Functional Characterization of CEBiP and CERK1 Homologs in
Arabidopsis and Rice Reveals the Presence of Different Chitin
Receptor Systems in Plants
Regular Paper
Tomonori Shinya, Noriko Motoyama, Asahi Ikeda, Miyuki Wada, Kota Kamiya, Masahiro Hayafune,
Hanae Kaku and Naoto Shibuya*
Department of Life Sciences, School of Agriculture, Meiji University, Kawasaki, Kanagawa, 214-8571 Japan
*Corresponding author: E-mail, [email protected]; Fax, +81-44-934-7039.
(Received July 4, 2012; Accepted July 27, 2012)
Chitin is a representative microbe-associated molecular
pattern (MAMP) molecule for various fungi and induces
immune responses in many plant species. It has been clarified that the chitin signaling in rice requires a receptor
kinase OsCERK1 and a receptor-like protein (Os)CEBiP,
which specifically binds chitin oligosaccharides. On the
other hand, Arabidopsis requires a receptor kinase
(At)CERK1 for chitin signaling but it is not clear whether
the plant also requires a CEBiP-like molecule for chitin
perception/signaling. To clarify the similarity/difference of
the chitin receptor in these two model plants, we first
characterized CEBiP homologs in Arabidopsis. Only one of
three CEBiP homologs, AtCEBiP (LYM2), showed a
high-affinity binding for chitin oligosaccharides similar to
rice CEBiP. AtCEBiP also represented the major chitinbinding protein in the Arabidopsis membrane. However,
the single/triple knockout (KO) mutants of Arabidopsis
CEBiP homologs and the overexpressor of AtCEBiP showed
chitin-induced defense responses similar to wild-type
Arabidopsis, indicating that AtCEBiP is biochemically functional as a chitin-binding protein but does not contribute to
signaling. Studies of the chitin binding properties of the
ectodomains of At/OsCERK1 and the chimeric receptors
consisting of ecto/cytosolic domains of these molecules indicated that AtCERK1 is sufficient for chitin perception by
itself.
Keywords: CEBiP CERK1 Chitin MAMP Plant immunity Receptor.
Abbreviations: ED, ectodomain; EGS, ethylene glycol bis[succinimidylsuccinate]; GN8, (GlcNAc)8; GN8-Bio, biotinylated
(GlcNAc)8; GPI, glycosylphosphatidylinositol; JM, juxtamembrane; KD, kinase domain; KO, knockout; LysM, lysin motif;
MAMP/PAMP, microbe/pathogen-associated molecular pattern; ORF, open reading frame; PRR, pattern-recognition receptor; ROS, reactive oxygen species; RT–PCR, reverse
transcription–PCR; TM, transmembrane.
Introduction
Plants are constantly exposed to potentially pathogenic
microbes in nature. As the first layer of defense machinery for
these microbes, plants have evolved the ability to detect them
through the perception of microbe/pathogen-associated molecular patterns (MAMPs/PAMPs) by pattern-recognition
receptors (PRRs), which then initiate various immune responses
(Boller and Felix 2009, Silipo et al. 2010, Dodds and Rathjen,
2011, Thomma et al. 2011). This system is also known to have
close similarity to the innate immune system in animals. On the
other hand, pathogenic microbes evolved to acquire various
effectors which perturb host immune systems to overcome
these barriers (Boller and He 2009, Dodds and Rathjen 2011,
Thomma et al. 2011). It has been shown that MAMP–PRR systems were targeted in various ways by microbial effectors. For
example, the fungal effectors Ecp6 and Avr4, secreted by
Cladosporium fulvum, inhibit the activation of chitin-triggered
host immunity (van Esse et al. 2007, de Jonge et al. 2010). Avr4
binds and protects chitin in the fungal cell walls from degradation by host chitinase, whereas Ecp6 binds chitin oligosaccharides released from the cell walls and prevents their recognition
by the host receptor. Other fungal effectors such as Slp1 and
Mg3LysM have also been shown to inhibit chitin-induced
immunity similarly to Ecp6 (Marshall et al. 2011, Mentlak
et al. 2012). The bacterial effector AvrPtoB is known to degrade
and abolish the function of plant PRRs, FLS2 and CERK1,
through the ubiquitination of these receptors (Gohre et al.
2008, Gimenez-Ibanez et al. 2009). To cope with the development of microbial effectors, plants also developed R-genes, the
products of which, R-proteins, specifically detect these effectors
and initiate effector-triggered immunity (Dodds and Rathjen
2011, Thomma et al. 2011). Such a co-evolution of host plants
and pathogenic microbes has been interpreted by the zigzag
model (Dangl and Jones 2001).
Chitin is a representative MAMP derived from fungal cell
walls and known to induce immune responses in various plants.
Plant Cell Physiol. 53(10): 1696–1706 (2012) doi:10.1093/pcp/pcs113, available online at www.pcp.oxfordjournals.org
! The Author 2012. Published by Oxford University Press on behalf of Japanese Society of Plant Physiologists.
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Chitin receptor in Arabidopsis and rice
We recently identified two types of plasma membrane
(glyco)proteins, OsCEBiP (Kaku et al. 2006) and OsCERK1
(Shimizu et al. 2010) in rice, and AtCERK1 (Miya et al. 2007)
in Arabidopsis as the essential components for chitin perception and signaling in these plants (herein, we designate the rice
CEBiP as OsCEBiP, Arabidopsis CERK1 as AtCERK1 and rice
CERK1 as OsCERK1 to avoid any confusion). OsCEBiP belongs to
the group of receptor-like proteins (Zhang et al. 2009) whereas
AtCERK1/OsCERK1 are receptor kinases. Ectodomains (EDs) of
these molecules contain a lysin motif (LysM), which has been
thought to bind to chitin. Knockdown of either OsCEBiP or
OsCERK1 resulted in the impairment of chitin-induced defense
responses in rice, indicating that both of these molecules are
required for chitin signaling (Shimizu et al. 2010). In addition, it
was observed that the addition of chitin oligosaccharides
induced transient hetero-oligomerization of OsCEBiP and
OsCERK1, indicating that these two molecules cooperatively
regulate chitin responses through the formation of a receptor
complex (Shimizu et al. 2010).
On the other hand, it is not clear whether CEBiP-like molecules are also involved in chitin perception/signaling in
Arabidopsis, though the presence of CEBiP homologs in the
Arabidopsis genome has been indicated (Zhang et al. 2009,
Willmann et al. 2011). Interestingly, Willmann et al. recently
reported that two of three Arabidopsis CEBiP homologs,
LYM1 and LYM3, are involved in the perception/signaling of
bacterial peptidoglycan together with AtCERK1, indicating the
presence of a two-component receptor system similar to the
rice chitin receptor OsCEBiP and OsCERK1 (Shimizu et al.
2010). This finding also showed that AtCERK1 is involved in
the perception of multiple MAMP molecules, chitin and peptidoglycan in Arabidopsis.
In this study, we tried to clarify whether the chitin receptor
system in Arabidopsis also requires CEBiP-like binding protein
similarly to the rice chitin receptor. Our results indicated, rather
unexpectedly, that the chitin receptor system in Arabidopsis is
significantly different from that of rice. In Arabidopsis, chitin
signaling does not require a CEBiP-like molecule and AtCERK1
itself seems enough for chitin perception/signaling, though an
Arabidopsis CEBiP homolog is biochemically fully functional as
a chitin-binding protein. Studies of the chitin binding properties of At/OsCERK1 and the chimeric receptors consisting of
EDs/cytosolic domains of these molecules indicated that the
differences in the properties of their EDs caused such a difference, ‘all-in-one’ or multiple component receptors for chitin
signaling in these plants.
Results
AtCEBiP, an ortholog of rice OsCEBiP, specifically
binds chitin oligosaccharides
For the identification of an Arabidopsis CEBiP homolog, we first
searched TAIR (The Arabidopsis Information Resource) website
with a keyword ‘LysM’ and found 14 Arabidopsis genes as
LysM-containing protein genes. Among them, three genes,
At1g21880 (LYM1), At2g17120 (LYM2) and At1g77630 (LYM3),
encoded LysM proteins with higher sequence similarity to
OsCEBiP than other genes. These three genes were also reported to form a distinct group of LysM receptor-like proteins
based on the systemic analysis of related LysM proteins (Zhang
et al. 2007). Thus, these three genes were thought to be the
candidates of functional CEBiP homologs in Arabidopsis and
were analyzed in the present study. These proteins were also
shown to be glycosylphosphatidylinositol (GPI)-anchored,
plasma membrane proteins by proteomic studies (Borner
et al. 2003, Elortza et al. 2003, Marmagne et al. 2007).
To clarify whether these Arabidopsis CEBiP homologs have
an ability to bind chitin oligosaccharides, tobacco BY-2 cells
were used for heterologous expression of these proteins. The
tobacco BY-2 cells used in this study were known to lack chitin
elicitor responsiveness as well as CEBiP-like binding proteins in
the plasma membrane, indicating their usefulness for the characterization of CEBiP-like proteins (Okada et al. 2002, Shinya
et al. 2006). For the validation of the expression system,
OsCEBiP was expressed in the BY-2 cells and analyzed for
chitin binding activity (Supplementary Fig. S1A). OsCEBiP expressed in the BY-2 cells showed a slightly lower molecular
weight on SDS–PAGE compared with OsCEBiP in rice membrane preparations. Such a difference in the molecular weight
seems to be explained by the different glycosylation between
the BY-2 cells and rice, as OsCEBiP was reported to be a highly
glycosylated protein (Kaku et al. 2006). The binding characteristics of OsCEBiP expressed in the BY-2 cells were evaluated by
analyzing the concentration dependency of the binding of biotinylated (GlcNAc)8 (GN8-Bio; Shinya et al. 2010) and its inhibition by various oligosaccharides. Binding of GN8-Bio to the
microsomal membrane was dependent on the ligand concentration and saturated within the range of 200–250 nM
(Supplementary Fig. S1B, C). The inhibitory potency of the
N-acetylchitooligosaccharides for the binding was dependent
on the size of the oligosaccharides, which is in good agreement
with the previous results obtained with the microsomal membrane from rice cultured cells (Supplementary Fig. S1D; Shinya
et al. 2010). Thus, the OsCEBiP expressed in BY-2 cells had
similar binding characteristics to the native OsCEBiP obtained
from rice cells, indicating that the expression system can be
applied for the analysis of Arabidopsis CEBiP homologs.
Using the BY-2 expression system and affinity labeling with
GN8-Bio, the three Arabidopsis CEBiP homologs were characterized for their chitin binding activity. Among them, the
microsomal membrane from the LYM2-expressing BY-2 cells
showed the presence of a high-affinity binding protein for
chitin oligosaccharides (Fig. 1A). LYM2 also showed the highest
sequence similarity to OsCEBiP and thus was designated as
AtCEBiP (Supplementary Fig. S2). AtCEBiP expressed in the
BY-2 cells showed a lower molecular weight compared with the
size of OsCEBiP expressed in the BY-2 cells (Fig. 1A), again most
possibly reflecting the difference in their glycosylation. On the
other hand, no GN8-Bio-tagged band was detected in the
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A
B
Fig. 1 Binding activity of three Arabidopsis CEBiP homologs expressed in tobacco BY-2 cells to chitin oligosaccharides. (A) AtCEBiP (LYM2)
showed specific binding to chitin oligosaccharides. Microsomal membranes (50 mg of protein) from tobacco BY-2 cells expressing the Arabidopsis
CEBiP homologs were mixed with GN8-Bio (0.4 mM) in the presence/absence of 40 mM unlabeled GN8 as a competitor and cross-linked with EGS.
Biotinylated proteins were detected with anti-biotin antibody after SDS–PAGE. The microsomal membranes from the OsCEBiP-expressing and
non-transformed (NT) BY-2 cells were used as a positive and negative control, respectively. (B) Relative expression of Arabidopsis CEBiP homolog
genes in each transformant. The expression levels of CEBiP homologs (relative to the expression of LYM2 in line #5 as 100) in these transformants
were comparable as proved by the quantitative RT–PCR with a primer set for the consensus sequence located at the 50 -non-coding region of
transcripts.
microsomal membrane from the LYM1- and LYM3-expressing
lines, although the expression levels of the transgenes in these
cell lines were similar to those of AtCEBiP and OsCEBiP in the
corresponding BY-2 cells (Fig. 1A, B). Recently, Willmann et al.
(2011) reported that LYM1 and LYM3 bind peptidoglycan but
the binding was not inhibited by chitin oligosaccharide, which
supports the inability of these proteins to bind chitin oligosaccharides. Further studies on the binding characteristics of
AtCEBiP showed that the binding of GN8-Bio to AtCEBiP was
saturated within the range of 200–300 nM (Fig. 2A, B) and
inhibited size dependently by a series of N-acetylchitooligosaccharides (Fig. 2C), whereas chitosan octasaccharide, (GlcN)8,
did not inhibit the binding of GN8-Bio (Fig. 2D). These characteristics indicated that AtCEBiP is biochemically very similar
to OsCEBiP.
AtCEBiP does not contribute to chitin signaling in
Arabidopsis, though it does represent the cell
surface binding site for chitin oligosaccharides
To analyze further the roles of these CEBiP homologs in chitin
signaling, we characterized their knockout mutants for GN8
binding and GN8-induced defense responses (Fig. 3A, B;
Supplementary Fig. S3). Although we could not detect the
presence of GN8-binding protein in Arabidopsis membrane
in a previous study (Miya et al. 2007), optimization of the
affinity labeling experiments enabled the detection of a
GN8-binding protein in the microsomal membrane of wildtype Arabidopsis (Supplementary Fig. S3D). The weaker
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band detected in the Arabidopsis membrane compared with
the band of OsCEBiP in the rice membrane indicated that the
amount of GN8-binding protein in Arabidopsis is much less
compared with rice. Importantly, the biotinylated band had
disappeared in the knockout mutant of AtCEBiP, whereas the
weak band was still detected in the mutants for two other
CEBiP homologs and the cerk1 mutant. These results indicated
that AtCEBiP is really a major GN8-binding protein in the
Arabidopsis membrane.
On the other hand, reactive oxygen specied (ROS) generation induced by GN8 was not impaired in the AtCEBiP-knockout (KO) mutant, similarly to LYM1-KO and LYM3-KO mutants
(Fig. 3A). Sensitivity to the chitin oligosaccharide, GN8, also did
not change (Fig. 3B). Moreover, AtCEBiP/LYM1/LYM3 tripleKO mutants also responded to GN8 normally for ROS generation and defense gene expression (Fig. 3C, D). These results
were quite different from the situation reported for rice chitin
receptor and Arabidopsis peptidoglycan receptor where the
absence of corresponding binding proteins, OsCEBiP or
LYM1/3, resulted in the suppression of cellular responses
induced by these MAMP molecules (Kaku et al. 2006,
Willmann et al. 2011).
As the expression level of AtCEBiP seemed much less compared with that of OsCEBiP (Supplementary Fig. S3D), we
speculated that the amount of AtCEBiP in the plasma membrane might not be enough for chitin signaling. To examine
such a possibility, AtCEBiP-overexpressing Arabidopsis plants
(AtCEBiPox) were established and analyzed for chitin response.
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Chitin receptor in Arabidopsis and rice
α-biotin
A
GN8-Bio 0
(nM)
50
75
100
150
200
250
300
competitor
(μ
μM)
(GlcNAc)8
B
Specific ligand binding (%)
C
(GlcNAc)5
100
(GlcNAc)3
80
α-biotin
60
D
40
competitor
20
0
0
100
200
α-biotin
300
Ligand concentration (nM)
Fig. 2 Binding characteristics of AtCEBiP to chitin oligosaccharides. (A) Saturation of the binding of GN8-Bio to AtCEBiP. Varying amounts of
GN8-Bio were mixed with the microsomal membrane (25 mg of protein) expressing AtCEBiP and cross-linked with EGS. (B) Intensities of the
biotinylated bands were quantified using Scion Image (Scion). Specific ligand binding to the AtCEBiP was calculated by subtracting the background intensity for the samples incubated in the presence of unlabeled GN8. Ligand binding was expressed as the percentage of the binding at
saturation. (C) Inhibition of GN8-Bio binding to AtCEBiP by chitin oligosaccharides. Binding of GN8-Bio (0.4 mM) to AtCEBiP was analyzed in the
presence of varying amounts of chitin oligosaccharides. (D) Specificity of the binding. Binding of GN8-Bio (0.4 mM) to AtCEBiP was analyzed in
the presence of GN8 (40 mM) or chitosan octasaccharide, (GlcN)8 (40 mM).
Two AtCEBiP-overexpressing lines showed the presence of
GN8-binding protein in their membrane preparations
(Fig. 3E, Supplementary Fig. S4), where the amount of
GN8-binding protein seemed even higher than OsCEBiP in
the rice membrane (Fig. 3E). However, the chitin-induced
ROS generation of the AtCEBiPox line was similar to that of
the wild-type Col-0, in which the amount of AtCEBiP was much
less compared with the overexpressors (Fig. 3E, F). Sensitivity to
GN8 was also not affected by AtCEBiP overexpression (Fig. 3F).
Overall, these results indicated that AtCEBiP does not contribute to chitin signaling, though it represents a major binding site
for GN8 in the Arabidopsis membrane.
Different properties of the ectodomains resulted
in an ‘all-in-one’ or multiple component chitin
receptor in Arabidopsis and rice
The observation that AtCEBiP does not contribute to chitin
signaling, though it represents a major chitin oligosaccharidebinding protein in the Arabidopsis membrane, led us to consider the possibility that AtCERK1 receptor kinase might serve
for both chitin perception and signaling. We thus compared
chitin binding properties of AtCERK1 and OsCERK1 to clarify
their function in chitin perception and signaling in these plants.
For this purpose, we transiently expressed At/OsCERK1HaloTag proteins, which were comprised of At/OsCERK1 ED,
transmembrane (TM) and juxtamembrane (JM) regions and
HaloTag (Promega) at the C-terminus, in Nicotiana benthamiana. Similar ED–TM–JM–HaloTag proteins were previously
used to prove CLV1–CLV3 interaction (Ogawa et al. 2008).
The HaloTag proteins solubilized from the microsomal membranes from the N. benthamiana leaves were used for a binding
assay with colloidal chitin (Fig. 4A). The results of the binding
assay showed that AtCERK1-HaloTag specifically binds to
chitin, confirming the previously reported results (Iizasa et al.
2010, Petutschnig et al. 2010). Direct binding of chitin oligosaccharides to AtCERK1 was very recently shown by the
co-crystallization of AtCERK1 with (GlcNAc)5 as well as the
binding studies with isothermal titration calorimetry
(Liu et al. 2012). These results seemed to support the possibility
that AtCERK1 serves for both chitin perception and signaling.
On the other hand, OsCERK1-HaloTag did not bind to colloidal chitin (Fig. 4A), again suggesting the difference between
rice and Arabidopsis. A difference in the function of At/
OsCERK1 was also suggested from the complementation
experiments of the cerk1 mutant with these genes. In the complementation experiments, OsCERK1 could not recover the
chitin-induced ROS generation in the cerk1 mutant, whereas
AtCERK1 completely recovered it (Fig. 4B; Supplementary
Fig. S5). As the differences in the chitin binding properties
between the ectodomains of At/OsCERK1 may explain such a
difference between AtCERK1 and OsCERK1 in complementing
the cerk1 mutant, we further compared the function of these
ectodomains in chitin signaling using the complementation
experiments with chimeric proteins consisting of each other’s
ED and kinase domain (KD). A chimeric construct encoding
AtCERK1-ED–OsCERK1-KD where the ED of OsCERK1 was
replaced by the ED of AtCERK1, partly recovered chitin-induced
ROS generation in the cerk1 mutant in the complementation
experiment (Fig. 4B; Supplementary Fig. S5). On the other
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Fig. 3 Chitin responses of knockout mutants and overexpressors of Arabidopsis CEBiP homologs. (A) ROS generation of lym mutants induced by
GN8. ROS generation was measured at 60 min after the treatment with 10 mg ml1 GN8. Data are shown as the average of 10 seedlings ± SD.
(B) ROS generation of a lym2 (AtCEBiP-KO) mutant induced by different concentrations of GN8. ROS generation was measured similarly to (A).
Similar results were also obtained with another mutant line (lym2-3). (C) ROS generation of a triple KO mutant for LYM1-3 (lym1-1/lym2-1/
lym3-1) induced by GN8. ROS generation was measured similarly to (A), except that nine seedlings were used for each experiment. (D) Expression
of defense-related genes induced by GN8 in a triple KO mutant for LYM1-3. Gene expression was analyzed 30 min after the treatment with
100 mg ml1 GN8. Data are the average of three independent biological replicates ± SD. (E) Detection of CEBiP-like proteins in the membrane
preparation from AtCEBiPox lines by affinity labeling. Microsomal membranes (10 mg of protein) from two AtCEBiPox lines were mixed with
GN8-Bio (0.8 mM) and cross-linked with EGS. The results with wild-type Arabidopsis (Col-0) and rice cells are shown for comparison. (F) ROS
generation of the AtCEBiP/LYM2ox line induced by GN8. ROS generation was measured at 30 min after treatment with GN8.
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Chitin receptor in Arabidopsis and rice
A
B
Fig. 4 Functional analysis of the ectodomains of AtCERK1 and OsCERK1. (A) Chitin binding properties of the ectodomains of At/OsCERK1.
AtCERK1-HaloTag and OsCERK1-HaloTag were expressed in N. benthamiana and used for binding experiments with colloidal chitin. Microsomal
membranes expressing the Halo-tagged proteins were solubilized with Triton X-100 and the resulting solutions were incubated with colloidal
chitin in the presence or absence of chitin (chitosan) oligosaccharides (1 mM). After separating the colloidal chitin by centrifugation, Halo-tagged
proteins in each fraction were detected by Western blotting with anti-HaloTag antibody. F, Flow-through; B, fraction bound to colloidal chitin; I,
input solution. (B) Functional analysis of the ectodomains by using a chimeric receptor approach. Chimeric constructs, OsCERK1-ED-TM–
AtCERK1-JM-KD (Chimera1) and AtCERK1-ED-TM–OsCERK1-JM-KD (Chimera2) were expressed in the AtCERK1-KO mutant (cerk1-2) and
evaluated for 10 mg ml1 GN8-induced ROS generation (0–30 min). AtCERK1 and OsCERK1 were also expressed in the cerk1 mutant and
examined similarly. An asterisk indicates a significant difference at P < 0.03. Data are shown as the average of nine seedlings ± SD.
Plant Cell Physiol. 53(10): 1696–1706 (2012) doi:10.1093/pcp/pcs113 ! The Author 2012.
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hand, another chimeric construct consisting of OsCERK1 ED
and AtCERK1 KD, OsCERK1-ED–AtCERK1-KD, did not complement the cerk1 mutant for chitin-induced ROS generation,
again indicating the importance of the ED for ligand recognition
and signaling in AtCERK1. These results were confirmed with
two independent transgenic lines, which showed much higher
expression of the transgenes compared with the expression of
the AtCERK1 gene in wild-type Col-0 plants (Supplementary
Fig. S5).
Discussion
The present study clearly indicated that rice and Arabidopsis
exploit different types of receptors for chitin signaling. The results obtained with the single/triple KO mutants and the overexpressor of AtCEBiP clearly indicated that AtCEBiP does not
contribute to chitin signaling in Arabidopsis, though it binds
chitin oligosaccharides with high affinity similar to OsCEBiP
that plays an important role in chitin signaling in rice.
Although we cannot exclude the possibility that some LysM
receptor-like proteins other than AtCEBiP are involved in chitin
signaling, it is difficult to imagine such a possibility because (i)
only AtCEBiP showed a high affinity binding to chitin oligosaccharides among the three tested LysM proteins which have
been recognized as closely related homologs of OsCEBiP
based on phylogenetic studies and (ii) the results of affinity
labeling with the microsomal membrane preparation indicated
that AtCEBiP is the major chitin-binding protein in the membrane. The fact that AtCERK1, which is essential for chitin signaling in Arabidopsis, binds chitin whereas OsCERK1 does not
indicated the difference between the chitin receptor systems in
rice and Arabidopsis from different aspects. These results also
indicated that the receptor kinase AtCERK1 serves both for
perception and transduction of chitin oligosaccharides in
Arabidopsis. The fact that the chimeric receptor consisting of
the AtCERK1 ED and the OsCERK1 KD partly recovered the
chitin response in the cerk1 mutant whereas OsCERK1 itself did
not, indicated the functionality of the AtCERK1 ED for the
perception of chitin oligosaccharides in the receptor kinase,
supporting the above notion. We think the limited recovery
of chitin response in the chimeric receptor was caused by the
incomplete fit of the OsCERK1 KD to the downstream signaling
system in Arabidopsis. A very recent finding that indicates the
homodimerization of AtCERK1 by (GlcNAc)8 also strongly supports such a possibility (Liu et al. 2012). Differences in the predicted number of LysMs in the ectodomains of AtCERK1 and
OsCERK1, three and one, respectively (Shimizu et al. 2010), may
give a clue to understanding the difference in the chitin binding
of these receptor kinases, as the deletion experiments indicated
the requirement of all three LysMs in AtCERK1 for high-affinity
chitin binding (Petutschnig et al. 2010).
We thus propose that the Arabidopsis chitin receptor consists of only AtCERK1 and the rice chitin receptor is a multicomponent receptor consisting of OsCEBiP and OsCERK1. If so,
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which receptor system is more general in plants? It is difficult to
answer this question at present but the fact that CEBiP-like
chitin-binding proteins were detected in the plasma membranes from various plants by affinity labeling (Okada et al.
2002), in contrast to the difficulty we originally encountered
for the detection of AtCEBiP in a similar experiment, may indicate the more general presence of complex-type receptor
systems similar to rice. A recent finding that a barley homolog
of CEBiP, HvCEBiP, contributes to basal resistance in barley
seems to support such a situation (Tanaka et al. 2010).
It seems rather strange that a biochemically functional
receptor-like protein such as AtCEBiP does not function in
chitin signaling. One possibility is that Arabidopsis abandoned
the use of a CEBiP-like molecule for chitin signaling along with
the evolution of AtCERK1. The presence of much lower level of
AtCEBiP protein in Arabidopsis compared with OsCEBiP in rice
may reflect the fact that AtCEBiP has been becoming a useless
molecule like a cecum in mammals. The reverse situation might
also be imagined. A multicomponent receptor system such as
OsCEBiP–OsCERK1 represents a more advanced system for signaling, though we could not find evidence that the multicomponent system in rice is more efficient for chitin signaling
compared with that of Arabidopsis. Finally, we cannot exclude
the possibility that AtCEBiP may have some still unidentified
function in plant immunity rather than chitin signaling, though
it is difficult to imagine what it is at present.
The recent finding that both LYM1 and LYM3, two other
CEBiP homologs of Arabidopsis, bind bacterial peptidoglycan
and serve for defense signaling together with AtCERK1
(Willmann et al. 2011) sheds new light on the function of
AtCERK1 and LysM receptor-like proteins in Arabidopsis.
These results indicated the presence of a two-component receptor system similar to the rice chitin receptor, OsCEBiP and
OsCERK1, in Arabidopsis. These results also showed that
AtCERK1 serves both for chitin and peptidoglycan signaling,
though AtCERK1 seems to contribute differently for signaling
in these systems. In the case of peptidoglycan signaling, the
binding proteins LYM1 and LYM3 not only bind ligand but
also contribute to the activation of AtCERK1 and downstream
signaling, similarly to the function of OsCEBiP in rice. On the
other hand, in the case of chitin signaling, AtCERK1 seems to
function for both ligand perception and signaling as discussed.
It is still unclear, however, how the ligand binding to LYM1/3 or
AtCERK1 leads to the activation of each receptor system,
though the ligand-dependent receptor complex formation for
OsCEBiP–OsCERK1 (Shimizu et al. 2010) and also autophosphorylation (Petutschnig et al. 2010) of AtCERK1 were
reported as initial steps of receptor activation.
Until now, several types of receptor systems have been identified for MAMP recognition in plant immunity. For example,
OsCEBiP–OsCERK1 for chitin recognition in rice and LYM1/3–
AtCERK1 for peptidoglycan recognition in Arabidopsis represent a multicomponent receptor system consisting of a
ligand-binding membrane protein and an RD receptor kinase
that carries a characteristic ‘RD’ sequence in the active site of
Plant Cell Physiol. 53(10): 1696–1706 (2012) doi:10.1093/pcp/pcs113 ! The Author 2012.
Chitin receptor in Arabidopsis and rice
the KD. FLS2–BAK1 and EFR–BAK1 for flagellin and EF-Tu recognition in Arabidopsis respectively, represent another multicomponent receptor system consisting of a ligand-binding,
non-RD receptor kinase and an RD receptor kinase. In these
multicomponent receptor systems, RD kinases such as At/
OsCERK1 and BAK1 have been indicated to have higher
kinase activity than the non-RD kinases and seem to play a
critical role in membrane signaling, as the lack of the kinase
activity of these RD kinases resulted in the suppression of
downstream responses (Schwessinger and Ronald 2012).
XA21, a receptor for Ax21, a conserved protein among
Xanthomonas bacteria, is known to bind a sulfated peptide
from AX21 and carry a cytoplasmic KD, indicating that it also
serves for both ligand perception and signaling similarly to
AtCERK1. However, the fact that XA21 carries a non-RD KD
similar to FLS2 and EFR may suggest the possibility that XA21
requires some unidentified RD kinase for full activation of defense responses (Schwessinger and Ronald 2012). On the other
hand, AtCERK1 seems to be unique as it serves both for ligand
perception and membrane signaling using the ED and RD KD
for chitin signaling but serves as a signaling molecule for a
multicomponent receptor system for peptidoglycan signaling
in Arabidopsis.
Intriguing questions remain as how common such an
‘all-in-one’ receptor system is in plant immune signaling
and also how AtCERK1 is activated when it functions as an
‘all-in-one’ molecule or a signaling unit of a multicomponent
receptor system.
Materials and Methods
Plant materials and growth conditions
Arabidopsis seeds were surface-sterilized and germinated on
Petri plates containing MGRL nutrients supplemented with
0.1% agarose and 1% sucrose, as reported (Albert et al. 2006).
Seedlings were grown for 8–10 d in a 16/8 h light/dark cycle at
22 C (light) or 15 C (dark) and used for elicitor treatment.
Arabidopsis plants were grown on soil in a growth chamber
at 22 C under a 12 h light–dark cycle (Miya et al. 2007).
Suspension-cultured tobacco BY-2 cells were maintained in
a modified N-6 medium as described previously (Shinya
et al. 2007).
The KO mutants of LysM proteins, lym1-1 and lym3-1, were
kindly provided by Thorsten Nürnberger (ZMBP, University
of Tübingen). The Ds-transposon insertion mutants,
lym2-2, lym2-3 and lym1-2, were obtained from the RIKEN
BioResource Center. The AtCERK1-KO mutant cerk1-2
(096F09) was obtained from GABI-Kat (Miya et al. 2007). The
T-DNA insertion mutant for lym2-1 and the seeds of ecotype
Nossen were obtained from the Arabidopsis Biological Resource
Center. To confirm the knockout of the target genes by reverse
transcription–PCR (RT–PCR), total RNA was extracted from
the Arabidopsis seedlings using an RNeasy Plant Mini kit
(Qiagen). Approximately 10–12 seedlings per treatment were
combined to obtain an ppropriate amount of total RNA.
For two-step RT–PCR, first-strand cDNA synthesis was performed using 1 mg of total RNA and a QuantiTect Reverse
Transcription Kit (Qiagen). PCR was performed with Takara
Ex Taq (TAKARA) using the gene-specific primers described
in Supplementary Table S1. The PCR amplicons were detected
with the MultiNA microchip electrophoresis system
(Shimadzu).
Generation of tobacco BY-2 cells expressing
Arabidopsis CEBiP homologs
Using full-length cDNAs as a template, the open reading frames
(ORFs) of Arabidopsis CEBiP homologs were amplified by PCR
by using the corresponding primer sets (Supplementary
Table S1). These fragments were subcloned into pENTR/
D-TOPO (Invitrogen) and then inserted into a binary vector
pMDC32 (Curtis and Grossniklaus 2003) by using the LR reaction (Invitrogen). The plasmids were then transformed into
Agrobacterium tumefaciens C58C1 by electroporation.
Tobacco BY-2 cells were co-cultivated with the transformed
A. tumefaciens for 2 d and the transformed cell lines were
selected on LSD agar medium containing 200 mg ml1 hygromycin and 12.5 mg ml1 cefotaxime sodium for 2–3 weeks.
Selected cell lines were transferred into LSD liquid medium
and used for the preparation of the microsomal fraction for
the binding assay. The expression level of each gene was measured by real-time quantitative RT–PCR using a primer set for
the consensus sequence located at the 50 -non-coding region of
transcripts from each plasmid (Supplementary Table S1). 18S
rRNA was used as an internal control for real-time quantitative
RT–PCR (Supplementary Table S1). OsCEBiP-expressing cell
lines were also established similarly.
Generation of transgenic Arabidopsis plants
To generate transgenic Arabidopsis overexpressing AtCEBiP,
flowering Arabidopsis plants (Col-0) were infected by A. tumefaciens carrying the expression vector of AtCEBiP by the
floral-dip method. The AtCEBiP expression levels in the overexpression lines were analyzed by real-time quantitative
RT–PCR using the primer set for AtCEBiP. Actin was used as
an internal control (Supplementary Table S1).
Expression vectors for complementation experiments with
the cerk1 mutant were constructed as follows. Subcloned
OsCERK1 in pENTR/D-TOPO (Shimizu et al. 2010) was inserted
into binary vector pMDC32. The vectors of chimeric receptors
were prepared according to previously described procedures
(Kishimoto et al. 2010). AtCERK1-ED-OsCERK1-KD, which contained the AtCERK1 ED and TM domain (1–762 bp of the ORF)
and the OsCERK1 KD (787–1,875 bp of the ORF), was subcloned into pENTR/D-TOPO and then inserted into
pMDC32. The expression vector for OsCERK1-ED-AtCERK1KD, which contained the OsCERK1 ED and TM domain
(1–786 bp of the ORF) and the AtCERK1 KD (763–1,854 bp of
the ORF), was constructed similarly. These expression vectors
Plant Cell Physiol. 53(10): 1696–1706 (2012) doi:10.1093/pcp/pcs113 ! The Author 2012.
1703
T. Shinya et al.
were introduced into cerk1 mutant plants as described.
The expression level of each gene was measured by real-time
quantitative RT–PCR using the primer set for the consensus
sequence located at the 50 -non-coding region of transcripts
from each plasmid (Supplementary Table S1).
Analysis of defense responses
The Arabidopsis seedlings were pre-incubated in fresh MGRL
medium in a 48-well microtiter plate for 2 h and then GN8 was
added. The ROS released by Arabidopsis seedlings were quantified by the luminol chemiluminescence method (Albert et al.
2006). Chemiluminescence counts of samples were corrected
for the background by subtracting the value of the water blank.
The data are presented as a means of 9–12 seedlings ± SD.
For the detection of defense gene expression, real-time
quantitative RT–PCR was performed using the synthesized
cDNA as a template according to the manufacturer’s protocol
(Life Technologies). The gene-specific primer sets used for the
experiments were described in Supplementary Table S1. Actin
was used as an internal control.
by inverse PCR with the primer set A for AtCERK1 or primer set
B for OsCERK1 (Supplementary Table S1) and the corresponding full-length cDNA as a template, by using the In-Fusion
Cloning Kit (TAKARA). Both PCR products encoding the
HatoTag and AtCERK1(ED–TM–JM) sequences were treated
with the Cloning Enhancer (TAKARA) before the infusion cloning reaction. The resulting AtCERK1(ED-TM-JM)-HaloTag
(abbreviated as AtCERK1-HaloTag) and OsCERK1(ED-TM-JM)HaloTag (abbreviated as OsCERK1-HaloTag) subcloned into
pENTR/D-TOPO were first digested with EcoRV or NruI to
disable the kanamycin resistance gene in AtCERK1-HaloTag or
OsCERK1-HaloTag, then transferred to a binary vector
pEAQ-DEST1 (Sainsbury et al. 2009) by using the LR reaction
(Invitrogen). The expression vectors thus obtained were transformed into A. tumefaciens LBA4404 by electroporation.
To express the Halo-tagged AtCERK1 or OsCERK1 in
N. benthamiana, the transformed A. tumefaciens was pressure-infiltrated into N. benthamiana leaves as reported by
Sainsbury et al. (2009). The leaves were collected at 6 d after
infiltration.
Affinity labeling with biotinylated-GN8
The microsomal membrane fraction was prepared from the
BY-2 cells harvested 6–7 d after transfer to the new medium
or seedling (Shibuya et al. 1993). GN8-Bio, the conjugate of
biocytin hydrazide and N-acetylchitooctaose, was prepared by
reductive amination (Shinya et al. 2010). Affinity labeling
of GN8-Bio was performed as described previously (Shinya
et al. 2010). The microsomal membrane preparation was
mixed with 0.4 mM GN8-Bio and adjusted to 30 ml with binding
buffer. After incubation for 1 h on ice, 3 ml of 3% EGS (ethylene
glycol bis[succinimidylsuccinate]) solution was added to the
mixture and left to stand for 30 min. The reaction was stopped
by the addition of 1 M Tris–HCl, mixed with SDS–PAGE sample buffer, boiled for 5 min, and used for SDS–PAGE.
Western blotting was performed on an Immun-Blot PVDF
Membrane (Bio-Rad). Detection of biotinylated proteins was
performed by using a rabbit antibody against biotin (Rockland
or Bethyl) as a primary antibody and horseradish peroxidaseconjugated goat anti-rabbit IgG (Chemicon) as a secondary
antibody. Biotinylated proteins were detected by chemiluminescence with Immobilon Western Detection reagents
(Millipore).
Expression of Halo-tagged AtCERK1 and OsCERK1
in N. benthamiana
For the construction of expression vector for At/OsCERK1HaloTag, HaloTag was inserted into the C-terminal region of
At/OsCERK1(ED–TM–JM) in pENTR/D-TOPO as follows. The
DNA fragment encoding the HaloTag was amplified from the
pFN18K vector (Promega) with the primer set C for AtCERK1 or
primer set D for OsCERK1, respectively (Supplementary
Table S1). The PCR product was ligated into the At/
OsCERK(ED–TM–JM) in pENTR/D-TOPO, which was amplified
1704
Measurement of chitin binding activity using
colloidal chitin
For the binding assay with colloidal chitin, microsomal membrane fractions were prepared from the AtCERK1-HaloTag- or
OsCERK1-HaloTag-expressing leaves (Shibuya et al. 1993). To
solubilize the membrane proteins, the microsomal membrane
was suspended with phosphate-buffered saline (PBS) containing 0.5% Triton X-100, 1 mM phenylmethylsulfonylfluoride
(PMSF) and 2 mM dithiothreitol (DTT), and kept overnight
on a shaker at 450 r.p.m. at 4 C. The suspension was ultracentrifuged at 100,000g for 30 min at 4 C. The solubilized protein
fraction was mixed with colloidal chitin, kept on ice for 2 h, and
centrifuged at 16,000g at 4 C. The supernatant contained
most of the proteins not bound to the colloidal chitin and
was named as the ‘flow-through’ fraction. The precipitate
was washed three times with PBS and then eluted with SDS–
PAGE loading buffer, giving the ‘bound’ fraction. Halo-tagged
proteins in each fraction were detected by Western blotting
with anti-HaloTag antibody (Promega). Colloidal chitin was
prepared as described previously (Huynh et al. 1992, Hon
et al. 1995).
Supplementary data
Supplementary data are available at PCP online.
Funding
This work was supported by the Program for Promotion of
Basic Research Activities for Innovative Biosciences
(PROBRAIN); the Ministry of Education, Culture, Sports,
Plant Cell Physiol. 53(10): 1696–1706 (2012) doi:10.1093/pcp/pcs113 ! The Author 2012.
Chitin receptor in Arabidopsis and rice
Science and Technology, Japan [grants No. 22248041 to N.S.,
No. 22570052 to H.K. and No. 24780334 to T.S.].
Acknowledgments
We thank Dr. Naoto Kawakami of Meiji University for the
valuable advice on generating the triple KO mutant. We
thank Drs. Hisakazu Yamane, Kazunori Okada and Koji
Miyamoto of University of Tokyo for the help in the heterogeneous expression experiments with N. benthamiana. We thank
Dr. Thorsten Nürnberger of University of Tübingen for the kind
donation of lym1-1 and lym3-1seeds. We are grateful to Dr.
Mark Curtis of University of Zurich and Dr. George
Lomonossoff of John Innes Center for pMDC32 and
pEAQ-DEST1 vectors, respectively. We thank Dr. Yoshitake
Desaki, Mr. Tomohiko Osada, Mr. Takuto Nakano, Mr.
Yosuke Sato, Mr. Takumi Tanimoto, Ms. Hikaru Shimada, Mr.
Maruya Suzuki and Mr. Masatoshi Shibuya of our laboratory for
their assistance in the experiments.
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