全 文 ：Received 8 Sept. 2003 Accepted 18 Nov. 2003
Supported by the State Key Basic Research and Development Plan of China (G2000016203) and the National Natural Science Foundation
of China (30070493).
* Author for correspondence. Tel: +86 (0)10 62893849; E-mail: .
http://www.chineseplantscience.com
Acta Botanica Sinica
植 物 学 报 2004, 46 (8): 955-964
Characterization of a Rice WRKY Gene Whose Expression Is Induced
upon Pathogen Attack and Mechanical Wounding
GUO Ze-Jian1, 2*, KAN Yun-Chao2, 3, CHEN Xu-Jun2, LI De-Bao2, WANG Dao-Wen4
(1. Department of Plant Pathology, China Agricultural University, Beijing 100094, China;
2. Biotechnology Institute, Zhejiang University, Hangzhou 310029, China;
3. Department of Biology, Nanyang Teaching College, Nanyang 473061, China;
4. Institute of Genetics and Developmental Biology, The Chinese Academy of Sciences, Beijing 100101, China)
Abstract: WRKY genes encode plant specific transcription factors that have been extensively studied
in dicotyledonous plant species. To investigate the function of these genes in monocots, we isolated a full
length cDNA clone of the rice WRKY gene which we tentatively named as OsiWRKY. The open reading
frame of OsiWRKY cDNA encoded a polypeptide of 482 amino acid residues. The deduced primary structure
of OsiWRKY contained a single WRKY domain of a typical C2-H2 zinc finger motif. A putative nuclear
localization signal was also predicted in OsiWRKY. Potential targeting of OsiWRKY to the nucleus was
confirmed by transient expression of an OsiWRKY::GFP fusion protein in rice cells. The recombinant
OsiWRKY protein bound specifically to the W-box element in electrophoretic mobility shift assay. In
inoculation experiments with Xanthomonas oryzae pv. oryzae, OsiWRKY transcription was increased more
dramatically in the resistant variety IR-26 than in the susceptible variety Jingang 30. Interestingly, an
increase in OsiWRKY transcription was also observed in the mock-inoculated control plants of both IR-26
and Jingang 30. However, the induction of OsiWRKY transcription occurred much earlier and more
substantial in pathogen-infected plants than in mock-inoculated controls. These results suggest that
OsiWRKY encodes a potential WRKY transcription factor that may function in responses of rice plants to
both pathogen attack and mechanical wounding. But the mechanisms underlying the function of OsiWRKY
in the two processes may be different.
Key words: transcription factor; WRKY; zinc finger; Oryza sativa; W-box
Higher plants have evolved sophisticated mechanisms
to defend themselves against both biotic and abiotic
stresses. A common feature of active plant responses to
environmental stresses is transcriptional activation or re-
pression of specific sets of genes (Yang et al., 1997; Singh
et al., 2002). Some of the plant genes induced by biotic
stress (e.g., pathogens) encode products with direct anti-
microbial activities (such as the pathogenesis-related pro-
teins glucanases and chitinases), whereas others code for
transcriptional factors that may regulate the transcription
of multiple downstream defense-related genes (Yang et al.,
1997; Singh et al., 2002). A better understanding of the
function of stress-inducible genes is of fundamental im-
portance in dissecting the molecular basis of plant/envi-
ronment interactions and developing novel strategies to
engineer stress-resistant crops.
The WRKY superfamily of transcription factors is
uniquely expressed in higher plants (Eulgem et al., 2000).
Members of this superfamily have been found involved in
plant responses to biotic and abiotic stresses (pathogen
infection, wounding) and plant developmental processes
(senescence, trichome and seed coat development) (Eulgem
et al., 2000; Hinderhofer and Zentgraf, 2001; Yu et al., 2001;
Robatzek and Somssich, 2002; Yoda et al., 2002; Dong
et al., 2003). Originally, WRKY proteins were isolated as
W-box (with the core sequence TGAC) binding proteins
(Meier et al., 1991). All WRKY proteins contain one (or
two) highly conserved WRKY domain of about 60 amino
acid residues (Eulgem et al., 2000). The N-terminus of the
WRKY domain possesses the strictly conserved amino
acid sequence WRKY whereas its C-terminus contains a
zinc finger motif. Based on differences in the primary
structures, WRKY proteins have been classified into three
groups, each of which may also contain subgroups. The
Group Ⅰprotein possesses two WRKY domains whereas
the protein belonging to Groups Ⅱ or Ⅲ contains only
one such domain (Eulgem et al., 2000). Whilst the compo-
sition of the zinc finger motif in the Groups Ⅰ and Ⅱ
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004956
WRKY proteins is C-X4-5-C-X22-23-H-X1-H (the underlined
residues form the potential C2-H2 zinc finger; X denotes
any amino acid residues), the one in Group Ⅲ WRKY pro-
teins is C-X7-C-X23-H-X1-C (the underlined residues form
the potential C2-HC zinc finger. Both the WRKY element
and the zinc finger structure are required for these tran-
scription factors to bind to the W-box (Eulgem et al., 2000).
W-box has been identified in the promoter regions of many
pathogenesis-related genes and a receptor-like kinase gene
functioning in leaf senescence (Eulgem et al., 2000;
Robatzek and Somssich, 2002). Interestingly, the promoter
regions of some WRKY genes also contain W-box elements
(Robatzek and Somssich, 2002).
WRKY genes have been isolated mostly from dicotyle-
donous plant species, including parsley, sweet potato,
tobacco, nightshade and Arabidopsis (Ishiguro et al., 1994;
Rushton et al., 1995; 1996; de Pater et al., 1996; Hara et al.,
2000; Huang et al., 2002). More than 70 different WRKY
genes have been found in the Arabidopsis genome (Eulgem
et al., 2000; Dong et al., 2003), their expression patterns in
response to Pseudomonas syringae infection have been
compared (Dong et al., 2003). Database search reveals the
presence of many potential WRKY genes in the rice ge-
nome (unpublished data). However, functional studies have
not been carried out for the great majority of these genes
except that one rice WRKY gene has been cloned in a dif-
ferential display experiment (Kim et al., 2000).
Based on the above discussions, we initiated a series
of studies aiming to investigate the potential functions of
WRKY genes in rice. In the present study, we report the
characterization of OsiWRKY (Oryza sativa inducible
WRKY), a gene whose transcription was induced by both
pathogen attack and mechanical wounding.
1 Materials and Methods
1.1 Isolation of OsiWRKY cDNA
In order to obtain a specific probe for isolating rice
WRKY genes, we designed a pair of degenerated primers
based on the conservation of the sequences encoding the
WRKY domains in several published WRKY genes, 5-tgg
(ac)g(agct)aa(ag)ta(ct)gg(agct)ca(ag)aa-3 (forward primer)
and 5-catg(ag)t(ct)(ag)tgc(ct)t(gct)(agct)cc(ct)tc(ag)ta-3
(reverse primer). The size of the expected fragment would
be about 160 bp. PCR was performed for 35 cycles with
parameters of 94 ℃ for 1 min, 50 ℃ for 45 s and 72 ℃ for 1
min. The template for PCR was a cDNA library that was
prepared using mRNAs extracted from the rice suspen-
sion-cultured cells (cultivar IR-72) treated with the cell wall
extracts of the rice blast pathogen Magnaporthe grisea
(strain P131). The desired PCR product was cloned into
the pGEM T-easy vector and sequenced. Using the cloned
fragment as a probe, the cDNA library was further screened
as described by Sambrook et al. (1989). The inserts in the
resultant positive clones were sequenced from both
strands. Bioinformatic analysis of the obtained cDNA se-
quences and the primary structure of the deduced protein
was carried out using the DNAstar software (DNAstar
Inc.), and programs in the MEGA website (http: //www.
oup-usa.org/sc/0195135857, Nei and Kumar, 2000) and the
EMBNET website (http:// www.ch.embnet.org/software/
COILS-form.html) for alignment analyses, and the PSORT
website (http://psort.nibb.ac.jp, Nakai and Hanekisa, 1992)
for analysis of protein structure.
1.2 Subcellular localization of OsiWRKY
For visualizing the location of OsiWRKY in rice cells,
the expression construct p35S-OsiWRKY::GFP was
prepared. The coding sequence of OsiWRKY was modified
by PCR amplification using the primers YiwBIF (5-
t taggatccggtt t tcct ttacaacgcaatg-3 , BamHⅠ site
u n d e r l i n e d ) a n d S i w N c o R ( 5 -
taaccatgggaggtccaaatgatggagg-3, NcoⅠ site underlined).
The PCR product was digested with BamHⅠ and NcoⅠ,
followed by ligating to pBS-GFP linearized with the same
enzymes. In-frame joining between the coding sequences
of OsiWRKY and GFP (green fluorescence protein) was
confirmed by DNA sequencing. Subsequently, the
OsiWRKY-GFP fusion cistron was placed in between the
CaMV35S promoter and termination sequence of the rice
Rubisco gene using standard DNA protocols (Sambrook
et al., 1989). Similarly, a control construct (p35S-GFP)
designed to express free GFP was also prepared.
Rice cells derived from the callus tissue of the variety
Xiushui 11 were bombarded with the particles coated with
the plasmid DNA of either p35S-OsiWRKY::GFP or p35S-
GFP using the PDS-1000/He system (Bio-Rad, USA). The
preparation of DNA-coated particles and the selection of
suitable parameters for the bombardment experiments were
followed with the instructions of the equipment supplier.
The treated cells were examined for GFP fluorescence at 48
h post bombardment using confocal microscopy (Olympus,
FV500). Fluorescent as well as bright field images of repre-
sentative cells were taken.
1.3 Expression of OsiWRKY cDNA in bacterial cells and
electrophoretic mobility shift assay
For bacterial expression experiments, the coding se-
quence of OsiWRKY was re-amplified by PCR, introducing
BamHⅠ and XhoⅠ sites to the 5 and 3 ends of the cod-
ing sequence, respectively. The PCR product was cloned
GUO Ze-Jian et al.: Characterization of a Rice WRKY Gene Whose Expression Is Induced upon Pathogen Attack and Mechanical
Wounding 957
in-frame into the pET-29b expression vector (Invitrogen)
digested with BamHⅠand XhoⅠ, and confirmed by DNA
sequencing. The construct pET-OsiWRKY would express
OsiWRKY polypeptide with a histidine tag at its C-
terminus. pET-OsiWRKY DNA was transformed into the
cells of Escherichia coli BL21. An overnight culture (2
mL) was added to 200 mL of LB medium and grown to an
OD600 of 0.5. Expression of the recombinant protein was
i n d u c e d w i t h 1 m m o l / L i s o p r o p y l b - D -
thiogalactopyranoside for 4 h at 37 ℃. Bacterial cells were
pelleted after the induction, suspended in an appropriate
volume of lysis buffer (100 mmol/L Na2HPO4 (pH 7.2), 300
mmol/L NaCl, 10% glycerol, and 1% Triton X-100), and
treated with sonication. Further purification of the His-
tagged protein was carried out using nickel agarose
(Amersham Biosciences) according to the manufactory’s
protocol. The purified protein was dialyzed overnight at 4
℃ against 1 × DNA binding buffer (4％ glycerol，1
mmol/L MgCl2，0.1 mmol/L EDTA，1 mmol/L
dithiothreitol, 50 mmol/L KCl, 0.025 g/L Poly(dI-dC), 10
mmol/L Tris-HCl (pH 7.5)) before being used for the elec-
trophoretic mobility shift assay (EMSA) as described pre-
viously (Dong et al., 2003).
For investigating the potential interaction between the
bacterially expressed OsiWRKY and the W-box element,
EMSA experiments were conducted using the oligonucle-
otides PitaF (5-ccagtcattgacaccgtcatttgttagctgact-3, the
italicized nucleotides constituted the core sequence of W-
box) and mPitaF (5-ccagttattgaaaccttcatttgttagctgaat-3, a
mutant of PitaF). The sequence of PitaF was derived from
the promoter region of the rice blast resistance gene Pi-ta
(Bryan et al., 2000). Assay reaction was performed in 20 mL
volume containing 2 mg of OsiWRKY recombinant protein,
1 ng of the oligonucleotide probe (end labeled with 32P
using T4 polynucleotide kinase), and 1 × DNA binding
buffer. The reaction was preceded at room temperature for
20 min before being electrophoresed in 10% native poly-
acrylamide gel in 0.5 × TBE buffer (45 mmol/L Tris borate,
1 mmol/L EDTA). Radioactive signal was detected using
the Typhoon imaging system (Amersham Biosciences,
USA).
1.4 Inoculation with the rice bacterial blight pathogen
Xanthomonas oryzae pv. oryzae strain Zhe 173 was
streaked on potato dextrose agar medium and incubated at
28 ℃ for 48 h in the dark. The bacterial cells were gently
scratched off the plate and resuspended in sterile water
(108 colony forming units/mL) for rice inoculation. Rice
seedlings (cultivars IR-26 and Jingang 30) were grown in
greenhouse (with a 16 h light period). Leaves of four-
week-old seedlings were inoculated by cutting leaf tips
with a pair of scissors dipped into the bacterial suspension.
The inoculated plants were kept in a growth chamber in
the dark at 28 ℃. A sample was immediately taken from the
inoculated leaves as the 0 h control; the remaining samples
were collected at 1, 3, 6, 12, 24 and 48 h post inoculation,
respectively. Mock inoculation was carried out similarly
except that sterile water rather than bacterial suspension
was used. An identical series of samples were also taken
from the mock inoculated plants. All samples were stored
at -70 ℃ prior to RNA extraction.
1.5 DNA and RNA analyses
For Southern analysis, the genomic DNA was extracted
from the young leaves of four rice varieties (IR-26, IR-72,
Jingang 30, Xiushui 11) as described previously (Murray
and Thompson, 1980). Aliquots of 10 mg genomic DNA
samples were digested with BamHⅠ, EcoRⅠ or SalⅠ.
After digestion, the DNA samples were separated on 0.8%
agarose gel. The resolved fragments were transferred onto
Hybond N+ membrane according to standard protocol
(Sambrook et al., 1989). For Northern analysis, total RNA
samples were prepared from the inoculated rice leaf tis-
sues using the Trizol reagent (Life Technologies, Germany).
Total RNA (20 mg) from each sample was separated in 1%
agarose gel containing formaldehyde followed by trans-
ferring onto the Hybond N+ nylon membrane. DNA and
RNA gel blots were hybridized with 32P-labeled probes
prepared using OsiWRKY cDNA and the Prime-A-Gene kit
(Promega, USA). To avoid cross hybridization, the DNA
fragment used for preparing the probe was derived from
the 3 end of the full length cDNA. This fragment did not
contain the coding sequence for the conserved WRKY
domain and is likely to be gene specific. Hybridization and
washing conditions were chosen as described previously
(Kan et al., 2001). The hybridization signals were recorded
using the Typhoon imaging system (Amersham
Biosciences, USA).
2 Results
2.1 Isolation of OsiWRKY cDNA and structural features
of its deduced protein
Using degenerated PCR primers designed according to
the conserved coding sequences of the WRKY domains
of several different WRKY proteins, a cDNA fragment of
expected size (about 160 bp) was amplified from the cDNA
library derived from elicitor-induced rice cells (Fig.1). The
DNA sequence of the 160 bp fragment specified a putative
polypeptide that was highly similar to the conserved
WRKY domain. By screening the elicitor-induced rice
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004958
cDNA library using the above fragment as a probe, three
positive colonies were obtained. DNA sequencing showed
that the inserts in the three positive clones represented
cDNAs from a single gene. The longest cDNA contained
1 847 nucleotides (Fig.2), which contained an open read-
ing frame encoding a predicted protein of 482 amino acid
residues. In addition to the WRKY domain, the predicted
OsiWRKY protein contained a putative nuclear localiza-
tion signal (Fig.2). The presence of these motifs indicated
that OsiWRKY contained the basic structures of a WRKY
transcription factor. Amino acid sequence comparison
showed that the most closely related region between
OsiWRKY and homologous proteins from other plant spe-
cies was the WRKY domain (Fig.3A). Within this domain,
OsiWRKY possessed a typical C2-H2 zinc finger structure
having the composition C-X4-C-X23-H-X1-H (Figs. 2, 3A),
which was identical to the one found in Group Ⅱ-c WRKY
proteins in A. thaliana (Eulgem et al., 2000) and NtWRKY9
from tobacco (Fig.3A) (Maeo et al., 2001). However, phy-
logenetic analysis based on the amino acid sequences of
the WRKY domains revealed that OsiWRKY was different
from homologous proteins from A. thaliana or tobacco
because it was not clustered with either AtWRKYs or
NtWRKY9 (Fig.3B).
The copy number of OsiWRKY in rice genome was esti-
mated by Southern blotting analysis using a gene specific
probe. Only one hybridizing band was detected in the ge-
nomic DNA samples of IR-72 that were digested with BamHⅠ,
EcoRⅠ, or SalⅠ (Fig.4, lanes 4-6). The same pattern of
hybridization signal was detected with EcoRⅠ -digested
genomic DNA samples of three other rice cultivars (IR-26,
Jingang 30 and Xiushui 11) (Fig.4, lanes 1-3), indicating
that OsiWRKY existed as a single copy gene in the ge-
nomes of the four rice varieties examined in this study.
2.2 Subcellular localization of OsiWRKY protein
PSORT analysis (Nakai and Kanehisa, 1992) indicated a
high likelihood of nuclear localization for the deduced
OsiWRKY (Fig.2), indicating that OsiWRKY may be local-
ized to the nucleus in rice cells. To examine this possibility,
we prepared the plant expression construct p35S-
OsiWRKY::GFP and used particle gun mediated transient
expression to investigate the subcellular localization of
the OsiWRKY::GFP fusion protein. Confocal microscopy
showed that in the cells bombarded with the DNA of p35S-
OsiWRKY::GFP, GFP fluorescence was concentrated in
the nucleus (Fig.5B). In contrast, in the cells bombarded
with the DNA of the control construct p35S-GFP, GFP
fluorescence was distributed more generally in the cyto-
plasm except in the central vacuole (Fig.5D).
2.3 DNA-binding property of the recombinant OsiWRKY
protein
It has been shown previously that WRKY proteins con-
taining the WRKY DNA-binding domain can specifically
bind to the W-box element possessing the TGAC core se-
quence (Rushton et al., 1995; Yang et al., 1995). To exam-
ine whether the protein product of OsiWRKY would bind
to W-Box element, we expressed OsiWRKY as a histidine
tagged protein in E. coli and used the purified protein to
conduct gel mobility shift assays. As shown in Fig.6 (lane
1), the recombinant OsiWRKY protein was able to bind
strongly to 32P-labeled PitaF fragment that contained two
TGAC core sequences. In contrast, OsiWRKY showed little
or no binding to 32P-labeled mPitaF fragment in which the
TGAC core sequences were obliterated by mutation (Fig.
6, lane 4). The specificity of the binding was further tested
by competition experiments. The addition of 100-fold ex-
cess of unlabeled PitaF to the binding reaction almost com-
pletely inhibited OsiWRKY interaction with the 32P-labeled
PitaF (Fig.6, lane 3). However, in a similar test, mPitaF failed
to compete with the binding of OsiWRKY to the 32P-la-
beled PitaF (Fig.6, lane 2).
2.4 Increase of OsiWRKY transcription in pathogen and
mock-inoculated rice plants
To examine whether OsiWRKY was involved in rice de-
fense response, we inoculated the seedlings of IR-26 and
Fig.1. Amplification of the coding sequence for the conserved
WRKY domain of WRKY transcription factors by PCR. The
size of the amplified fragment was approximately 160 bp (lane
2, arrowed) by comparing to DNA markers (lane 1). The band
indicated by the arrowhead (lane 2) represents excess primers.
GUO Ze-Jian et al.: Characterization of a Rice WRKY Gene Whose Expression Is Induced upon Pathogen Attack and Mechanical
Wounding 959
Jingang 30 with the Zhe 173 strain of X. oryzae pv. oryzae.
The two cultivars had previously been shown to be resis-
tant and susceptible to the Zhe 173 strain, respectively
(unpublished results). As shown in Fig.7 (panel A), the
expression of OsiWRKY was rapidly induced in both IR-26
and Jingang 30 at 1 h post pathogen inoculation. However,
the kinetics of transcription accumulation over time dif-
fered between the two varieties. The level of OsiWRKY
transcription induced in IR-26 from 3 h onwards was much
higher than that in Jingang 30 (Fig.7, panel A). It was inter-
esting to note that OsiWRKY transcription was also
induced in the mock-inoculated plants of both varieties
(Fig.7, panel C). However, the timing of the induction was
delayed and the level of the induction was also lower com-
pared to those of OsiWRKY induced by pathogen infec-
tion (Fig.7, panel C).
3 Discussion
In higher plants, the data currently available on WRKY
genes support the view that they play important roles in
pant responses to biotic and abiotic stresses as well as in
plant developmental processes. Because higher plant
Fig.2. The nucleotide sequence of OsiWRKY cDNA and its deduced amino acid sequence. The 5 and 3 untranslated regions are italicized.
The putative nuclear localization signal is boxed. The residues forming the WRKY domain are represented by bold letters. The cysteine
and histidine residues making up the potential C2-H2 type zinc finger structure are underlined. The WRKY element, which is universally
conserved in all WRKY transcription factors identified so far, is marked beneath by a double line. Asterisk indicates the stop codon. The
OsiWRKY cDNA has been deposited in GenBank with the accession number AF459793.
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004960
genomes contain multiple WRKY genes that encode pro-
teins with various levels of similarities, systematic analy-
ses on the expression and function of WRKY genes in the
future would best be accomplished through using model
species (such as A. thaliana or rice) for which there are
more genetic resources and tools available. Important
progress has been made in A. thaliana in terms of anno-
tating a complete list of potential WRKY genes and evalu-
ating their transcriptional responses to the infection of a
bacterial pathogen. In contrast, less is known about WRKY
genes in rice. We searched the database of published rice
sequences and found more than 100 potential WRKY genes
in rice (unpublished data). As a prelude for more compre-
hensive studies of rice WRKY genes in the near future, we
conducted cDNA cloning and expression pattern analysis
of OsiWRKY.
The conservation in the coding sequences of the
WRKY domain allowed us to design a pair of degenerate
primers, with which we isolated by PCR a DNA fragment
coding for the WRKY domains of rice WRKY proteins.
Further screening of a cDNA library using the probe de-
rived from the amplified DNA fragment yielded three posi-
tive clones. Surprisingly, the inserts of the three clones all
came from the same gene (OsiWRKY). Theoretically, the
probe should cross-hybridize with cDNA clones represent-
ing additional rice WRKY genes. It is possible that in the
cDNA library used for our screening, the cDNA clones of
OsiWRKY is over represented.
Fig.3. Phyogenetic tree of OsiWRKY with nine closest matching proteins from a BLAST search. A. Comparison of the WRKY domains
in OsiWRKY and homologous Group Ⅱ-c WRKY proteins using multiple alignment of amino acid sequences. The WRKY element is in
bold form. The cysteine and histidine residues forming the potential C2-H2 type zinc finger structure are underlined. B. Phylogenetic
relationships of representative Group Ⅱ-c WRKY proteins from three plant species by clustering analysis using the neighbor joining
method (Nei and Kumar, 2000). During the clustering analysis, the Poisson correction (PC) distance and the pairwise deletion option
(with respect to gaps present in the aligned sequences) are adopted. The scale bar indicates the calculated distances in among the
compared sequences. The GenBank accession numbers for the WRKY proteins are AF404855 (AtWRKY8), AF404857 (AtWRKY12),
AF421153 (AtWRKY13), AY052647 (AtWRKY23), AF442393 (AtWRKY28), AF442397 (AtWRKY48), AF421155 (AtWRKY68),
AF421158 (AtWRKY71), AB063576 (NtWRKY9) and AF459793 (OsiWRKY).
GUO Ze-Jian et al.: Characterization of a Rice WRKY Gene Whose Expression Is Induced upon Pathogen Attack and Mechanical
Wounding 961
The deduced OsiWRKY protein contained structural
motifs reminiscent of previously studied WRKY transcrip-
tion factors. However, only one WRKY domain with a typi-
cal zinc finger structure (C-X4-C-X23-H-X1-H) was found
in OsiWRKY, suggesting that it belongs to Group Ⅱ-c
WRKY transcription factors. A putative NLS was predicted
in front of the WRKY domain in OsiWRKY and the
OsiWRKY::GFP fusion protein was localized to the nucleus
of the rice cell. However, further mutagenesis experiments
are needed to verify if the predicted NLS is indeed in-
volved in targeting OsiWRKY to the nuclear compartment.
We did not find potential coiled coil region or leucine zip-
per motif within the primary structure of OsiWRKY, indi-
cating that the mechanism of OsiWRKY function may dif-
fer from those WRKY proteins that contain coiled coil re-
gions or leucine zippers.
Our EMSA experiments demonstrated that the
Fig.4. Determination of the copy number of OsiWRKY in the
genomes of four rice varieties by Southern blotting analysis. The
genomic DNA samples from IR-26 (lane 1), Jingang 30 (lane 2) or
Xiushui 11 (lane 3) were digested with EcoRⅠ, whereas the
samples from IR-72 were digested with BamHⅠ (lane 4), EcoRⅠ
(lane 5), or SalⅠ (lane 6). DNA markers are indicated.
Fig.5. Subcellular localization of OsiWRKY using particle gun-
mediated transient expression. The bright field and fluorescent
images of a representative rice cell expressing the OsiWRKY::
GFP fusion protein are shown in A and B, respectively. In this
cell, GFP fluorescence is localized to the nucleus (arrowed). In
contrast, in a representative cell expressing the GFP fusion pro-
tein alone C, GFP fluorescence is distributed more generally in
the cytoplasm D except for the central vacuole (indicated by the
letter “V”). Bar: 7 mm.
Fig.6. Interaction between recombinant OsiWRKY and W-box
element as assessed by electrophoretic mobility shift assay
(EMSA). OsiWRKY bound strongly to the 32P-labeled, W-box
containing DNA fragment PitaF (lane 1). This binding was not
substantially affected by the presence of an excess (×100) amount
of the mPitaF fragment in which the W-box element had been
mutated (lane 2). However, the inclusion of an excess (×100)
amount of cold PitaF diminished significantly the binding of
OsiWRKY to 32P-labeled PitaF (lane 3). There was no interaction
between OsiWRKY and 32P-labeled mPitaF (“#” indicates 32P-
labeled mPitaF which was used for EMSA). + and – indicate the
reagent which was used or was not used in the reaction,
respectively.
Acta Botanica Sinica 植物学报 Vol.46 No.8 2004962
OsiWRKY recombinant protein bound specifically to the
W-box cis-element having the TGAC core sequence. Al-
though originally identified as an elicitor-responsive ele-
ment in parsley PR1-1, a member of PR-10 class of genes,
the W-box element has also now been found in the pro-
moters of many defense-related genes (Eulgem et al., 2000).
WRKY proteins are demonstrated to bind with the DNA
fragments containing the W-box core sequence in vitro,
although the binding activity is dependent on the protein
structure, the number of W-box, and the space between
the core sequences (Hara et al., 2000; Maeo et al., 2001;
Yu et al., 2001). Mutations in the W-box sequences of
NPR1 promoter of Arabidopsis compromise the ability of
NPR1 gene to complement npr1 mutants for salicylic acid-
induced defense gene expression and disease resistance
(Yu et al., 2001), indicating in vivo the importance of W-
box elements in the positive regulation of NPR1 expres-
sion during the activation of plant defense responses. The
PitaF probe used in this study was from the promoter of
the Pi-ta resistance gene of rice against M. grisea (Bryan
et al., 2000), whether OsiWRKY involves in the regulation
of Pita expression in vivo needs to be verified.
Evidence is accumulating to indicate that wounding
and pathogen responses in plants share a number of
components in their signal transduction pathways
(Cheong et al., 2002). The expression of OsiWRKY was
induced by both rice bacterial blight pathogen and me-
chanical wounding caused by mock inoculation (Fig.7),
indicating the possibility of OsiWRKY is involved in the
two signaling pathways. However, the following
observations suggest the mechanisms of OsiWRKY tran-
script induction in response to pathogen attack and me-
chanical wounding may be different. First, the level of
OsiWRKY expression was higher in the resistant rice vari-
ety (IR-26) than in the susceptible one (Jingang 30).
Second, induction of OsiWRKY transcription by pathogen
attack occurred much earlier and stronger than by mechani-
cal wounding. In this respect, a dual function for OsiWRKY
in regulating plant defense responses against pathogen
and mechanical wounding using differing mechanisms may
also be alike. To distinguish the different functions and
mechanisms of OsiWRKY in future research, it will be nec-
essary to prepare knock-out or knock-down mutants of
OsiWRKY, and to examine the phenotypes of the mutants.
Acknowledgements: We thank Prof. PENG You-Liang
(China Agricultural University, Beijing) for providing the
rice cDNA library used for isolating OsiWRKY cDNA in
this work. In addition, we are grateful to Dr. ZHAO Xin-
Hua (China National Rice Research Institute, Hangzhou)
and Prof. CHAI Rong-Yao (Zhejiang Agricultural Institute,
Hangzhou) for providing Xanthomonas oryzae pv. oryzae
strain and help with the bacterial inoculation.
References:
Bryan G T, Wu K S, Farrall L, Jia Y, Hershey H P, McAdams S
A, Faulk K N, Donaldson G K, Tarchini R, Valent B. 2000. A
single amino acid difference distinguishes resistant and sus-
ceptible alleles of the rice blast resistance gene Pi-ta. Plant
Cell, 12: 2033-2045.
Fig.7. Induction of OsiWRKY expression by Xanthomonas oryzae pv. oryzae and mock inoculations. Total RNA samples were
extracted from the resistant variety IR-26 and the susceptible variety Jingang 30 treated with pathogen or mock inoculations at seven time
points (0, 1, 3, 6, 12, 24, 48 h). They were separated on agarose gels, blotted onto nylon membrane filters (by capillary transfer) and
hybridized with 32P-labeled OsiWRKY specific probe. Panels A and C depict the results of the hybridization signals. Panels B and D show
ribosome RNA bands of separated total RNA samples in the ethidium bromide-stained agarose gels before capillary transfer, demonstrat-
ing equal loading of total RNA samples in the gel blotting analysis.
GUO Ze-Jian et al.: Characterization of a Rice WRKY Gene Whose Expression Is Induced upon Pathogen Attack and Mechanical
Wounding 963
Cheong Y H, Chang H S, Gupta R, Wang X, Zhu T, Luan S. 2002.
Transcriptional profiling reveals novel interactions between
wounding, pathogen, abiotic stress, and hormonal responses
in Arabidopsis. Plant Physiol, 129: 661-677.
de Pater S, Greco V, Pham K, Memelink J, Kijne J. 1996.
Characterization of a zinc-dependent transcriptional activator
from Arabidopsis. Nucleic Acids Res, 24: 4624-4631.
Dong J, Chen C, Chen Z. 2003. Expression profiles of the
Arabidopsis WRKY gene superfamily during plant defense
response. Plant Mol Biol, 51: 21-37.
Du L, Chen Z. 2000. Identification of genes encoding receptor-
like protein kinases as possible targets of pathogen- and sali-
cylic acid-induced WRKY DNA-binding proteins in
Arabidopsis. Plant J, 24: 837-847.
Eulgem T, Rushton P J, Robatzek S, Somssich I E. 2000. The
WRKY superfamily of plant transcription factors. Trends
Plant Sci, 5: 199-206.
Huang T, Duman J G. 2002. Cloning and characterization of a
thermal hysteresis (antifreeze) protein with DNA-binding
activity from winter bittersweet nightshade, Solanum
dulcamara. Plant Mol Biol, 48: 339-350.
Ishiguro S, Nakamura K. 1994. Characterization of a cDNA en-
coding a novel DNA-binding protein, SPF1, that recognizes
SP8 sequences in the 5 upstream regions of genes coding for
sporamin and β-amylase from sweet potato. Mol Gen Genet,
244: 563-571.
Hara K, Yagi M, Kusano T, Sano H. 2000. Rapid systemic accu-
mulation of transcripts encoding a tobacco WRKY transcrip-
tion factor upon wounding. Mol Gen Genet, 263: 30-37.
Hinderhofer K, Zentgraf U. 2001. Identification of a transcrip-
tion factor specifically expressed at the onset of leaf senescence.
Planta, 213: 469-473.
Kan Y-C, Liu S-W , Guo Z-J, Li D-B. 2002. Characterization of a
cyclophilin cDNA from soybean cells. Acta Bot Sin, 44: 173-
176.
Kim C Y, Lee S H, Park H C, Bae C G, Cheong Y H, Choi Y J, Han
C, Lee S Y, Lim C O, Cho M J. 2000. Identification of rice
blast fungal elicitor-responsive genes by differential display
analysis. Mol Plant Microbe In, 13: 470-474.
Maeo K, Hayashi S, Kojima-Suzuki H, Morikami A, Nakamura
K. 2001. Role of conserved residues of the WRKY domain in
the DNA-binding of tobacco WRKY family proteins. Biosci
Biotechnol Biochem, 65: 2428-2436.
Meier I, Hahlbrock K, Somssich I E. 1991. Elicitor-inducible and
constitutive in vivo DNA footprints indicate novel cis-acting
elements in the promoter of a parsley gene encoding patho-
genesis-related protein 1. Plant Cell, 3: 309-315.
Murray M G, Thompson W F. 1980. Rapid iso1ation of high
mo1ecular weight plant DNA. Nucleic Acids Res, 8: 4321-
4325.
Nei N, Kumar S. 2000. Molecular Evolution and Phylogenetics.
Oxford, UK: Oxford University Press.
Robatzek S, Somssich I E. 2002. Targets of AtWRKY6 regulation
during plant senescence and pathogen defense. Gene Dev, 16:
1139-1149.
Rushton P J, Macdonald H, Huttly A K, Lazarus C M, Hooley R.
1995. Members of a new family of DNA-binding proteins
bind to a conserved cis element in the promoters of a-Amy2
genes. Plant Mol Biol, 29: 691-702.
Rushton P J, Torres J T, Parniske M, Wernert P, Hahlbrock K,
Somssich I E. 1996. Interaction of elicitor-induced DNA-bind-
ing proteins with elicitor response elements in the promoters
of parsley PR1 genes. EMBO J, 15: 5690-5700.
Sambrook J, Fritsch E F, Maniatis T. 1989. Molecular Cloning: a
Laboratory Manual. 2nd ed. New York: Cold Spring Harbor
Laboratory Press.
Singh K B, Foley R C, Onate-Sanchez L. 2002. Transcription
factors in plant defense and stress responses. Curr Opin Plant
Biol, 5: 430-436.
Yang Y, Shah J, Klessig D F. 1997. Signal perception and trans-
duction in plant defense responses. Gene Dev, 11: 1621-
1639.
Yoda H, Ogawa M, Yamaguchi Y, Koizumi N, Kusano T, Sano H.
2002. Identification of early-responsive genes associated with
the hypersensitive response to tobacco mosaic virus and char-
acterization of a WRKY-type transcription factor in tobacco
plants. Mol Gen Genet, 267: 154-161.
Yu D, Chen C, Chen Z. 2001. Evidence for an important role of
WRKY DNA binding proteins in the regulation of NPR1 gene
expression. Plant Cell, 13: 1527-1539.
(Managing editor: ZHAO Li-Hui)