RAR1 and HSP90 form a complex with Rac/Rop GTPase and function in innate-immune responses in rice.
ABSTRACT A rice (Oryza sativa) Rac/Rop GTPase, Os Rac1, is involved in innate immunity, but its molecular function is largely unknown. RAR1 (for required for Mla12 resistance) and HSP90 (a heat shock protein 90 kD) are important components of R gene-mediated disease resistance, and their function is conserved in several plant species. HSP90 has also recently been shown to be important in mammalian innate immunity. However, their functions at the molecular level are not well understood. In this study, we examined the functional relationships between Os Rac1, RAR1, and HSP90. Os RAR1-RNA interference (RNAi) rice plants had impaired basal resistance to a compatible race of the blast fungus Magnaporthe grisea and the virulent bacterial blight pathogen Xanthomonas oryzae. Constitutively active Os Rac1 complemented the loss of resistance, suggesting that Os Rac1 and RAR1 are functionally linked. Coimmunoprecipitation experiments with rice cell culture extracts indicate that Rac1 forms a complex with RAR1, HSP90, and HSP70 in vivo. Studies with Os RAR1-RNAi and treatment with geldanamycin, an HSP90-specific inhibitor, showed that RAR1 and HSP90 are essential for the Rac1-mediated enhancement of pathogen-associated molecular pattern-triggered immune responses in rice cell cultures. Furthermore, the function of HSP90, but not RAR1, may be essential for their association with the Rac1 complex. Os Rac1 also regulates RAR1 expression at both the mRNA and protein levels. Together, our results indicate that Rac1, RAR1, HSP90, and HSP70 form one or more protein complexes in rice cells and suggest that these proteins play important roles in innate immunity in rice.
- SourceAvailable from: Yoji Kawano[Show abstract] [Hide abstract]
ABSTRACT: Nucleotide-binding domain and leucine-rich repeat (NLR)-containing family proteins function as intracellular immune sensors in both plants and animals. In plants, the downstream components activated by NLR family proteins and the immune response mechanisms induced by these downstream molecules are largely unknown. We have previously found that the small GTPase OsRac1, which acts as a molecular switch in rice immunity, is activated by Pit, an NLR-type resistance (R) protein to rice blast fungus, and this activation plays critical roles in Pit-mediated immunity. However, the sites and mechanisms of activation of Pit in vivo remain unknown. To clarify the mechanisms involved in the localization of Pit, we searched for consensus sequences in Pit that specify membrane localization and found a pair of potential palmitoylation sites in the N-terminal coiled-coil region. Although wild-type Pit was localized mainly to the plasma membrane, this membrane localization was compromised in a palmitoylation-deficient mutant of Pit. The palmitoylation-deficient Pit displayed significantly lower affinity for OsRac1 on the plasma membrane, thereby resulting in failures of the Pit-mediated cell death, the production of reactive oxygen species, and disease resistance to rice blast fungus. These results indicate that palmitoylation-dependent membrane localization of Pit is required for the interaction with and the activation of OsRac1, and that OsRac1 activation by Pit is vital for Pit-mediated disease resistance to rice blast fungus.The Journal of biological chemistry. 05/2014;
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ABSTRACT: Serotonin is a well known neurotransmitter in mammals and plays an important role in various mental functions in humans. In plants, the serotonin biosynthesis pathway and its function are not well understood. The rice sekiguchi lesion (sl) mutants accumulate tryptamine, a candidate substrate for serotonin biosynthesis. We isolated the SL gene by map-based cloning and found that it encodes CYP71P1 in a cytochrome P450 monooxygenase family. A recombinant SL protein exhibited tryptamine 5-hydroxylase enzyme activity and catalyzed the conversion of tryptamine to serotonin. This pathway is novel and has not been reported in mammals. Expression of SL was induced by the N-acetylchitooligosaccharide (chitin) elicitor and by infection with Magnaporthe grisea, a causal agent for rice blast disease. Exogenously applied serotonin induced defense gene expression and cell death in rice suspension cultures and increased resistance to rice blast infection in plants. We also found that serotonin-induced defense gene expression is mediated by the RacGTPase pathway and by the G alpha subunit of the heterotrimeric G protein. These results suggest that serotonin plays an important role in rice innate immunity.Journal of Biological Chemistry 02/2010; 285(15):11308-13. · 4.65 Impact Factor
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ABSTRACT: Rac GTPase is involved in the transduction and regulation of various cellular processes in eukaryotes such as the plant defense system. This regulatory gene has been reported to produce a broad spectrum resistance against pathogens in plants. The purpose of this study is to isolate and characterize the Rac GTPase gene from UKMRC9, a locally developed cultivar that shows tolerance to major rice diseases in Malaysia. Total RNA of infected young rice leaves with low disease index was extracted, reverse transcribed into cDNA and sequenced. The nucleotide sequence was translated into a 307 amino acid Rac GTPase protein and was subjected to in silico analysis to defer protein characteristics, evolutionary relations and structural characteristics. This protein was found to be 99% similar to the Rho GTPase in Oryza sativa and contains actin and actin-like protein domains that have been implicated in cytoskeletal modelling and remodelling in cells with a function in structure and primary defence against host infiltration. The 3D structure of the protein contains a 6 β-sheet structure that is surrounded by helixes, with an insert region and two switches that are characteristic of ATP binding Rac/Rho proteins. As Rac/Rho is diverse in plants, this isolated gene will be used in future mutational and transgenic studies that will enable us to elucidate the functional role of this protein in rice and compare the observed function with the reported functions observed in plants as well as other eukaryotic organism.04/2014;
RAR1 and HSP90 Form a Complex with Rac/Rop GTPase and
Function in Innate-Immune Responses in Rice
Nguyen Phuong Thao,aLetian Chen,aAyako Nakashima,aShin-ichiro Hara,aKenji Umemura,bAkira Takahashi,c,1
Ken Shirasu,c,2Tsutomu Kawasaki,aand Ko Shimamotoa,3
aLaboratory of Plant Molecular Genetics, Nara Institute of Science and Technology, Ikoma 630-0101, Japan
bAgricultural and Veterinary Research Lab, Meiji Seika Kaisha, Kohoku-ku, Yokohama 222-8567 Japan
cSainsbury Laboratory, John Innes Centre, Norwich NR4 7UH, United Kingdom
A rice (Oryza sativa) Rac/Rop GTPase, Os Rac1, is involved in innate immunity, but its molecular function is largely unknown.
RAR1 (for required for Mla12 resistance) and HSP90 (a heat shock protein 90 kD) are important components of R gene–
mediated disease resistance, and their function is conserved in several plant species. HSP90 has also recently been shown
to be important in mammalian innate immunity. However, their functions at the molecular level are not well understood. In
this study, we examined the functional relationships between Os Rac1, RAR1, and HSP90. Os RAR1-RNA interference (RNAi)
rice plants had impaired basal resistance to a compatible race of the blast fungus Magnaporthe grisea and the virulent
bacterial blight pathogen Xanthomonas oryzae. Constitutively active Os Rac1 complemented the loss of resistance, sug-
gesting that Os Rac1 and RAR1 are functionally linked. Coimmunoprecipitation experiments with rice cell culture extracts
indicate that Rac1 forms a complex with RAR1, HSP90, and HSP70 in vivo. Studies with Os RAR1-RNAi and treatment with
geldanamycin, an HSP90-specific inhibitor, showed that RAR1 and HSP90 are essential for the Rac1-mediated enhancement
of pathogen-associated molecular pattern–triggered immune responses in rice cell cultures. Furthermore, the function of
HSP90, but not RAR1, may be essential for their association with the Rac1 complex. Os Rac1 also regulates RAR1 expression
at both the mRNA and protein levels. Together, our results indicate that Rac1, RAR1, HSP90, and HSP70 form one or more
protein complexes in rice cells and suggest that these proteins play important roles in innate immunity in rice.
Plants use two innate immune systems to respond to pathogen
infection, known as pathogen-associated molecular pattern
(PAMP)–triggered innate immunity and effector-triggered immu-
nity (Zipfel and Felix, 2005; Chisholm et al., 2006; Jones and
Dangl, 2006). PAMP-triggered innate immunity is induced by
recognition of PAMPs by transmembrane receptors and gives
early responses to pathogen infection. Effector-triggered immu-
nity involves resistance (R) proteins as receptors that specifically
recognize pathogen effectors, either directly or indirectly, and
usually induces hypersensitive cell death (HR). The major R
proteins are intracellular receptors and contain nucleotide bind-
ing site (NBS) and leucine-rich repeat (LRR) domains. Although
there has been extensive research on plant innate immunity, the
molecular mechanisms of these two immune responses are not
RAR1 (for required for Mla12 resistance) is an important
component of R gene–mediated disease resistance (Shirasu
et al., 1999; Tornero et al., 2002; Muskett et al., 2002), which
contains two zinc binding finger motifs termed CHORD-I and
CHORD-II (Cys- and His-rich domains) (Shirasu et al., 1999). In
plants, RAR1 interacts directly with SGT1 (for suppressor of the
G2 allele of skp1) and HSP90 (Azevedo et al., 2002; Liu et al.,
2002, Takahashi et al. 2003). The CHORD-I domain of RAR1
interacts directly with the N terminus of HSP90, and SGT1 binds
the CHORD-II domain of RAR1. SGT1 is required for disease
resistance mediated by diverse R proteins (Austin et al., 2002;
required for the function of an SCF (for Skp1/Cullin/F-box pro-
tein) (Kitagawa et al., 1999; Matsuzawa and Reed, 2001). HSP90
is an abundant, highly conserved, ATP-dependent molecular
chaperone that is essential for eukaryotic cell viability (Picard,
2002). More recently, RAR1, SGT1, and HSP90 were shown to
play an important role in regulating the stability of R proteins that
contain the NBS-LRR domain (Bieri et al., 2004; Holt et al., 2005;
Azevedo et al., 2006).
HSP90 and SGT1 play a key role in the activation of mamma-
lian immune responses induced by the Nod-like receptor (NLR)
protein family, a group that contains an NBS-LRR domain (Hahn,
2005; da Silva Correia et al., 2007; Mayor et al., 2007). SGT1 and
HSP90 interact with NLR proteins to form an inflammasome
complex and are required for activation of the complex. There-
fore, regulatory networks for immune responses in plants and
mammals share at least some common components, and
1Current address: Department of Molecular Genetics, National Institute
of Agrobiological Sciences, Tsukuba, Ibaraki 305-8602, Japan.
2Current address: RIKEN Plant Science Center, 1-7-22 Suehiro,
Tsurumi, Yokohama 230-0045, Japan.
3Address correspondence to firstname.lastname@example.org.
The author responsible for distribution of materials integral to the
findings presented in this article in accordance with the policy described
in the Instructions for Authors (www.plantcell.org) is: Ko Shimamoto
WOnline version contains Web-only data.
OAOpen Access articles can be viewed online without a subscription.
The Plant Cell, Vol. 19: 4035–4045, December 2007, www.plantcell.org ª 2007 American Society of Plant Biologists
understanding the molecular mechanisms involved in protein
complexes containing R proteins or NLR proteins, SGT1, and
HSP90 is becoming crucial in the study of innate immunity in
Rac/Rop small GTPases are highly conserved in the plant
kingdom and constitute the sole group of Rho family small
GTPases in plants (Gu et al., 2004). There are seven Rac/Rop
thaliana (Guetal.,2004).OsRac1,which islocatedin theplasma
membrane, is involved in ROS production through an NADPH
(Kawasaki et al., 1999; Ono et al., 2001). Os Rac1 functions as a
positive regulator of NADPH oxidase activation in PAMP signal-
ing (Kawasaki et al., 1999) and at the same time suppresses
expression of a scavenger metallothionein (Wong et al., 2004) in
transient accumulation of reactive oxygen species (ROS). Ex-
pression of constitutively active (CA)-OsRac1 enhances resis-
Suharsono et al., 2002), whereas a dominant negative (DN)
version of Os Rac1 (DN-OsRac1) suppresses HR induced by
incompatible races of rice blast fungus, indicating that Os Rac1
is one of the key regulators of rice innate immunity (Ono et al.,
2001). Os Rac1 regulates the stability and activation of Os
MAPK6, a rice mitogen-activated protein kinase, by sphingolipid
elicitors (Lieberherr et al., 2005). Cinnamoyl-CoA reductase, a
key enzyme in lignin biosynthesis, is activated by Rac1 in rice
innate immunity (Kawasaki et al., 2006), and DN-OsRac1 inhibits
N-mediated resistance to tobacco mosaic virus infection in
tobacco (Nicotiana tabacum; Moeder et al., 2005). There are a
number of other studies aimed at understanding the molecular
mechanisms of rice innate immunity (Yang et al., 2004; Gu et al.,
2005; Wangetal.,2006; Takahashietal.,2007),but thedetails of
how these pathways are regulated remain unknown.
GTPase. We show that both RAR1 and HSP90 play a critical role
in rice innate immunity and that they apparently form a complex
with Rac GTPase. Furthermore, loss of HSP90 function impairs
immune responses as well as the formation of a protein complex
with Rac GTPase-RAR1-HSP90. These results indicate a critical
role for the functional protein complex containing Rac GTPase,
RAR1, and HSP90 in rice innate immunity.
Os RAR1 Is Involved in Basal Resistance against Rice Blast
and Bacterial Blight Infection in Rice
RAR1 is required for the function of multiple R genes, such as
Mla, RPM1, RPS2, RPS5, and N genes (Shirasu et al., 1999;
Tornero et al., 2002; Muskett et al., 2002; Austin et al., 2002;
Azevedo etal., 2002;Liu etal.,2002; Holt etal., 2005).Therefore,
we were interested in determining whether RAR1 is involved in
disease resistance in rice.
To investigate the function of RAR1 in rice disease resistance,
we knocked down RAR1 expression with RNA interference
(RNAi) (Figures 1A and 1B; see Supplemental Figure 1A online).
Generally, Os RAR1 expression was greatly decreased in Os
RAR1-RNAi lines (Figure 1; see Supplemental Figure 2 online).
Japonica rice var Kinmaze, which was used for the production of
transgenic plants, carries the Pi-a blast resistance gene that is
incompatible with race 031 but is compatible with race 007 of
Magnaporthe grisea. Two T1 transgenic lines carrying the Os
RAR1-RNAi construct (Figure 1B, lines 18 and 61) showed no
difference in response to infection with race 031 from the wild-
type plant or a T1 segregant without a transgene (Figure 1B, line
15), making it unlikely that Pi-a–mediated blast resistance re-
quires RAR1. To further test for possible Os RAR1 requirements
in R gene–mediated blast resistance, we performed the same
infection analysis using the Os RAR1-RNAi transformants in the
Kanto IL 5 and Kanto IL 14 backgrounds that carry Pi-z and Pi-b
blast resistance genes, respectively. These experiments clearly
showed that RAR1 is not required by either of these two R genes
for the three R genes for rice blast resistance is consistent with
previous results indicating that RAR1 is required for many but not
all R genes examined (Shirasu and Schulze-Lefert, 2003).
To determine whether RAR1 is involved in basal resistance to
virulent blast isolates, Os RAR1-RNAi plants were infected with
compatible race 007. Leaves of untransformed control plants
developed typical disease symptoms (Figures 1C and 1D, race
carried the Os RAR1-RNAi transgene had larger lesions (Figures
1C and 1D, lines 26 and 28), whereas the two segregants that
carried no transgenes had lesion lengths similar to the wild-type
control (Figures1Cand1D,lines 15and27).Resultsofinfections
assays with T0 transgenic plants are shown in Supplemental
Figures 1A to 1C online. Furthermore, infection of T1 generation
plants with the rice bacterial blight pathogen Xanthomonas
oryzae pv oryzae race 1 (T7174), which is also compatible with
var Kinmaze, gave results similar to blast infection (Figures 1E
and 1F). Results of infection assays with T0 transgenic plants are
shown in Supplemental Figures 1D and 1E online. These results
demonstrate that RAR1 is involved in basal resistance to both
rice blast fungus and bacterial blight. This is consistent with the
previous reports that RAR1 functions in the basal resistance of
Arabidopsis to Pseudomonas syringae pv tomato (Holt et al.,
2005) and of mlo barley (Hordeum vulgare) to M. grisea (Jarosch
et al., 2005).
Os Rac1 and RAR1 Are Functionally Linked in Basal
Resistance to Rice Blast
Our previous studies have shown that Rac1 is an important
component of disease resistance in rice (Kawasaki et al., 1999;
Ono et al., 2001; Suharsono et al., 2002). Therefore, to examine
whether Rac1 and RAR1 are functionally linked in rice disease
resistance, we generated transgenic rice carrying both CA-
OsRac1 and Os RAR1-RNAi by introducing the Os RAR1-RNAi
construct into CA-OsRac1 transgenic calli and regenerating
plantsfor infection experiments (Figure 2). As previouslyreported,
CA-OsRac1 rice plants were more resistant to a compatible race
of rice blast (Ono et al., 2001). Transgenic plants carrying both
the CA-OsRac1 and Os RAR1-RNAi constructs were more re-
sistant to rice blast than Os RAR1-RNAi plants and had the
4036 The Plant Cell
Figure 1. RAR1 Is Involved in Basal Resistance against Rice Blast and Bacterial Blight Infection in Rice.
(A) Diagram of the Os RAR1-RNAi construct.
(B) Transgenic plants expressing Os RAR1-RNAi were inoculated with an incompatible race 031 of the rice blast fungus M. grisea. Lesions are shown on
leaf blades 12 d after inoculation. WT indicates untransformed wild type plant, and 18, 61, and 15 are T1 segregants derived from an Os RAR1-RNAi
plant. Response of leaf blades of the wild type Kinmaze with infection by the compatible race 007 was used as a control for this experiment. Total RNA
was extracted from leaves of the wild type Kinmaze and Os RAR1-RNAi plants. Os RAR1 mRNA was amplified by RT-PCR using specific primers. The
17S rRNA primers were included in the PCR reactions as an internal control.
(C) Response of transgenic plants expressing Os RAR1-RNAi to infection with the compatible race 007 of M. grisea. WT indicates untransformed control
plant, and 26, 28, 15, and 27 are T1 segregants derived from an Os RAR1-RNAi plant. Photographs were taken 12 d after infection. Presence or absence
of the Os RAR1-RNAi transgene are indicated by þ and ?, respectively. RNA analysis was performed as indicated in (B).
(D) Quantitative analysis of disease lesions induced by infection with a compatible race of rice blast fungus shown in (C). Leaves were inoculated with
compatible race 007 of M. grisea. Bars indicate SE obtained from 6 to 20 measurements.
(E) Response of transgenic plants expressing Os RAR1-RNAi to infection with X. oryzae pv oryzae race1 (T7174). WT indicates untransformed control
plants, and 26, 28, 15, and 27 are T1 segregants derived from an Os RAR1-RNAi plant. Photographs were taken 12 d after infection. Presence or
absence of the Os RAR1-RNAi transgene are indicated by þ and ?, respectively.
(F) Quantitative analysis of disease lesions induced by infection with a compatible race of bacterial blight shown in (E). Leaves were inoculated with the
X. oryzae race 1. Bars indicate SE obtained from four to nine measurements.
RAR1 and HSP90 in Rice Innate Immunity4037
same level of resistance as untransformed control plants (Figure
2), indicating that RAR1 is required for Rac1-mediated basal
resistance to rice blast. Similar results were obtained for basal
resistance to bacterial blight (data not shown).
Attempts to generate Os SGT1-RNAi rice cell cultures and
whole plants by the same transformation protocol used for gen-
erating Os RAR1-RNAi cell culture and plants were unsuccessful.
This is likely due to the fact that Os SGT1 is an essential single-
copy gene in rice. Therefore, the function of SGT1 in rice innate
immunity has not as yet been determined by genetic methods.
Os Rac1 Forms a Complex with RAR1, HSP90, and
HSP70 in Vivo
Because Os Rac1 and Os RAR1 are functionally linked in basal
resistance, we examined whether RAR1 and Rac1 interact in
vivo. For an analysis of RAR1 interaction with Rac1 in vivo,
suspension cell cultures expressing myc-tagged CA-OsRac1,
DN-OsRac1, and CS-OsRac1 driven by the maize (Zea mays)
Ubiquitin promoter (Lieberherr et al., 2005) were used for
coimmunoprecipitation (co-IP) experiments. Results of co-IP
experiments using anti-myc and anti-RAR1 antibodies indicate
that CA-OsRac1 and DN-OsRac1 associate with endogenous
Os RAR1, but there is no association in extracts from CS-
OsRac1, which has impaired membrane localization, suggesting
that membrane localization of Rac1 isrequiredfor its association
experiments, we always detected two protein bands for Rac1 by
anti-myc antibody (Figure 3A, bottom panel; Lieberherr et al.,
2005). However, the reasons for this observation are not known.
To confirm the association of Os Rac1 and Os RAR1, we
immunoprecipitated RAR1 in protein extracts from three trans-
genic Rac1 cell cultures with anti-RAR1 antibody and then
Figure 2. Quantitative Analysis of Disease Lesions Induced by Infection
with a Compatible Race of M. grisea.
Leaves of the nontransgenic wild type, CA-OsRac1, Os RAR1-RNAi, and
double transgenic CA-OsRac1/Os RAR1-RNAi plants were used in three
independent experiments (mean 6 SE; n ¼ 12 to 61). Double transgenic
plants were made by introducing the Os RAR1-RNAi construct into the
CA-OsRac1 transgenic line. The double transgenic plants were more
resistant to race 007 of M. grisea than Os RAR1-RNAi plants and more
susceptible than CA-OsRac1 plants.
Figure 3. Os Rac1 Forms a Complex with RAR1, HSP90, and HSP70
(A) Co-IP of OsRac1 and RAR1, HSP90, and HSP70. Total protein
extracts from Os Rac1 transgenic mutants were incubated with anti-myc
antibody and protein A Sepharose beads. Precipitates were washed,
collected by centrifugation, and separated by SDS-PAGE. Total ex-
tracted and immunoprecipitated samples from wild type cell culture were
used as a control. Immunoblot analyses were performed with anti-RAR1
antibody. Os RAR1 was detected in CA and DN-OsRac1 immune
complexes but not in C212S-OsRac1 (top panel). All three Os Rac1
mutants contained HSP90 (second panel). HSP70 was also detected in
all three Os Rac1 mutants. Os SGT1 was not detected in Os Rac1 protein
complexes (bottom panel).
(B) Immunoprecipitation with anti-RAR1 antibody and immunobloting
with Os Rac1 indicates the association of RAR1 and Rac1 in complexes
from CA- and DN-OsRac1 cells but not from CS-OsRac1 cells. Signals
were also detected from crude extracts of transgenic but not of the
nontransgenic wild-type cell culture (top panel). As a positive control,
RAR1 was detected in crude extracts as well as in precipitated
complexes from all cell cultures (middle panel).
(C) Confirmation of HSP90 association with Rac1 in vivo. Immunopre-
cipitation was performed with anti-HSP90 antibody and immunoblotted
with anti-Os Rac1 (top panel) and anti-HSP90 (bottom panel) antibodies.
4038The Plant Cell
examined the precipitate with anti-myc antibody (Figure 3B). Os
extracts but not in the CS-OsRac1 mutant, whereas RAR1
protein was detected in co-IPs from three Rac1 mutants and
the untransformed control (Figure 3B, top two panels). These
results indicate that RAR1 and Rac1 are part of the same protein
complex in rice cell cultures.
Because RAR1 interacts with HSP 90 and we confirmed
interaction of rice RAR1 and HSP90 in yeast two-hybrid assays
(data not shown), we tested whether HSP90 is part of the Rac1
complex in vivo. The co-IP experiments showed that HSP90
coprecipitated withRac1 in extracts from the three myc-OsRac1
transgenic cell cultures but not from untransformed cultured
cells (Figure 3A, second panel). The association between Rac1
and HSP90 was confirmed by co-IP with anti-HSP90 antibody
followed by immunoblots with anti-myc antibody (Figure 3C).
These results demonstrate that Rac1 and HSP90 associate with
each other in vivo.
HSP70 is known to participate with HSP90 in almost all
cochaperone complexes studied in eukaryotes (Pratt and Toft,
2003). Therefore, we examined whether HSP70 also coprecipi-
tated with Rac1 by anti-myc antibody (Figure 3A, third panel).
Os Rac1 complex. These data suggest that Rac1 forms a
multiprotein complex containing RAR1, HSP90, and HSP70,
which is mainly localized at the plasma membrane, although
other possibilities remain to be studied.
SGT1 interacts with RAR1 and HSP90 (Shirasu and Schulze-
Lefert, 2003), and the SGT1-HSP90 association was recently
shown to be crucial in NLR-mediated immune responses in
mammals (da Silva Correia et al., 2007; Mayor et al., 2007).
However, repeated experiments clearly indicate that SGT1 does
not coimmunoprecipitate with Os Rac1 (Figure 3A), Os RAR1
(Figure 3B), or HSP90 (data not shown) under the conditions
used. Because two-hybrid assays with rice RAR1, SGT1, and
HSP90 showed that SGT1 is able to interact with RAR1 and
HSP90 (data not shown), it is possible that SGT1 interaction with
the Rac1 complex may be unstable and transient in rice cell
cultures. Alternatively, SGT1 may complex with RAR1 and
HSP90 in some other subcellular localizations.
RAR1 and HSP90 Are Essential for Rac1-Mediated
Enhancement of Sphingolipid-Triggered Immune
Responses in Rice Cell Cultures
Because RAR1 and HSP90 complex with Os Rac1, we investi-
gated whether PAMP-triggered immune responses, such as PR
gene expression and ROS production, require RAR1. For this
purpose, we analyzed PR gene expression in cultured cells in
response to sphingolipid elicitors (SEs). SEs were isolated from
the membranes of rice blast fungus and were shown to induce
the accumulation of phytoalexins, cell death, increased resis-
(Koga etal.,1998;Umemuraetal.,2000;Suharsono etal.,2002).
was induced in the wild type and was strongly enhanced in CA-
OsRac1 cells (Figure 4A). Os RAR1-RNAi did not affect induction
of PBZ1 or Chitinase1 expression. Interestingly, however, in the
double transgenic CA-OsRac1/Os RAR1-RNAi cell line, PR gene
expression was lower than in the CA-OsRac1 cell culture and
close to wild-type levels (Figure 4A), indicating that the enhance-
ment of PR gene expression by CA-OsRac1 was counteracted
by Os RAR1-RNAi. Thus, RAR1 is essential for Rac1-mediated
enhancement of PR gene expression in rice cell cultures,
suggesting a functional link between Rac1 and RAR1 in PR
SE induces H2O2production in rice cell cultures (Suharsono
et al., 2002; Wong et al., 2004; Lieberherr et al., 2005), raising the
possibility that RAR1 is involved. H2O2production was much
higher in the CA-OsRac1 cell culture (Figure 4B). However, this
Figure 4. RAR1 and HSP90 Are Essential for Enhancement of PAMP-
Triggered Immune Responses Mediated by Rac1 in Rice Cell Cultures.
(A) Suppression of SE-induced PBZ1 and Chitinase 1 by Os RAR1-RNAi
in CA-OsRac1 cell cultures. SE induction of PBZ1 and Chitinase 1 was
examined in the wild type, CA-OsRac1, Os RAR1-RNAi, and Os RAR1-
RNAi/CA-OsRac1 double mutants after 3, 6, or 9 h of treatment with SE.
Activation of SE-induced PBZ1 and Chitinase1 expression in the CA-
OsRac1 cell cultures (left) was suppressed by Os RAR1-RNAi (right).
(B) Suppression of SE-induced hydrogen peroxide production by Os
RAR1-RNAi in the CA-OsRac1 cell culture. Levels of H2O2were mea-
sured 3, 6, or 9 h after SE treatment.
(C) SE-induced PBZ1 and Chitinase 1 expression in rice cell cultures in
the presence or absence of GDA treatment. Cell cultures of wild type and
CA-OsRac1 were treated with GDA (right) or control DMSO (left) over-
night with a time course of SE treatment. Expression of PBZ1 and
Chitanase1 was strongly enhanced in CA-OsRac1 cells and reduced by
GDA treatment (right).
RAR1 and HSP90 in Rice Innate Immunity4039
increase in H2O2 production was diminished in the double
transgenic CA-OsRac1/Os RAR1-RNAi cell cultures (Figure
4B), indicating that, like PR gene induction, RAR1 is involved in
Rac1-mediated H2O2production in rice cell cultures.
Because HSP90 is essential for disease resistance in plants
(Schulze-Lefert, 2004) and for some NLR-mediated immune
responses in mammals (da Silva Correia et al., 2007; Mayor
et al., 2007), its role in PAMP-triggered immune responses
mediated by Os Rac1 was determined using geldanamycin
(GDA), an HSP90-specific inhibitor. Wild type and CA-OsRac1
cell cultures were pretreated with GDA overnight and then
treated with SE for 3, 6, and 9 h. PBZ1 and Chitinase1 were
induced by SE in wild-type cells, but expression was much
greater in CA-OsRac1 cells. A high level of PR gene expression
was observed even at time point 0 (i.e., without SE treatment) in
CA-OsRac1 transgenic cells, peaking by 6 h after SE treatment.
Treatment of cell cultures with GDA resulted in a substantial
decrease in PBZ1 and Chitinase1 mRNA in CA-OsRac1 cell
culture (Figure 4C). These results indicate that HSP90 function is
essential for Rac1-mediated enhancement of PAMP signaling.
RAR1 and HSP90 are essential for Rac1-mediated enhancement
of PAMP-triggered immune responses in rice cell cultures.
HSP90 but Not RAR1 May Be Essential for Association with
the Rac1 Complex
The nucleotide-bound state of HSP90 is known to be essential
for function of its client adaptor proteins in the complex (Catlett
such as NOD1 and NALP3 and activation of immune responses
in mammals (da Silva Correia et al., 2007; Mayor et al., 2007).
and RAR1, rice cell cultures were treated with 10 mM GDA, and
There was no HSP90 signal with GDA treatment, indicating that
5A). GDA treatment also caused dissociation of RAR1 from the
Rac1 complex (Figure 5B). Furthermore, anti-RAR1 antibody
was used to test whether intact HSP90 is required for HSP90-
RAR1 associations. GDA treatment reduced precipitation with
anti-RAR1 to nearly undetectable levels, suggesting that HSP90
is essential for association HSP90 and RAR1 (Figure 5C). Finally,
we tested whether intact Os RAR1 is required for association of
HSP90 in the Rac1 complex using Os RAR1-RNAi/myc-CA-
OsRac1 cell culture and found that RAR1 is not required for the
association of HSP90 in the Rac1 complex (Figure 5D). Together,
these results indicate that HSP90 function is essential for associ-
Os RAR1 Expression Is Regulated by Os Rac1
Os RAR1 protein was apparently present in higher concentra-
tions in CA-OsRac1 cell cultures, suggesting that Os RAR1
mRNA accumulation was measured by RT-PCR in wild-type and
CA-OsRac1 cell cultures. Os SGT1 mRNA was also measured
because SGT1 transcripts are induced by pathogen infection in
rice and Arabidopsis (Cooper et al., 2003; Zimmermann et al.,
2004). CA-OsRac1 overexpression cultures had much higher
Figure 5. HSP90 Function but Not Os RAR1 May Be Essential for Their Association with the Os Rac1 Complex.
(A) and (B) Wild-type and CA-OsRac1 cell cultures were treated with 10 mM GDA or DMSO overnight. Total protein extracts were incubated with anti-
myc antibody and protein A Sepharose beads. Precipitates were washed, collected by centrifugation, and separated by SDS-PAGE. Immunoblot
analyses were performed with anti-HSP90 (A) or anti-RAR1 (B) antibody to examine the interactions of Rac1 with HSP90 and RAR1. Signals were
detected only from immunocomplexes without GDA treatment. Immunoblots with anti-myc antibody were used as a control ([A], bottom panel).
(C) Immunoprecipitation performed with anti-RAR1 antibody after GDA treatment. Immunoblot analyses were performed with anti-HSP90 antibody.
HSP90 was not detected in RAR1-precipitated complex after GDA treatment.
(D) RAR1 is not required for association of HSP90 in the Rac1 complex. Total protein extracts from the wild type, CA-, DN-, or CS-OsRac1, Os RAR1-
RNAi, and the CA-OsRac1/Os RAR1-RNAi double transgenic mutant were incubated with anti-myc antibody. Precipitates were washed, collected by
centrifugation, and separated by SDS-PAGE. Total extracted and immunoprecipitated samples from wild-type cultured cells were used as control.
Immunoblot analyses were performed with anti-HSP90 antibody to examine Os Rac1-HSP90 association. Os RAR1-RNAi in the CA-OsRac1/Os RAR1-
RNAi double transgenic mutant had no effect on Rac1-HSP90 association (last column).
4040The Plant Cell
mRNA levels of both Os RAR1 and Os SGT1 (Figure 6A),
suggesting that Os Rac1 regulates RAR1 and SGT1 expression.
To further examine the regulatory relationship between Os Rac1,
Os RAR1, and Os SGT1, a cell culture in which CA-OsRac1 can
be induced (ICA) by treatment with dexamethasone (DEX) (Wong
et al., 2004) and an Os Rac1-RNAi cell culture (Miki et al., 2005)
were examined. Induction of CA-OsRac1 with DEX treatment
clearly increased both transcript (Figure 6B) and protein (Figure
6B) accumulation levels of RAR1 compared with the ethanol-
treated control (ICA/DEX and ICA/ethanol), and RAR1 protein
mRNA accumulation correlated with Rac1 expression, but SGT1
protein levels did not change in these cell cultures (Figure 6B).
These results indicate that RAR1 could be transcriptionally
regulated by Rac1, but any control of SGT1 by Rac1 would be
at the posttranscriptional level. The presence of some RAR1
that there is another regulatory component besides Rac1 or that
Rac1 also has some posttranscriptional roles in RAR1 expres-
sion. Nevertheless, these results suggest a close regulatory link
between Rac1 and RAR1 at the transcriptional and possibly
In barley, Arabidopsis, and tobacco, RAR1, together with HSP90
and SGT1, plays a key role in R gene–mediated resistance
(Shirasu and Schulze-Lefert, 2003). Our results demonstrate that
RAR1 is required for basal resistance against compatible races
of rice blast and bacterial blight (Figures 1D to 1F) and that this
resistance is mediated by Os Rac1 (Figure 2). Interestingly, Os
resistance. Recently, it was shown that Os RAR1 is not required
for Pish-mediated blastresistance (Takahashi etal.,2007).Since
RAR1 is not required for all R genes (Shirasu and Schulze-Lefert,
2003), these results in rice are not unexpected. We demon-
strated thatRAR1 iscritical forOsRac1–mediated enhancement
of PAMP signaling (Figures 4A and 4B). Therefore, RAR1 activity
in rice innate immunity may be on a broader scale than in those
Our results are consistent with the involvement of RAR1 in
basal resistance in Arabidopsis and mlo barley (Holt et al., 2005;
response to virulent pathogens, and its molecular mechanism is
weak R proteins that recognize cognate effectors (Jones and
Dangl, 2006). In this model, RAR1 function associated with basal
resistance is also mediated by R proteins. More recently, RAR1
was shown to be a target of the P. syringae effector AvrB (Shang
et al., 2006). Therefore, considering the observation that there
are many PAMP receptors in plant cells and that RAR1 could
potentially form complexes containing such receptors and other
key signaling proteins, the extent of RAR1 involvement in various
defense signaling pathways remains to be studied.
Our yeast two-hybrid analysis showed that Os RAR1 and
HSP90 do not directly interact with Os Rac1, although Os RAR1
interacts directly with HSP90 (data not shown). Thus, it seems
that Os Rac1 forms a complex with Os RAR1 and HSP90 by
indirect interactions. We recently obtained evidence that Os
Rac1 interacts with Sti1/Hop in vivo and in vitro (L. Chen and
K. Shimamoto, unpublished data). Sti1/Hop is a cochaperone in
the HSP90 complex with HSP90 and HSP70 and is highly con-
served in eukaryotes (Pratt and Toft, 2003; Zhang et al., 2003).
Thus, Os RAR1 and HSP90 are likely to form a complex with Os
Rac1 through Sti1/Hop.
Results of RT-PCR and immunoblotting showed that the
overexpression of Os Rac1 strongly increased Os RAR1 mRNA
and protein accumulation (Figure 6), suggesting that Rac1 may
function upstream of RAR1 in defense signaling and that the
presence of a regulatory link between Os Rac1 and Os RAR1 in
SE-induced signaling. Interestingly, treatment with GDA did not
alter mRNA or protein accumulation of either Os RAR1 or Os
SGT1 (data not shown). These results may suggest the presence
of multiple regulatory pathways in which various components
interact with each other at multiple levels of regulation.
HSP90 Is a Critical Regulator in Os Rac1–Mediated PAMP
Signaling in Rice
Heat shock proteins are well known for regulating the maturation
of protein complexes, for degrading damaged or misfolded
peptides, and for involvement in the activity of many signal
Figure 6. Os RAR1 Expression Is Regulated by Os Rac1.
(A) Correlation of mRNA expression levels of Os Rac1, Os RAR1, and Os SGT1. Overexpression of CA-OsRac1 strongly increased mRNA accumulation
of RAR1 and SGT1 in rice cell cultures. Levels of mRNAs were measured by RT-PCR.
(B) Levels of mRNA (RT-PCR) and protein accumulation (protein gel blot [WB]) for Rac1, RAR1, and SGT1. DEX-inducible CA-OsRac1 (ICA) and an
OsRac1-RNAi cell culture were examined by RT-PCR and immunoblotting after DEX induction and ethanol as a control.
RAR1 and HSP90 in Rice Innate Immunity 4041
transduction proteins (Pratt and Toft, 2003; Rutherford, 2003).
Previous studies have demonstrated that HSP90 plays a general
role in R protein–mediated immunity in plants and that it acts
physically close to R proteins (Hubert et al., 2003; Kanzaki et al.,
2003; Lu et al., 2003; Takahashi et al., 2003; Liu et al., 2004).
However, no specific role for HSP90 in protein complex forma-
tion or specific immune responses in plants has been identified.
The close association of HSP90 with Os Rac1 (Figure 3)
prompted an investigation into the possible involvement of
HSP90 in Rac1-mediated enhancement of PAMP signaling in
rice cell cultures. To examine the function of HSP90, we used
GDA, which binds the N-terminal structural domain of HSP90
binding pocket, and it has little effect on prokaryotic pathogens
(Stebbins et al., 1997; Picard, 2002). GDA experiments on rice
cell cultures demonstrated the requirement of HSP90 for Rac1-
mediated enhancement of PAMP signaling to induce PR genes
(Figure 4C). These results could be explained by the dissociation
of RAR1 and HSP90 from the Rac1 complex caused by GDA
(Figure 5), since it has been demonstrated that GDA treatment
alters the conformation and dissociation of cochaperones, such
as p23, Hop, and HSP70, from the steroid receptor complex in
mammalian cells (Whitesell et al., 1994; Bagatell et al., 2001;
Waza et al., 2006). These findings are similar to those in recent
studies of innate immunity in mammals (da Silva Correia et al.,
2007; Mayor et al., 2007), which demonstrate that GDA causes
inactivation of NLR-containing protein complexes and that
HSP90 is required for stability of the signaling complex.
We demonstrated the critical role of HSP90 in Os Rac1–
mediated enhancement of PAMP signaling; thus, we tested
whether HSP90 is also required for SE-dependant ROS pro-
duction. We examined H2O2production after GDA treatment
followed by SE treatment. However, GDA failed to suppress
SE-induced H2O2production in CA-OsRac1 cell cultures (data
not shown). One possible interpretation of this result is that GDA
itself increases ROS generation (Dikalov et al., 2002).
Withregardtothe possiblefunctionofHSP70 ininnateimmune
responses in rice, we have no information at the moment. The
three groups (N.P. Thao and K. Shimamoto, unpublished data),
much like Arabidopsis, which also contains 14 genes for HSP70
(Sung et al., 2001). We attempted to knock down expression
of nine genes in group 1 and three genes in group 2 by RNAi
methods employing conserved coding sequences as targets
(Miki et al., 2005). However, we were not able to obtain trans-
was clearly decreased (N.P. Thao and K. Shimamoto, unpub-
lished data). A different approach would therefore be required to
determine the function of HSP70, if any, in rice innate immunity.
A Network of Proteins Involved in Innate Immunity in Rice
CA-OsRac1 activates ROS production and HR in rice and
confers resistance to rice blast and bacterial blight (Kawasaki
et al., 1999; Ono et al., 2001; Suharsono et al., 2002). However,
the molecular mechanisms of Os Rac1 immune response reg-
ulation are largely unknown. In this study, we present evidence
suggesting that Os Rac1 associates with well-studied compo-
nents of plant innate immunity, such as RAR1 and HSP90, and
HSP70. We have previously demonstrated that Os Rac1 also
a WD repeat–containing receptor of activated C kinase homolog
(RACK1/RWD), which is involved in hormone signaling and
development (Chen et al., 2006), specifically binds CA-OsRac1
and is essential for basal resistance to rice blast and PAMP-
triggered immunity in rice (A. Nakashima and K. Shimamoto,
unpublished data). RACK1/RWD is likely to be a second link
between Rac1 and RAR1 because RWD interacts directly with Os
(A. Nakashima and K. Shimamoto, unpublished data), thus con-
firming the observation that RAR1 is part of the Rac1 complex.
These results connect Rac/Rop GTPase with known players in
plant defense pathways and reveal the general importance of
overexpression of CA-OsRac1 increased transcript levels of Os
RAR1 and Os SGT1 and that CA-OsRac1 complements the loss
of Os RAR1 in basal resistance to rice blast (Figure 2) suggest
that Rac1 is a central mediator of disease resistance in rice and
that it coordinates the activity of other important factors, such as
RAR1, HSP90, and the MAPK cascade. Recently, wehave found
a direct interaction between CA-OsRac1 and the N terminus of
the NADPH oxidases Os RBOHs and St RBOHB (Wong et al.,
2007). Thus, it is possible that Os RAR1 and HSP90 are required
for full activation of the Os Rac1 complex, at least partially
through regulation of NADPH oxidase.
With respect to the intracellular localization of the Os Rac1
complex, the results obtained in this study and previous studies
(Ono et al., 2001) suggest that the primary location of Rac1
complex is likely to be at the plasma membrane. However, since
recent studies indicate that some R proteins also function in the
nucleus (Shen and Schulze-Lefert, 2007), whether the Rac1
complex could be also located in the nucleus or the cytoplasm
remains to be studied.
SGT1 plays diverse roles in plants, whereas RAR1 has much
more specialized resistance functions (Shirasu and Schulze-
Lefert, 2003). SGT1 was not detected in immunoprecipitated
RAR1 complexes in rice cell cultures under the conditions we
used, whereas yeast two-hybrid analysis showed that rice SGT1
and RAR1 directly interact (data not shown). One explanation for
this is that the interaction between Os SGT1 and Os RAR1 is
a single complex or whether they simultaneously coregulate
other complexes required for downstream signaling events. One
current model derived from these observations is that a network
of proteins including some known components of plant innate
immunity, such as Os Rac1, RAR1, SGT1, HSP90, HSP70, Os
MAPK6, and RBOH (NADPH oxidase), can form one or more
mutants Os Rac1-G19V (CA-OsRac1), Os Rac1-T24N (DN-OsRac1), and
4042The Plant Cell
Os Rac1-C212S (CS-OsRac1) were described previously (Kawasaki
et al., 1999; Ono et al., 2001). Os Rac1 protein was tagged with the
myc epitope at the N terminus, and the expression of each construct was
under the control of maize (Zea mays) Ubiquitin promoters. To make an
Os RAR1-RNAi construct, a cDNA fragment amplified by PCR using
two primers, Os RAR1-F (59-TCTGAGTGAGCCTAGGGTTTG-39) and
Os RAR1-R (59-GACCGAAGTCTCCACACACA-39), was inserted into
pANDA developed as a vector for RNAi (Miki and Shimamoto, 2004).
Agrobacterium tumefaciens–mediated transformation of rice calli was
performed according to a published method (Hiei et al., 1994). Plants
regenerated from transformed calli were selected by hygromycin resis-
tance. Rice suspension cell cultures expressing Os RAR1-RNAi and
CA-OsRac1/Os RAR1-RNAi were also produced.
Infection of Rice Plants with Blast Fungus and Bacterial Blight
Growth conditions for Magnaporthe grisea and methods for leaf punch
infection have been described previously (Takahashi et al., 1999). For
bacterial blight infection, leaves were inoculated by the clipping method
(Ono et al., 2001) with the Japanese Xanthomonas oryzae pv oryzae race
1 (T7174), which is compatible with var Kinmaze. Disease lesions were
measured 12 d after inoculation.
Rice Cell Cultures and Elicitor Treatment
Rice cell cultures expressing CA- and DN-OsRac1 were generated as
described previously (Kawasaki et al., 1999; Ono et al., 2001; Suharsono
et al., 2002). For analysis of gene expression, rice cell cultures were
collected after treatment with 5 mg/mL of an SE prepared from rice blast
Total RNA was extracted from cultured cells and seedlings using RNeasy
plant RNA extraction kit (TAKARA). One mg RNA was digested with
PCR reactions were performed with specific primer sets: Os Rac1
(59-AGATAGGGCCTATCTTGCTGATCATC-39 and 59-ACAAGCGCTTC-
59-TGCAAAGGAGTGAGGCTTTT-39), PBZ1 (59-GGGCACCATCTACAC-
CATGAA-39 and 59-GTCGCACACCGCCACC-39), Chitinase1 (59-TCT-
TAACATCACTGCAACTCAG-39 and 59-CTGCGAGCTCTGGACAC-39),
Ubiquitin (59-CCAGGACAAGATGATCTGCC-39 and 59-AAGAAGCTGAA-
GCATCCAGC-39), Os SGT1 (59-ATGGATCCATATGGCAACCGCCGC-
CGCG-39 and 59-ATGCGGCCGCTTAGTACTCCCATTTCTTAAGC-39),
and Os RAR1 (59-TGCAAAACTGGAAAGCACAC-39 and 59-GGAACTG-
Rice suspension cell cultures expressing CA-OsRac1, DN-OsRac1, and
C212S-OsRac1 were ground in cold extraction buffer (137 mM NaCl, 8.1
mM Na2HPO4anhydrous, 1.47 mM Na2HPO4, pH 7.4, 10% sucrose, and
complete protein inhibitor tablets [Roche]). Cell debris was removed by
centrifugation at 12,000g for 25 min. Protein concentrations were deter-
mined using Bradford protein assay with BSA as the standard. Proteins
wereseparated in10 or12.5%SDSpolyacrylamidegelsandblotted onto
nitrocellulose membranes (Millipore) with a semidry electroblotting-Trans
blot SD cell (Bio-Rad).
For immunodetection, membranes were incubated for 1 h with primary
antibodies against the myc epitope (mouse monoclonal [Invitrogen] or
rabbit polyclonal [Santa Cruz Biotechnology]). Hv RAR1 (Takahashi et al.,
2003), Hv HSP90 (Takahashi et al., 2003), SGT1 (Takahashi et al., 2003),
and HSP70 (Stressgen Biotechnologies), followed by anti-mouse/rat/
rabbit/IgG were conjugated to horseradish peroxidase (Sigma-Aldrich).
Specific protein bands were visualized with the ECL chemiluminescent
Western blotting detection reagent (GE Healthcare) and Hyperfilm ECL
For immunoprecipitations, 1 g extract (in a 1-mL volume) was incu-
48C for 3 h. Supernatants were collected and combined with 20 mL anti-
myc antibody and rotated end-over-end at 48C for 1 h. Fifty microliters of
protein A or G were added, and the incubation was continued overnight.
Immunocomplexes were washed three times with 1 mL ice-cold washing
buffer (extraction buffer plus 150 mM NaCl and 0.5% Triton X-100),
and separated by SDS-PAGE as described.
Quantification of H2O2
QuantificationofSE-induced H2O2production was performed withmodi-
cultured cells were precultured in a fresh R2S medium at 308C for 16 h.
Cells were transferred to 2mL offresh medium containingSE (10 mg/mL).
Aliquots were collected following incubation and filtered through a 0.22-
mmfilter.Thefiltered aliquot (100mL)wasmixed with1mLxylenol orange
buffer [0.25 mM FeSO4, 0.25 mM (NH4)2SO4, 25 mM H2SO4, 10 mM
sorbitol, and 12.5 mM xylenol orange] and incubated for 2 h at room tem-
perature. Absorbance was measured in a spectrophotometer (Beckman)
at 650 nm, and H2O2levels were determined based on a standard curve
made from known concentrations of H2O2dissolved in the R2S medium.
ForGDA treatment, GDA was diluted froma 10mMstockin DMSOinto
fresh R2S medium in the preculture step (described above) to a concen-
tration of 10 mM. DMSO alone was added into the R2S medium as a
Sequence data from this article can be found in the GenBank/EMBL data
libraries under the following accession numbers: AB029508 (Os Rac1),
The following materials are available in the online version of this article.
Supplemental Figure 1. Basal Resistance to Rice Blast and Bacterial
Blight Infection in Os RAR1 RNAi T0 Plants.
Supplemental Figure 2. Os RAR1 Transcript Levels Are Consistent
with Protein Levels in Os RAR1-RNAi Rice Cultured Cell Lines.
We thank Mika Nobuhara, Yuko Tamaki, and Masako Hamane for
technical assistance. This research was supported by Grants-in-Aid
from the Ministry of Agriculture, Forestry, and Fisheries of Japan (Rice
Genome Project IP4001) and the Japan Society for Promotion of
Science (13G0023) to K.S.
Received September 5, 2007; revised November 22, 2007; accepted
December 7, 2007; published December 21, 2007.
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