pepr2 is a second receptor for the pep1 and pep2 ...pepr2 is a second receptor for the pep1 and pep2...
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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
References /content/22/2/508.full.html#ref-list-1
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