 h after supplying Pep1 (Figures 3A to 3C). The amount of PEPR1

and PEPR2 mRNA gradually decreased by 4 h (Figures 3B and

3C). PROPEP1 mRNA remained at an elevated level compared

with PEPR1 and PEPR2mRNA and did not decrease to a twofold

level compared with controls until 12 h (Figure 3A).

The effects of all of the Pep peptides (Pep1-6) on PEPR1 and

PEPR2 transcript levels were also analyzed by qRT-PCR (Figures

3D to 3F). The peptides were supplied at a final concentration of

10 nM for 1 h to 2-week-oldArabidopsis seedlings grown in liquid

medium. Among the Pep peptides, Pep1, Pep2, and Pep3

were strong inducers of PROPEP1 (6- to 12-fold), PEPR1 (6- to

10-fold), and PEPR2 (4- to 7-fold) transcript levels. Pep4 and

Pep5 caused a weaker induction (2- to 4-fold) of transcript levels

for all three genes compared with Pep1-3. Pep6 induced an

increase in the levels of PROPEP1 and PEPR1 transcripts

weakly (3-fold) but failed to affect PEPR2. Tomato systemin

(Sys), an 18–amino acid peptide involved in defense responses

to herbivores in Solanaceae plants (Ryan and Pearce, 2003;

Schilmiller and Howe, 2005), did not cause any accumulation of

transcripts of PROPEP1, PEPR1, or PEPR2, indicating that the

response to Pep peptides was not an artifact of our experimental

procedure (Figures 3D to 3F). Pep peptides are involved in innate

immunity in Arabidopsis, and PROPEP1-3 genes were differen-

tially induced by PAMPs (Huffaker et al., 2006). Analysis of

previously published microarray data revealed that PEPR1 and

PEPR2 were also induced by flg22 and elf18 (data not shown)

(Zipfel et al., 2004, 2006). These results were confirmed by

qRT-PCR analysis. A fivefold induction of PROPEP1 and PEPR1

was observed 1 h after supplying flg22 and elf18 (10 nM each),

while twofold induction of PEPR2 was observed after supplying

elf18 (Figures 3D to 3F). Together, these results indicate that

PEPR1 and PEPR2 transcript levels are sensitive to both Pep

peptide and PAMP signaling pathways.

pepr1pepr2DoubleMutantsAreUnable toRespond toPep1

Since the expression patterns of PEPR2 are correlated with

those of PROPEP1 and PEPR1 in the experiments described

above, it is possible that PEPR2 is also involved in Pep1 per-

ception. To clarify the involvement of PEPR2 in Pep1 signaling,

T-DNA insertional mutants, SALK_059281 (pepr1-1) and

SALK_014538 (pepr1-2) for PEPR1 and SALK_036564 (pepr2-1)

and SALK_004447 (pepr2-2) for PEPR2, were obtained from the

ABRC. Both pepr1-2 and pepr1-1 have T-DNA insertions in

regions that encode the extracellular LRRs of PEPR1 (Figure 4A).

The pepr2-1 and pepr2-2 have T-DNA insertions in regions that

encode the cytoplasmic juxtamembrane and the extracellular

LRR domains of PEPR2, respectively (Figure 4A). After selecting

homozygous single lines, two sets of double mutants were

generated by crossing. RT-PCR was performed to confirm that

the T-DNA insertions prevented accumulation of full-length tran-

scripts in all cases (Figure 4B). The pepr1-1 and pepr1-2mutant

lines did not express PEPR1, whereas PEPR2 expression was

normal. The pepr2-1 and pepr2-2 mutant lines did not express

PEPR2, whereas PEPR1 expression was normal. In double

mutant plants, pepr1-1 pepr2-1 and pepr1-2 pepr2-2, no ex-

pression was observed for either PEPR1 or PEPR2. The T-DNA

single and double mutants showed normal growth and fertility

phenotypes (data not shown).

When wild-type Arabidopsis seedlings were supplied with

Pep1, the expression of the PROPEP1 gene was induced (Figure

3A). If both PEPR1 and PEPR2were to perceive Pep1 and lead to

the induction of downstream genes, the induction of PROPEP1

gene expression by Pep1 would be partially reduced in the single

mutants and lost in the doublemutants. Two-week-old seedlings

grown in liquid medium were incubated with 10 nM Pep1 for 30

min and then subjected to qRT-PCR analysis using PROPEP1

gene-specific primers (Figure 4C). In wild-type Arabidopsis

seedlings, Pep1 induced PROPEP1 gene expression by 7- to-8

fold (Figure 4C). In pepr1-1 and pepr1-2 seedlings, only a 50%

average induction was observed compared with the wild type,

suggesting the presence of other receptors for Pep1 in addition

to PEPR1. In pepr2-1 and pepr2-2 seedlings, the reduction of

PROPEP1 induction was not as evident. However, the induction

of PROPEP1 by Pep1 was completely abolished in both double

mutant lines, suggesting that PEPR2 also perceived Pep1.

Pep peptides are thought to amplify PAMP signals to induce

defense-related genes (Huffaker and Ryan, 2007; Ryan et al.,

2007; Boller and Felix, 2009). To examine the effect of Pep1 on

early response gene expression in the PEPR mutants, we chose

mitogen-activated protein kinase-3 (MPK3) and WRKY tran-

scription factor genes, WRKY22, WRKY29, WRKY33, WRKY53,

and WRKY55, which were induced by the fungal PAMP chitin

Figure 1. (continued).

analyzed using the ClustalW program (see Supplemental Data Set 1 online) and the PHYLIP program using full-length amino acid sequences and

visualized by the TreeView program. The numbers on the branches indicate the bootstrap value calculated from 1000 bootstrap sets. The LRR receptor

protein kinases with known biological function are indicated on the right.

(B) The amino acid sequence alignment of PEPR1 and PEPR2 using the ClustalW program. Identical amino acid residues are shaded black, and similar

residues are shaded gray using the BioEdit program. Predicted protein domains are indicated on the right side and the two Cys pairs are indicated by

asterisks.

PEPR2, a Receptor for Pep Peptides 511

(Wan et al., 2008), and the bacterial PAMPs flg22 and elf18 (Zipfel

et al., 2006). Induction of these genes was sometimes reduced

in the single mutants but remained high (50 to 100% of the

induction seen in the wild type). However, in the pepr1 pepr2

double mutant plants, induction of all the genes was reduced to

levels close to those seen in water controls (Figures 4D and 4E;

see Supplemental Figures 3A to 3D online). Since Pep peptides

are known to induce several defense-related genes (Huffaker

and Ryan, 2007), pepr1 pepr2 double mutant plants treated

with Pep1 were also assayed for the expression of the gene

encoding a defensin (PDF1.2) (Figure 4F). In this study, 4-week-

old Arabidopsis plants grown in soil were sprayed with 1 mM

Pep1 and subjected to qRT-PCR analysis after 6 h. The PDF1.2

gene was induced by 30-fold in wild-type plants and was not

induced by Pep1 in the double mutants (Figure 4F). A similar

induction pattern was observed for a pathogenesis-related pro-

tein (PR-1) gene (see Supplemental Figure 3E online).

Preinfiltration of Pep Peptides Reduces Symptom

Development by P. syringae

Plants overexpressing PROPEP1 showed increased resistance

to the root pathogen P. irregulare (Huffaker et al., 2006). To

elucidate the importance of PEPR1 and PEPR2 in defense

responses, pepr1 and pepr2 single and double mutants were

infected with P. irregulare and the necrotrophic pathogen, Alter-

naria brassicicola. A clear difference in symptom development

between wild-type and mutant lines was not observed (data not

shown). Infection with the biotrophic pathogen, Pseudomonas

syringae pv tomato DC3000 (Pst DC3000), of 5-week-old wild-

type and mutant plants produced necrotic regions of the same

size. However, we found that preinfiltration of a 10 nM solution of

Pep1 reduced Pst DC3000 growth in leaves of wild-type plants

and that this reduction was concentration dependent with a

maximal effect at 1mM(Figure 5A). This result was comparable to

leaves preinfiltrated with flg22, which was reported to cause

growth reduction of Pst DC3000 (Zipfel et al., 2004) (Figure 5A).

The reduction of Pst DC3000 growth was not observed when

[A17] Pep1(9-23), an inactive derivative of Pep1 (Pearce et al.,

2008), was preinfiltrated (Figure 5B). Other Pep peptides also

enhanced the resistance to Pst DC3000 with differential inten-

sities (Figure 5C). Pep4 and Pep6 treatment produced slightly

weaker effects on Pst DC3000 proliferation compared with other

Pep peptides.

To assess the relative contributions of PEPR1 and PEPR2 in

the resistance to Pst DC3000 after infiltration of Pep1, the T-DNA

insertional mutants were inoculated. When 1 mM Pep1 was

infiltrated into leaves 1 d before Pst DC3000 inoculation, the size

of the necrotic regions was reduced in both wild-type and single

mutant lines, pepr1-1 and pepr2-1 (Figure 5D). On the other

Figure 2. Transcriptional Induction of PEPR1 and PEPR2 by Wounding and MeJA in 4-Week-Old Arabidopsis.

(A) and (B) Relative expression of PEPR1 (A) and PEPR2 (B) in wounded leaves and unwounded upper leaves. Error bars indicate SE from four

independent experiments. The number of asterisks indicates samples that are significantly different from corresponding samples at 0 h (t test: one

asterisk, P < 0.05; two asterisks, P < 0.02; three asterisks, P < 0.01).

(C) and (D) Relative expression of PEPR1 (C) and PEPR2 (D) after MeJA and water spraying. Error bars indicate SE from five independent experiments.

The number of asterisks indicates samples that are significantly different from corresponding samples sprayed with water (t test: one asterisk, P < 0.05;

two asterisks, P < 0.02; three asterisks, P < 0.01).

512 The Plant Cell

hand, Pep1 preinfiltration did not affect symptom development

by Pst DC3000 infection in double mutants, pepr1-1 pepr2-1

(Figure 5D). Bacterial growth was reduced to 1/100 in the wild

type and pepr2-1 and to 1/25 in pepr1-1 by Pep1 preinfiltration,

but nodecrease in growthwasobservedwith the doublemutants

(Figure 5E). Similar results were obtained when another set of

mutant lines, pepr1-2, pepr2-2, and pepr1-2 pepr2-2, was used

(see Supplemental Figure 4 online). Taken together, these results

indicate that Pep1 induces defense against Pst DC3000 as

strong as flg22 and that Pep1-mediated defense (but not flg22-

mediated defense) is dependent on presence of the PEPR1 or

PEPR2 receptors.

PEPR2 Binds to Pep1

The results shown above strongly suggested that PEPR2 also

could be a receptor for Pep1. In previous experiments, photo-

affinity labeling using microsomal fractions of the T-DNA inser-

tional mutants of PEPR1, pepr1-1, and pepr1-2 did not show any

additional Pep1 binding proteins (Yamaguchi et al., 2006). How-

ever, if the protein level of PEPR2 or binding capacity of PEPR2 to

Pep1 is much lower than PEPR1, it might be difficult to detect

binding between PEPR2 and 125I-azido-Cys-pep1. To clarify

whether PEPR2 binds to Pep1, the PEPR2 coding region was

fused to the cauliflower mosaic virus 35S promoter and trans-

formed into tobacco suspension-cultured cells (Nicotiana taba-

cum). Tobacco cells expressing PEPR1 and b-glucuronidase

(GUS) geneswere also created as positive and negative controls,

respectively. RT-PCR analysis revealed that transgenic cells

selected on kanamycin-containing medium expressed each

transgene (Figure 6A). The transgenic cells were incubated with

0.25 nM 125I1-azido-Cys-Pep1 (Yamaguchi et al., 2006) and

irradiated with UV-B to cross-link the Pep1 binding proteins.

After separation of extracted proteins by SDS-PAGE, the labeled

proteins were detected on x-ray film (Figure 6B). A major protein

band of 170 kD, consistent with the size of PEPR1 in Arabidopsis

(Yamaguchi et al., 2006), and a 150-kD band was labeled in

PEPR1- and PEPR2-expressing cells, respectively, but not in the

Figure 3. The Effect of Supplying Various Peptides to Wild-Type Arabidopsis Plants on Transcription of Target Genes.

(A) to (C) Two-week-old Arabidopsis seedlings grown in liquid medium were incubated with either Pep1 (10 nM) or water for the indicated time period,

and the expression patterns of PROPEP1 (A), PEPR1 (B), and PEPR2 (C) were analyzed by qRT-PCR. Expression levels are indicated relative to the

expression at 0 h.

(D) to (F) Two-week-old Arabidopsis seedlings grown in liquid medium were incubated with 10 nM Pep1, Pep2, Pep3, Pep4, Pep5, Pep6, tomato

systemin (Sys), flg22, or elf18, and the expression of PROPEP1 (D), PEPR1 (E), and PEPR2 (F) was analyzed after 1 h by qRT-PCR. Expression levels

are indicated relative to the expression in water supplying seedlings.

Error bars indicate SE from three different experiments. The number of asterisks indicates samples that are significantly different from corresponding

samples at 0 h or supplied with water (t test: one asterisk, P < 0.05; two asterisks, P < 0.02; three asterisks, P < 0.005).

PEPR2, a Receptor for Pep Peptides 513

cells incubated with 50 nM unlabeled Pep1 as a competitor of125I1-azido-Cys-Pep1 (Figure 6B). Minor radioactive bands at

lower molecular masses probably represent partial degradation

of the PEPR1 and PEPR2 proteins (Figure 6B).

To elucidate the different binding properties with Pep1 between

PEPR1 and PEPR2, substrate saturation analysis was con-

ducted using radiolabeled Pep1 (125I1-Tyr-Pep1) and transgenic

tobacco cells (Figure 6C). The binding between 125I1-Tyr-Pep1

and PEPR1-expressing cells was almost saturated at 1.5 nM,

whereas the binding between 125I1-Tyr-Pep1 and PEPR2-

expressing cells was saturated at ;2 to 3 nM (Figure 6C).

Scatchard analysis of these data revealed that dissociation

constants between 125I1-Tyr-Pep1 and PEPR1- and PEPR2-

expressing cellswere 0.56 and 1.25 nM, respectively, suggesting

that PEPR1 has a slightly higher affinity for Pep1 than does

PEPR2 (Figure 6D).

PEPR1 Is a Receptor for Pep1-6 and PEPR2 for Pep1

and Pep2

The binding properties of each Pep peptide to PEPR1 and

PEPR2were analyzed by competition assayswith 125I1-Tyr-Pep1

Figure 4. Analysis of the T-DNA Mutants in PEPR1 and PEPR2.

(A) T-DNA insertion sites in the PEPR1 and PEPR2 genes are indicated by black triangles. Black regions represent the signal peptides, dark-gray

regions represent transmembrane domains, light-gray regions represent the kinase domains, and striped regions represent the LRR domain.

(B)RT-PCR analysis of the PEPR1 and PEPR2 transcripts in wild-type and T-DNA insertional mutants. The T-DNAmutants from (A)were analyzed along

with two double mutants (pepr1-1 pepr2-1 and pepr1-2 pepr2-2). b-TUBULIN (TUB2) gene was amplified as an internal control.

(C) to (E) The effect of Pep1 on the expression patterns of the early response genes PROPEP1, MPK3, and WRKY33, respectively, for the T-DNA

mutants. Two-week-old Arabidopsis seedlings grown in liquid mediumwere incubated with 10 nM Pep1 for 30 min, and the expression was analyzed by

qRT-PCR.

(F) The effect of Pep1 on the expression pattern of the late response gene PDF1.2 for the T-DNA mutants. Four-week-old Arabidopsis plants grown in

soil were sprayed with 1 mM Pep1 in 0.01% Silwet L-77, and total RNA was extracted after 6 h. The expression was analyzed by qRT-PCR.

Expression levels are indicated relative to expression in wild-type seedlings supplied with water. Error bars indicate SE from three different experiments.

The letters indicate groupings by the one-way analysis of variance with Tukey multiple comparison test (P < 0.05).

514 The Plant Cell

(Figures 7A to 7D). Just before addition of 0.5 nM 125I1-Tyr-Pep1

to the transgenic tobacco cells, 10 nM unlabeled Pep1-6 was

added, and the remaining radioactivitywasmeasured.Unlabeled

Pep1, Pep2, Pep5, and Pep6 reduced binding of 125I1-Tyr-Pep1

to PEPR1-expressing cells to 30 to 40% of water control, Pep3

reduced binding to 70% of control levels, and Pep4 did not

reduce binding (Figure 7A). Whereas unlabeled Pep1 and Pep2

reduced binding of 125I1-Tyr-Pep1 to PEPR2-expressing cells to

10 and 50%of control levels, respectively, Pep3-6 did not reduce

binding (Figure 7B). PEPR1- and PEPR2-expressing tobacco

cells responded in the alkalinization assay to each of thePeps in a

manner consistentwith the results obtained from the competition

assay (Figures 7C and 7D). All Pep peptides caused alkalinization

of the medium in PEPR1-expressing cells with the weakest

Figure 5. P. syringae pv Tomato DC3000 (Pst DC3000) Infection Assay of Wild-Type Arabidopsis and T-DNA Mutants Pretreated with Peptides.

(A) Pst DC3000 proliferation in wild-type plants with flg22 and Pep1. Light-gray bars and dark-gray bars indicate samples just after inoculation and 4 d

after inoculation (DAI), respectively. Values presented are the average 6 SE from three independent experiments. The asterisks indicate samples that

are significantly different from the sample supplied with water (t test, P < 0.02). cfu, colony-forming units.

(B) Pst DC3000 proliferation in wild-type and pepr1-1 pepr2-1 plants with flg22 (1 mM), Pep1 (1 mM), and [A17] Pep1(9-23) (1 mM). Values presented are

the average 6 SE from three independent experiments. The asterisks indicate samples that are significantly different from corresponding samples

supplied with water (t test, P < 0.02).

(C) Pst DC3000 proliferation in wild-type plants with indicated peptides (1 mM). Light-gray bars and dark-gray bars indicate samples just after

inoculation and 4 d after inoculation, respectively. Values presented are the average 6 SE from three independent experiments. The asterisks indicate

samples that are significantly different from the sample supplied with water (t test, P < 0.04).

(D) Symptoms of the wild type, pepr1-1, pepr2-1, and pepr1-1 pepr2-1 preinfiltrated with water or Pep1 (1 mM) 4 d after infection with Pst DC3000.

(E) Pst DC3000 proliferation in the wild type, pepr1-1, pepr2-1, and pepr1-1 pepr2-1 with or without Pep1. Values presented are the average6 SE from

nine plants from three independent experiments. The asterisks indicate samples that are significantly different from corresponding samples supplied

with water (t test, P < 0.0008). In all experiments, peptides or water were infiltrated 1 d prior to infection.

PEPR2, a Receptor for Pep Peptides 515

activity found for Pep4 (Figure 7C). These results were compa-

rable to the results obtained using wild-type Arabidopsis cells

(Yamaguchi et al., 2006). On the other hand, only Pep1 and Pep2

caused medium alkalinization to PEPR2-expressing tobacco

cells (Figure 7D). These results obtained from competition and

alkalinization assays using transgenic tobacco cells indicated

that PEPR1 binds to Pep1-6 and that PEPR2 binds to Pep1 and

Pep2. These results also revealed that the levels of alkalinization

of the medium caused by each Pep peptide reflect the binding

properties of PEPR1 and PEPR2 to each Pep peptide.

The preferences for each Pep peptide for PEPR1 and PEPR2

were confirmed inArabidopsis T-DNAmutant lines bymonitoring

the transcript levels of MPK3 and WRKY33 as marker genes.

Two-week-old seedlings grown in liquid medium were supplied

with Pep1-6 for 30 min, and gene induction was analyzed by

qRT-PCR (Figure 7E; see Supplemental Figure 5 online). Both the

MPK3 andWRKY33 genes were induced by Pep1-6 in wild-type

and pepr2-1 seedlings with the lowest induction by Pep4. On the

other hand, these genes were induced only weakly by Pep1 and

Pep2 in pepr1-1 seedlings and were not induced by any Pep

peptides in pepr1-1 pepr2-1 seedlings.

DISCUSSION

PEPR1 and PEPR2 Together Perceive Peps

In this study, we found that PEPR1 and PEPR2 together con-

tribute to the perception of Pep1-6 and elicitation of the defense

responses induced by these peptides. However, only PEPR1

was purified previously as the Pep1 binding protein from Arabi-

dopsis suspension cultured cells, and other Pep1 binding pro-

teins had not been found by photoaffinity labeling using

microsomal proteins of the T-DNA insertional lines of PEPR1,

pepr1-1, and pepr1-2 (Yamaguchi et al., 2006). These results

implicated PEPR1 as the primary receptor for Pep1, and this

conclusion is further supported by the results obtained in this

study. The induction of early responsive genes, such as PRO-

PEP1 and MPK3, by Pep1 was reduced to 50% in the PEPR1

single mutants, whereas this induction was affected less in the

PEPR2 single mutants (Figures 4D and 4E). The suppression of

the Pst DC3000 growth by Pep1 preinfiltration in wild-type

Arabidopsis was reduced to a greater degree in the pepr1 single

mutants than in the pepr2 single mutants (Figure 5D). A plausible

Figure 6. Binding of Radiolabeled Pep1 to PEPR1- and PEPR2-Expressing Tobacco Cells.

(A) Confirmation of transgene (GUS, PEPR1, and PEPR2) expression of transgenic tobacco cells by RT-PCR. The tobacco elongation factor 1a (EF-1a)

gene was amplified as an internal reference transcript.

(B) Photoaffinity labeling of transgenic tobacco cells using 0.25 nM 125I-azido-Cys-Pep1 with or without 50 mM of unlabeled Pep1 as a competitor. Ten

microliters of sample solution from wild-type, PEPR2, and GUS-expressing cells and 1 mL of a sample solution from PEPR1-expressing cells were

separated by SDS-PAGE and exposed to x-ray film.

(C) Saturation analysis of 125I1-Tyr-Pep1 binding to transgenic tobacco cells expressing PEPR1 and PEPR2. Error bars indicate SE for three different

experiments.

(D) Scatchard analysis of the data from (C). The Kd is calculated as the negative inverse of the slope of the plot.

516 The Plant Cell

explanation for the activity difference between PEPR1 and

PEPR2 was the difference in their binding affinities for Pep1.

However, the difference in binding affinities might not be the sole

reason, since the calculated Kds of PEPR1 and PEPR2 for Pep1

are within the same range (Figure 6D). Further investigations,

such as comparisons of their relative protein levels and their

intracellular kinase activities in Arabidopsis, will be needed to

understand how these two receptors have different roles.

However, PEPR2 contributes significantly to the perception of

Peps and to downstream responses. The specific contributions

of PEPR2 are revealed through studies that emphasize the

overlapping functions of PEPR1 and PEPR2. For example, either

Figure 7. Binding Preference of PEPR1 and PEPR2 for Pep1-6.

(A) and (B) Competition assay of Pep peptides with 125I1-Y-Pep1 (0.5 nM) for binding to transgenic tobacco cells expressing PEPR1 (A) and PEPR2 (B).

Remaining specific binding of 125I1-Y-Pep1 to cells in the presence of unlabeled competitor peptide (Pep1-6) (10 nM) is indicated as a percentage of

specific binding of 125I1-Y-Pep1 to the cells without competitor.

(C) and (D) Medium alkalinization of transgenic tobacco cells expressing PEPR1 (C) and PEPR2 (D) by the Pep peptides, assayed at the five

concentrations shown.

(E) The effect of Pep peptides on the expression pattern of MPK3 gene for the T-DNA mutants. Two-week-old Arabidopsis seedlings grown in liquid

medium were incubated with 10 nM peptide for 30 min, and the expression was analyzed by qRT-PCR. Expression levels are indicated relative to the

expression in wild-type seedlings supplied with water.

Error bars indicate SE for three ([A] to [D]) and five (E) different experiments. The number of asterisks indicates samples that are significantly different

from corresponding samples supplied with water ([A], [B], and [E]) and with Pep1 ([C] and [D]) (t test: one asterisk, P < 0.05; two asterisks, P < 0.01;

three asterisks, P < 0.001).

PEPR2, a Receptor for Pep Peptides 517

receptor will provide for transcriptional responses of both early-

and late-acting defense response genes (Figures 4C to 4F). Both

single mutants were capable of demonstrating resistance to

bacterial proliferation in Pep1 preinfiltrated leaves. Both recep-

tors bind strongly to Pep1-2, but PEPR1 shows a higher affinity to

ep3-6.

Pep1 Shares Some Signaling Components with the PAMPs

flg22, elf18, and Chitin to Amplify Innate Immune Response

Pep1 induces the defense-related genes PR-1 and PDF1.2

(Huffaker et al., 2006; Huffaker and Ryan, 2007). Here, we also

found transcriptional induction of MPK3, WRKY22, WRKY29,

WRKY33, and WRKY53 by Pep1 (Figures 5D and 5E; see

Supplemental Figures 3A to 3D online), which are important in

defense signaling (Eulgem and Somssich, 2007; Colcombet and

Hirt, 2008) and have been reported to be induced by a fungal

PAMP, chitin (Wan et al., 2004; Wan et al., 2008), and bacterial

PAMPs, flg22 and elf18 (Zipfel et al., 2004, 2006). Based on the

comparative analysis of microarray data using Arabidopsis sup-

plied with flg22, elf18, and chitin (Zipfel et al., 2004, 2006; Wan

et al., 2008), Wan et al. (2008) concluded that flg22, elf18, and

chitin signaling share a conserved downstream pathway leading

to basal resistance. Similarly, Pep/PEPR signaling likely works

through some of the same signaling components as PAMPs

because Pep1 both induced transcription of MPK3, PDF1.2,

PR-1, and WRKY genes, which are also induced by flg22, elf18,

and chitin, and enhanced resistance to fungal and bacterial

pathogens (Figures 4 and 5; Huffaker et al., 2006).

Like Pep1 receptors, the receptors for flg22 and elf18, FLS2

and EFR, respectively, are plasma membrane LRR receptor

kinases (Chinchilla et al., 2006; Zipfel et al., 2006). After percep-

tion by FLS2, flg22 induces WRKY22 and WRKY29 through

activation of a MAPK cascade composed of MEKK, MKK4/

MKK5, and MPK3/MPK6 (Asai et al., 2002; Valerie et al., 2009).

Chitin also activates MPK3 and MPK6 activity (Wan et al., 2004),

and the receptor for chitin is probably an RLK, CERK1/LysM-

RLK1, with an extracellular chitooligosaccharide binding motif

(LysM) and an intercellular kinase domain (Miya et al., 2007; Wan

et al., 2008). It is possible that the induction of defense-related

genes and enhancement of basal resistance by Pep1 occurs

through activating the same MAPK cascade after perception by

PEPR1 and PEPR2. There is precedence for MAPK signaling

downstreamof LRRRLKs from studies of the roles of HAESA and

HAESA LIKE2, which also belong to LRR XI subfamily (Cho et al.,

2008; Stenvik et al., 2008).

Possible Contribution of WRKYs to Pep-PEPR System

In Arabidopsis, there are 72 expressed WRKY genes (http://

www.Arabidopsis.org/browse/genefamily/WRKY.jsp), and

many of them are implicated in the regulation of the plant

immune response positively and negatively via modulation of

the JA/SA signaling pathways (Eulgem and Somssich, 2007).

WRKY29, WRKY33, and WRKY53, which are induced by Pep1,

are reported to be positive regulators of defense responses for

bacterial and/or fungal pathogens, such as P. syringae, Botritis

cinerea, and A. brassicicola (Asai et al., 2002; Zheng et al., 2006;

Murray et al., 2007). WRKY transcription factors bind to W-box

DNA elements (C/TTGACC/T) that are found in the promoters of

many defense-related genes, including PR-1 and NPR1 (Maleck

et al., 2001; Yu et al., 2001; Eulgem and Somssich, 2007). WRKY

transcription factors also regulate the expression of their own

genes and/or other WRKY genes in addition to the defense-

related genes, composing the positive and negative feedback

loops and feed-forward modules (Eulgem and Somssich, 2007).

Interestingly, multiple W-box DNA elements were predicted in

the promoter region of PEPR1 and PROPEP1-5 genes in the

AtcisDB (Arabidopsis thaliana cis-regulatory database; http://

Arabidopsis.med.ohio-state.edu) (Palaniswamy et al., 2006).

Therefore, theWRKY transcription factors may play an important

role in the amplification of the Pep peptide signal.

The Pep-PEPR System Is One Component of Multiple

Amplification Mechanisms

Pep peptides are considered to be endogenous amplifiers of

innate immunity after perception of PAMPs by PRRs based on

the following results: (1) overexpression of PROPEP1 and

PROPEP2 enhanced resistance to P. irregulare; (2) supplying

Pep peptides differentially induced defense related genes, such

asPDF1.2 andPR-1; (3) supplying Pep peptides also induced the

expression of their own precursor genes, except Pep6; and (4)

supplying PAMPs and inoculation with pathogens both dramat-

ically induced the PROPEP2 and PROPEP3 genes (Huffaker

et al., 2006; Huffaker and Ryan, 2007; Ryan et al., 2007). In this

study, we confirmed the Pep1 effects on defense responses,

such as transcriptional induction of MPK3 and WRKY gene and

enhancement of resistance to Pst DC3000 upon Pep1 applica-

tion. However, wild-type Arabidopsis and the pepr1 pepr2 dou-

ble mutants did not show obvious differences upon inoculation

with several pathogens without Pep1 preinfiltration in our exper-

imental conditions. The plant defense response is a very sophis-

ticated mechanism and affected by many factors, including

specific pathogen-plant combinations, pathogen concentra-

tions, and environmental factors, such as temperature, light,

and humidity. Therefore, it is possible that PEPR1 andPEPR2 are

important for the resistance to specific pathogens or specific

inoculation conditions that we have not identified yet.

Considering the multilayered amplification mechanisms of

PTI, it is also not surprising that pepr1 pepr2 doublemutants did

not show any obvious difference in their resistance to patho-

gens compared with wild-type plants because other amplifica-

tionmechanisms still exist inArabidopsis, including the salicylic

acid, jasmonic acid, and ethylene signaling pathways. How-

ever, overexpression of PROPEP1 and PROPEP2 and applica-

tion of Pep peptides can enhance defense responses, including

H2O2 generation and transcriptional induction of defense-

related genes (Huffaker et al., 2006; Huffaker and Ryan, 2007;

this study). It will be interesting to determine how many

enodogenous elicitors and receptors, as well as additional

amplification loops and crosstalk, will be identified that protect

plants from surrounding microorganisms. It will also be inter-

esting to determine the relative contribution of these endoge-

nous regulators to PTI.

518 The Plant Cell

METHODS

Plant Material and Growth Conditions

Arabidopsis thaliana plants, ecotype Columbia, were grown on twice

autoclaved soil (1208C, 20 min) with four plants per pot (83 83 7 cm) at

22 to 258C with a 9-h photoperiod for 4 to 5 weeks. For experiments

conducted under sterile conditions, Arabidopsis seedlings were grown in

Petri dishes containing half-strength Murashige and Skoog (MS) salts

(Sigma-Aldrich), 1% sucrose, and 0.6% agar for a week at 258C under

constant light. Three seedlings were transferred into a flat-bottom glass

tube (10 3 2.5 cm diameter) containing 3 mL of liquid medium (half-

strength MS salts and 1% sucrose) and incubated on an orbital shaker at

160 rpm for a week at 258C under constant light. T-DNA insertional lines

(see Accession Numbers section) were obtained from the ABRC at Ohio

State University. The double mutants pepr1-1 pepr2-1 and pepr1-2

pepr2-2 were obtained by crossing, with homozygous lines screened by

two sets of PCR analyses, one using the gene-specific primer pair and the

other using the gene-specific primer and the T-DNA left border of the

vector as a primer. The primers used in this study are listed in Supple-

mental Table 1 online.

Computer Analyses

Full-length amino acid sequences of all members in the subfamily LRR XI

(Shiu et al., 2004) were aligned using the ClustalW program (Thompson

et al., 1994) using the BioEdit program (http://www.mbio.ncsu.edu/

BioEdit/bioedit.html) (Hall, 1999). Phylogenetic analysis (unrooted) was

performed using the PHYLIP program (PHYLIP 3.68; http://evolution.

genetics.washington.edu/phylip.html). In the PHYLIP program, SEQ-

BOOT, PROTPARS, and CONSENSE programs were used for making

the phylogenetic tree (unrooted). The resulting tree was drawn with

the TreeView program (http://taxonomy.zoology.gla.ac.uk/rod/treeview.

html) (Page, 1996). Domain predictions were performed using the SignalP

program (http://www.cbs.dtu.dk/services/SignalP/) (Bendtsen et al.,

2004) for the signal sequence and the TopPred program (http://mobyle.

pasteur.fr/cgi-bin/MobylePortal/portal.py?form=toppred) (von Heijne,

1992) for the transmembrane region. LRR and protein kinase domains

were based on the data from The Arabidopsis Information Resource

(http://www.Arabidopsis.org/).

Plant Treatments

The leaves of 4-week-old Arabidopsis grown in soil were mechanically

wounded across the main vein with a hemostat. At the indicated time

points, the wounded leaves and the unwounded upper leaves were

collected for extraction of total RNA by the method of de Vries et al.

(1988). Four-week-old Arabidopsis plants grown in soil were sprayed

with MeJA (625 mM in 0.1% Triton X-100) and kept in a closed plexiglas

box under light conditions until harvesting. For expression analysis of

the PROPEP1, PEPR1, PEPR2, MPK3, WRKY22, WRKY29, WRKY33,

WRKY53, and WRKY55 genes, 2-week-old Arabidopsis plants grown in

liquid medium were incubated with 10 nM peptides, and whole plants

were harvested. For expression analysis of PDF1.2 and PR-1 genes,

4-week-old Arabidopsis grown in soil was sprayed with At Pep1 (1 mM in

0.01% Silwet L-77), and the leaves were collected after 6 h because

background expression of PDF1.2 and PR-1 was high in the seedlings

grown in liquid medium.

RT-PCR

Total RNA was isolated from Arabidopsis as described by de Vries et al.

(1988). For analysis of expression ofPEPR1 andPEPR2 in theArabidopsis

T-DNA insertion lines and the transgenic tobacco (Nicotiana tabacum)

cells, total RNAwas treatedwith DNase I (NewEnglandBiolabs), and 2mg

of total RNA was reverse transcribed by Superscript III (Invitrogen) using

an oligo(dT) primer (Invitrogen). Subsequently, 0.2 mL of the reverse

transcription reaction was used as a template for PCR amplification.

Arabidopsis b-tubulin (TUB2) gene and the tobacco elongation factor 1a

(EF-1a) gene were amplified as internal controls. The number of PCR

cycles was 35 for PEPR1 and PEPR2 and 30 for TUB2 and EF-1a. For the

quantitative analysis of gene expression, total RNA treated with DNaseI

was reverse transcribed using the DyNAmo cDNA synthesis kit (Finn-

zyme) with random hexamer, and qPCR was performed using the

DyNAmo HS SYBR Green qPCR kit (Finnzyme) and Mx3005P QPCR

systems (Stratagene). Ubiquitin 5 (UBQ5) was amplified as an internal

control. The primers used in this study are listed in Supplemental Table

1 online.

Pseudomonas syringae Infection

Twenty four hours prior to bacterial inoculation, leaves were infiltrated

with peptides or water (1mL). Syringe inoculation of P. syringae pv tomato

DC3000 (Pst DC3000) was performed as described (Zipfel et al., 2004).

Pst DC3000 was grown at 288C on low-salt Luria-Bertani (1 g of NaCl/L)

agar medium containing 100 mg/L of rifampicin (Sigma-Aldrich) for 24 h,

resuspended in sterile water to 5 3 105 colony-forming units/mL, and

pressure infiltrated into leaves of 5-week-old Arabidopsis plants with a

needleless syringe. The infiltrated area was ;5 mm in diameter. The

plants were covered with a clear plastic lid after bacterial solution was

completely absorbed. Leaf discs (0.28 cm2) from two different leaves

were ground in 100 mL of 10 mM MgCl2 in a 1.5-mL tube. The samples

were thoroughly vortex mixed with 900 mL of water and diluted 1:10

serially. The samples (8 mL) were plated on low-salt Luria-Bertani agar

medium containing 100 mg/L of rifampicin. Plates were placed at room

temperature for 2 d, after which the colony-forming units were counted.

Transgenic Tobacco Cells

The expression vector for PEPR1 was previously created (Yamaguchi

et al., 2006). The coding region of PEPR2was amplified by PCR using the

primers attached bySmaI sites, ligated intoSmaI sites ofmodified pBI121

vector (Yamaguchi et al., 2006) between the 35S promoter and the nos

terminator. The expression vectors for PEPR1 and PEPR2, and the

original pBI121 vector, which contains the GUS gene between the 35S

promoter and the nos terminator, were introduced into tobacco BY2

suspension-cultured cells using Agrobacterium tumefaciens by the

method of Nakayama et al. (2000). The transgenic cells were assayed

using the alkalinization assay as previously described (Pearce et al.,

2001).

Photoaffinity Labeling

Photoaffinity labeling using radioiodinated Pep1 was performed as pre-

viously described (Scheer and Ryan, 1999; Yamaguchi et al., 2006).

Briefly, Cys-Pep1 was coupled to the azido-photoaffinity cross-linker,

N-(4-[p-azidosalicylamido]butyl)-39-(29-pyridyldithio)propionamide (Pierce

Biotechnology) and iodinated by Na125I using an IODO-GEN iodination

tube (Pierce Biotechnology) to create 125I-azido-Cys-Pep1. One milliliter

of tobacco cells was incubated with 125I-azido-Cys-Pep1 (0.25 nM) for 10

min under red light at room temperature and then irradiated with a UV-B

lamp (F15T8.UV-B, 15 W) for 10 min on ice. After washing the cells with

MS medium, the cells were sedimented by centrifugation at 12,000g and

disrupted in 500 mL of 5% SDS by boiling for 30 min. The cell debris was

removed by centrifugation at 12,000g. Proteins in the supernatant were

PEPR2, a Receptor for Pep Peptides 519

precipitated by addition of 400 mL of methanol and 200 mL of chloroform

andpelleted in amicrofuge. The pellet was dissolved in 100mL of Laemmli

sample buffer containing 5% SDS at 658C for 1 h, and 1 or 10 mL was

separated by 8% SDS-PAGE. The gels were dried and exposed to x-ray

film for 50 h.

Binding and Competition Assays Using Radiolabeled-Pep1

Radioiodination was performed using 2 mCi of Na125I with 12.5 nmol of

Tyr-Pep1, followed by purification by HPLC as previously described

(Scheer and Ryan, 1999; Yamaguchi et al., 2006). The specific radioac-

tivity of 125I1-Tyr-Pep1 was calculated to be 2 mCi/nmol. Radioligand

binding assays were performed as previously described (Yamaguchi

et al., 2006) with minor modifications. Suspension-cultured transgenic

tobacco cells were used for assays 5 to 6 d after subculturing. The cells

were separated from the medium using Miracloth (Calbiochem), washed

twice with 40 mL of culture medium, and adjusted to a fresh weight of 0.2

g/mLwith freshmedium. A 2-mL aliquot of cells was pipetted into awell of

a 12-well culture plate and allowed to equilibrate for 1 h at room temper-

ature while agitated on an orbital shaker (160 rpm). 125I1-Tyr-Pep1 was

added to the medium to give a final concentration of 0.05 to 3.0 nM. To

assay binding, 500mL of cells was removed and filtered through a 2.5-cm

Type A/EGlass Fiber Filter (Pall) using a 12-well vacuumfiltrationmanifold

(Millipore). The cells were washed three times with 5 mL of cold MS

medium containing 3% sucrose, suspended in 1 mL of MS medium

containing 3% sucrose, transferred to a test tube, and then analyzed for

total radioactivity in a g-ray counter (Isodata 2020). For the competition

assay, the competitor peptide (10 nM) was added to 2 mL cells just be-

fore addition of 0.5 nM 125I1-Tyr-Pep1 and incubated for 1 min. Specific

binding was calculated by subtracting nonspecific binding (binding in the

presence of 200-fold excess native Pep1) from total binding.

Accession Numbers

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

Initiative or GenBank/EMBL databases under the following accession

numbers: PEPR1, At1g73080; PEPR2, At1g17750; PROPEP1,

At5g64900; PROPEP2, At5g64890; PROPEP3, At5g64905; PROPEP4,

At5g09980; PROPEP5, At5g09990; PROPEP6, At2g22000; IKU2,

At1g09970; HSL2, At5g65710; HSL1, At1g28440; HAESA, At4g28490;

GSO1, At4g20140; GSL2, At5g44700; PXY/TDR, At5g61480; BAM1,

At5g65700; BAM2, At3g49670; BAM3, At4g20270; BRI1, At4g39400;

CLV1, At1g75820; MPK3, At3g45640; WRKY22, At4g01250; WRKY29,

At4g23550; WRKY33, At2g38470; WRKY53, At4g23810; WRKY55,

At2g40740; PDF1.2, At5g44420; PR1, At2g14610; TUB2, At5g62690;

UBQ5, At3g62250; At1g08590; At1g17230; At1g34110; At1g72180;

At2g33170; At3g19700; At3g24240; At4g26540; At4g28650;

At5g48940; At5g49660; At5g56040; At5g63930; EF-1a, D63396; and

pBI121, AF485783. Germplasm information for the T-DNA insertion lines

used in this study can be found in the ABRC at Ohio State University

(Columbus, OH) under the following accession numbers: pepr1-1,

SALK_059280; pepr1-2, SALK_014538; pepr2-1, SALK_036564; and

pepr2-2, SALK_004447.

Supplemental Data

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

Supplemental Figure 1. Conserved Regions of the Kinase Domain of

PEPR1, PEPR2, CLV1, and BRI1.

Supplemental Figure 2. The Effect of MeSA and ACC on the

Expression Patterns of PEPR1 and PEPR2.

Supplemental Figure 3. The Effect of Pep1 on the Expression

Patterns of WRKY22, WRKY29, WRKY53, WRKY55, and PR-1 Genes

for the T-DNA Insertional Mutants.

Supplemental Figure 4. P. syringae pv Tomato DC3000 (Pst

DC3000) Infection Assay of T-DNA Insertional Mutants Pretreated

with Either Water or Pep1 (1 mM).

Supplemental Figure 5. The Effect of Pep Peptides on the Expres-

sion Pattern of the WRKY33 Gene in the T-DNA Mutants.

Supplemental Table 1. Primers Used in This Study.

Supplemental Data Set 1. Alignment of Arabidopsis LRR-XI Sub-

family Proteins Used for Phylogenetic Analysis in Figure 1A (FASTA

Format).

ACKNOWLEDGMENTS

We thank G. Pearce (Washington State University) and for critical

discussion and reading of this manuscript, J.A. Browse (Washington

State University) for critical discussion and providing Pst DC3000, J.

Gothard and S. Vogtmann (Washington State University) for growing our

plants, G. Barona (Washington State University) for maintaining the cell

cultures, D. Deavila (Washington State University) for help and advice in

radiolabeling, and G. Munske (Washington State University) for peptide

synthesis. This work was supported by Monsanto, National Science

Foundation Grant IBN 0418946 to F.E.T., National Science Foundation

Grant IBN 0623029 to C.A.R., the Charlotte Y. Martin Foundation, and

the Washington State University College of Agriculture, Human, and

Natural Resources Sciences.

Received June 2, 2009; revised January 17, 2010; accepted January 31,

2010; published February 23, 2010.

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522 The Plant Cell

DOI 10.1105/tpc.109.068874; originally published online February 23, 2010; 2010;22;508-522Plant Cell

Yube Yamaguchi, Alisa Huffaker, Anthony C. Bryan, Frans E. Tax and Clarence A. RyanArabidopsisResponses in

PEPR2 Is a Second Receptor for the Pep1 and Pep2 Peptides and Contributes to Defense

 This information is current as of May 23, 2021

Supplemental Data /content/suppl/2010/02/10/tpc.109.068874.DC1.html

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