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Plant Physiology 132:1961-1972 (2003) © 2003 American Society of Plant Biologists BWMK1, a Rice Mitogen-Activated Protein Kinase, Locates in the Nucleus and Mediates Pathogenesis-Related Gene Expression by Activation of a Transcription Factor1Division of Applied Life Science (BK21 Program) and Plant Molecular Biology and Biotechnology Research Center, Gyeongsang National University, Jinju 660–701, Korea
Mitogen-activated protein kinase (MAPK) cascades are known to transduce plant defense signals, but the downstream components of the MAPK have as yet not been elucidated. Here, we report an MAPK from rice (Oryza sativa), BWMK1, and a transcription factor, OsEREBP1, phosphorylated by the kinase. The MAPK carries a TDY phosphorylation motif instead of the more common TEY motif in its kinase domain and has an unusually extended C-terminal domain that is essential to its kinase activity and translocation to the nucleus. The MAPK phosphorylates OsEREBP1 that binds to the GCC box element (AGCCGCC) of the several basic pathogenesis-related gene promoters, which in turn enhances DNA-binding activity of the factor to the cis element in vitro. Transient co-expression of the BWMK1 and OsEREBP1 in Arabidopsis protoplasts elevates the expression of the  -glucuronidase reporter gene driven by
the GCC box element. Furthermore, transgenic tobacco (Nicotiana
tabacum) plants overexpressing BWMK1 expressed many
pathogenesis-related genes at higher levels than wild-type plants with an
enhanced resistance to pathogens. These findings suggest that MAPKs contribute
to plant defense signal transduction by phosphorylating one or more
transcription factors.
Mitogen-activated protein kinase (MAPK) cascades are known to play essential roles in the signal transduction pathways involved in numerous eukaryotic cellular processes from cell division to cell death (Davis, 2000  ;
Ligterink and Hirt, 2001). In
the last few years, it has become apparent that MAPK cascades also play vital
roles in signal transduction pathways of plants, including plant defense
signaling (Innes, 2001;
Tena et al., 2001;
Zhang and Klessig, 2001). The
Arabidopsis genome sequence has revealed the presence of 23 MAPK genes in the
genome, which suggests that the MAPK cascades in plants may be quite
complex.
Accumulating lines of evidence indicate that plants rapidly activate MAPKs
when exposed to a variety of abiotic and biotic stress stimuli
(Ligterink et al., 1997
Understanding the transcriptional regulation of defense-associated genes is
important in helping improve the disease resistance mechanisms in plants
(Dangl and Jones, 2001
To date, some MAPKs have been identified and characterized from rice
(Oryza sativa; He et al.,
1999
Here, we report our molecular and functional analysis of a rice MAPK,
BWMK1. This protein phosphorylates the rice transcription factor OsEREBP1
(rice ethylene-responsive element-binding protein 1; accession no. AF193803).
Such EREBPs are known to bind to the GCC box DNA motif (AGCCGCC) that is
located in the promoter of several PR genes. BWMK1 is localized in the nucleus
and is activated by a fungal elicitor. In vitro phosphorylation of OsEREBP1 by
BWMK1 enhanced its ability to bind to the GCC box. Transient co-expression of
BWMK1 with OsEREBP1 in Arabidopsis protoplasts improved the expression of the
BWMK1 Belongs to a New Family of Plant MAPKs
To isolate the rice MAPKs that are induced by a fungal elicitor, we
screened the full-length cDNA from fungal elicitor-treated rice cDNA library
using the MAP kinase fragment that resulted from reverse transcriptase-PCR
using degenerate oligonucleotide primers corresponding to highly conserved
regions found in plant Ser/Thr protein kinases, namely, LREIKLCRM and
DVWSVGCIF. The longest cDNA consisted of 2,032 bp that contained full-length
cDNA (EMBL/GenBank accession no. AF194415) encoding a 55.7-kD protein. The
deduced amino acid sequence of the protein contains MAPK motifs, and we
designated it as OsMAPK1 (rice MAPK isoform 1). After we registered the
OsMAPK1 clone, an identical rice blast- and wounding-activated MAP kinase
(BWMK1, accession no. AF177392) was reported by He et al.
(1999
BWMK1 is composed of an N-terminal kinase domain (KD) and an unusually long
C-terminal extension domain (CD) that contains a putative Leu zipper motif
(He et al., 1999
The phylogenetic tree resulting from comparisons of deduced amino acid
sequences indicates that plant MAPKs can be grouped into at least five
distinct families (Fig. 1).
Among them, the MAPKs in families I and II are mostly involved in pathogen and
abiotic stress signalings, whereas some family III MAPKs are involved in cell
cycle regulation (Zhang and Klessig,
2001
To characterize the BWMK1, we produced glutathione S-transferase
(GST) fusion proteins containing full-length, KD or CD in Escherichia
coli and then purified the GST fusion proteins
(Fig. 2A). Before we performed
the kinase activity assay with purified GST fusion proteins, amounts of
purified proteins was checked by loading with 10% (w/v) SDS-PAGE (data not
shown). As shown in Figure 2B, the full-length protein was able to phosphorylate both MBP and itself.
However, both phosphorylation activities were completely destroyed when the CD
was deleted, indicating that it is essential for the kinase activity of BWMK1.
Similar observations have been made for other protein kinases, such as Nlk
(Brott et al., 1998
To determine the subcellular localization of the MAPK in vivo, the green
fluorescent protein (smGFP) gene
(Davis and Vierstra, 1998
To determine whether BWMK1 is activated in response to pathogen signals, we
performed immunocomplex kinase assays using Ab-pNBWMK1, an antibody that was
raised against a synthetic peptide representing the N-terminal 15 amino acids
of BWMK1. Before using the antibody for the further analysis, specificity of
antibody was assessed by immunoblot analysis against a panel of different
GST-fused BWMK1 proteins as described in
Figure 2A. The anti-pNBWMK1
antibody recognized the GST-BWMK1 and GST-BWMK1 KD but not the GST-BWMK1 CD
and GST protein (Fig. 3B). To
verify the antibody specificity, we tested the immunoblot analysis using the
plant crude extract from rice (Fig.
3C). As shown in Figure
3C, the major band (more than 90%) that was recognized with
antibody was similar in molecular size, which was estimated by deduced amino
acid of BWMK1. Thus, we used the antibody for immunocomplex kinase assay. The
kinase activity of BWMK1 was rapidly and transiently activated by several
defense signals, including a fungal elicitor, hydrogen peroxide
(H2O2), salicylic acid (SA), jasmonic acid (JA), and
ethephon (Fig. 4A). The
activity peaked 5 to 30 min after treatment. Several plant MAPKs, including
SIPK (Zhang and Klessig, 1997
To examine the biological functions of the BWMK1 gene in plant
defense responses, we constructed transgenic tobacco plants that
constitutively express BWMK1 under the control of the cauliflower
mosaic virus (CaMV) 35S promoter. Transgenic plant lines expressing
BWMK1 were first selected by northern blotting and assayed for
phenotypic changes and PR gene expression. We obtained eight BWMK1-expressed
transgenic plants, and five lines among them showed the lesion mimic phenotype
and the enhancement of PR gene expression. We then selected two lines for
further analysis to test the resistance against pathogens. As shown in
Figure 5A, hypersensitive
response (HR)-like necrotic lesions formed on the rosette leaves of the
transgenic lines. The earliest cell death in the transgenic plants was
observed on the first rosette leaf 4 to 5 d after leaf emergence, and the
succeeding true leaves also exhibited cell death. HR-like cell death is
usually correlated with a variety of biochemical reactions, such as the
accumulation of autofluorescent compounds, callose deposition, and
lignification at and around the lesion sites
(Dixon et al., 1994
It has been shown that plants with spontaneous lesions often show an
elevated expression of the genes that encode PR proteins and an increased
resistance to pathogens (Dangl et al.,
1996
Because the transgenic plants showed elevated expression of PR genes with spontaneous lesions, we assayed whether the transgenic plants are better able to resist fungal and bacterial pathogens. We inoculated transgenic and wild-type plants with the oomycete pathogen Phytophthora parasitica var nicotianae. Five days later, the wild-type plants exhibited disease symptoms, but the transgenic plants did not. Seven days after inoculation, the wild-type plants had severe disease symptoms, including leaf wilting and stem rot, and most were dead 8 d after inoculation. However, the transgenic plants remained healthy without any appreciable disease symptoms (Fig. 6A). The transgenic plants also showed enhanced resistance to the virulent bacterial pathogen Pseudomonas syringae pv tabacci (Pst). At 5 d after inoculation, the in planta growth of Pst in transgenic plants was 100-fold less than in the wild-type plants (Fig. 6B). Thus, constitutive overexpression of BWMK1 appears to enhance resistance to a range of pathogens, probably because the expression of many PR genes is elevated.
It has been suggested that potential substrates for the protein kinases
activated in response to pathogen signals include DNA-binding proteins
(Droge-Laser et al., 1997
To further confirm that BWMK1 interacts with OsEREBP1, OsEREBP1 was fused to the GAL4 activation domain of pGAD424 (CLONTECH Laboratories, Palo Alto, CA), and the prey construct, AD-OsEREBP1, was introduced into the yeast strain pJ69-4A carrying the bait construct, BD-BWMK1. Yeast cells could grow on synthetic drop-out plates lacking Trp, Leu, and adenine, and expression of the LacZ reporter gene was observed only when they were provided with both AD-OsEREBP1 and BD-BWMK1. Yeast cells expressing either AD-OsEREBP1 or BD-BWMK1 alone could not grow on selection media, indicating specific interaction between AD-OsEREBP1 and BD-BWMK1 (Fig. 7B).
Recombinant BWMK1 protein has both auto- and MBP substrate phosphorylation activities (Fig. 2) and directly interacts with OsEREBP1. To examine whether the MAPK can phosphorylate OsEREBP1, we produced a recombinant OsEREBP1 fused to GST in E. coli. We then assessed whether GST-BWMK1 could phosphorylate GST-OsEREBP1. As shown in Figure 7C, BWMK1 did phosphorylate OsEREBP1. To test whether OsEREBP1 specifically binds to the GCC box (AGCCGCC) motif found in the promoter of several PR genes, we performed an electrophoresis mobility shift assay (EMSA) using a 32P-labeled GCC box (2xAGCCGCC) or a mutant GCC box (2xATC-CTCC) motif. OsEREBP1 specifically binds to the GCC box motif (Fig. 7D) but not to the GCC box mutant (data not shown). To determine if the phosphorylation of OsEREBP1 by BWMK1 affects its DNA-binding activity to the corresponding cis elements, we first phosphorylated OsEREBP1 with the BWMK1 protein and then examined its ability to bind to the GCC box (2xAGCCGCC) by EMSA. As shown in Figure 7D, the phosphorylation of OsEREBP1 by BWMK1 strongly enhanced its DNA-binding activity to the synthetic GCC box (2xAGCCGCC) motif in vitro. In addition, phosphorylation of GST-OsEREBP1 by the kinase in the presence of ATP also strongly enhanced its ability to bind to the GCC box motif (Fig. 7D, lane 5) relative to its activity in absence of ATP (Fig. 7D, lane 4). These observations strongly suggest that BWMK1 may regulate PR gene expression via the phosphorylation of one or more transcription factors.
To demonstrate that the enhanced in vitro DNA-binding activity of the
phosphorylated OsEREBP1 is also observed in vivo, we performed transient
transactivation assays using Arabidopsis leaf mesophyll protoplasts
(Abel and Theologis, 1994
Here, we report several important findings that provide new insights into the MAPK downstream signaling that is involved in plant defense mechanisms. First, we have isolated a unique MAPK, BWMK1, which is activated in rice by defense-related signals. Second, we have also isolated the substrate that BWMK1 probably phosphorylates in vivo, namely, OsEREBP1, which is a transcription factor that binds to the GCC box DNA motif found in the promoters driving several PR genes. The OsEREBP1 that is involved in plant defense signaling is supported by our experiments showing that BWMK1 phosphorylates OsEREBP1 in vitro and that this enhances the ability of OsEREBP1 to bind to the GCC box DNA motif.
The KD of BWMK1 contains a TDY phosphorylation motif instead of the more
common TEY sequence and a putative Leu zipper motif in its CD
(He et al., 1999
The protein kinase activity of BWMK1 was rapidly and transiently activated
by various pathogen signals, including a fungal elicitor,
H2O2, SA, JA, and ethylene. Peak activity was reached 5
to 30 min after treatment, after which BWMK1 transcript levels
increased. However, the protein levels of the kinase did not alter similarly
during this period (Fig. 4). It
is likely that the plant maintains a certain steady-state protein level of
MAPKs to allow it to rapidly respond to an unexpected pathogen attack and that
upon activation, the kinase is consumed, causing transcription of the kinase
gene to be stepped up to maintain the protein levels. Therefore, these
observations indicate that the pathogen signals activate the kinase by
posttranslational modification, which is also observed for other plant MAPKs
involved in defense responses, including SIPK, WIPK, and ERMK
(Ligterink et al., 1997
Because BWMK1 is activated in response to various defense signals
(Fig. 4) and is localized to
the nucleus (Fig. 2C), it is
possible that this kinase is involved in the transcriptional regulation of PR
gene expression by phosphorylating one or more transcription factors. We
isolated one possible transcription factor that could interact with BWMK1 by
yeast two-hybrid screening of a rice cDNA expression library. The
transcription factor belongs to the EREBP family, and we designated it as
OsEREBP1. The EREBP family consists of transcription factors that recognize
the GCC box DNA element present in the promoters of several PR genes
(Buttner and Singh, 1997 To further verify the relationship between BWMK1-mediated phosphorylation of the OsEREBP1 and PR gene expression, we transiently transfected Arabidopsis leaf mesophyll protoplasts with a GUS reporter gene fused to a synthetic tandem dimer of the GCC box DNA motif and the minimal promoter pDel.151-8. Co-expression with the OsEREBP1 effector plasmid driven by the CaMV 35S promoter transactivated GUS reporter gene expression about 2-fold. When the protoplasts were cotransfected with the reporter construct and both the OsEREBP1 and BWMK1 effector plasmids, the reporter gene expression was further elevated by 4-fold. These observations strongly suggest that BWMK1 enhances the expression of GCC box-driven PR genes by phosphorylating the OsEREBP1 transcription factor.
The GCC box motif is present in the PR2 and PR5 gene
promoter that is stimulated by both ethylene and JA
(Zhou et al., 1997
The Pto kinase in tomato plants phosphorylates the Pti4 protein. The
phosphorylation of Pti4 enhances its ability to bind to the GCC box contained
in several PR gene promoters (Gu et al.,
2000 In summary, BWMK1, which can be classified into family V MAPK, is activated by SA-associated defense signals and by ethylene/JA-associated plant defense signals. The MAPK phosphorylates the OsEREBP1, which in turn enhances the DNA-binding activity of the factor to the corresponding cis-acting element, GCC box motif (AGCCGCC), in several basic PR gene promoters. Transient cotransfection assays using Arabidopsis leaf protoplast support the in vitro DNA-binding result by elevating expression level of the GUS reporter gene about 4-fold when the reporter plasmid was cotransfected with both BWMK1 and OsEREBP1. Ectopic overexpression of the BWMK1 in transgenic tobacco plants causes HR-like cell death and increased resistance to pathogens with elevated level of PR gene expression. Thus, these results suggest that BWMK1 is involved in MAPK cascades in plant defense signal transduction via direct phosphorylation of a transcription factor(s).
Rice (Oryza sativa L. Milyang 117) Suspension Cell Culture and Treatments
Suspension cell lines of rice were cultured and maintained as described by
Kyozuka et al. (1990
The yeast two-hybrid screening method was employed to isolate transcription
factors that interact with BWMK1. We digested the BWMK1 cDNA with
SmaI and BclI and ligated the fragments into the pGBT9
plasmid, which contains the Trp1 selection marker (CLONTECH). The
prey library containing cDNA from rice suspension cells was constructed in
plasmid pAD-GAL4 (Stratagene, La Jolla, CA), which harbors the Leu2
selection marker. The yeast strain pJ69-4A
(James et al., 1996
Total RNA was isolated as described by Lee et al.
(1995
For expression in bacteria, full-length BWMK1, the KD (BWMK1 KD), and the CD (BWMK1 CD) were fused to the C terminus of GST. The GST-BWMK1 fusion construct was generated by digesting the full-length BWMK1 in pBluescript SK– with SmaI/BclI and inserting the excised fragment into the corresponding sites of the GST expression vector pGEX-2T (Amersham, Buckinghamshire, UK). An EcoRI fragment of BWMK1 in pBluescript SK– that encodes only the KD of BWMK1 was subcloned into the GST expression vector to generate GST-BWMK1 KD, whereas an EcoRI-XhoI fragment encoding only the CD was subcloned into the same expression vector to create GST-BWMK1 CD. The OsEREBP1 cDNA in pBluescript SK– was also digested with SmaI/XhoI and ligated into the pGEX-5X vector to generate GST-OsEREBP1. The resulting constructs were then introduced into Escherichia coli strain BL21 (pLysS), and the GST fusion proteins were expressed and purified using glutathione-agarose beads according to the manufacturer's instructions (Amersham).
Autophosphorylation activities of the full-length, KD and CD of BWMK1 were
assayed at different protein concentrations in 20 µL of reaction buffer (20
mM Tris-HCl [pH 7.5], 1 mM dithiothreitol, 10
mM MgCl2, and 100 µM ATP) containing 5
µCi [
A polyclonal antibody recognizing BWMK1 (Ab-pNBWMK1) was raised by immunizing rabbits with a synthetic peptide representing the NH2 terminus (MEFFTEYGEAASQYQ) of BWMK1. For immunoblot analysis, 50 µg of total protein per lane was resolved by 10% (w/v) SDS-PAGE. The separated proteins were then transferred to a nitrocellulose membrane (Amersham) by semidry electroblotting. After blocking the membrane at room temperature for 1 h in Tris-buffered saline containing 0.1% (v/v) Tween 20 buffer (20 mM Tris [pH 7.5], 150 mM NaCl, and 0.1% [v/v] Tween 20) with 6% (w/v) nonfat dry milk (Carnation, Glendale, CA), the membrane was incubated with 0.2 µg mL–1 Ab-pNBWMK1 for 1 h. The blot was then washed four times in TTBS buffer, incubated with a horseradish peroxidase-conjugated secondary antibody (1:5,000 [v/v] dilution), and developed by using an enhanced chemiluminescence kit (Amersham).
Preparation of protein extracts from rice suspension cells treated with
fungal elicitor or chemicals and immunocomplex kinase assay were performed as
described by Zhang and Klessig
(1997
For construction of transgenic tobacco (Nicotiana tabacum cv
Xanthi-nc) plants, BWMK1 cDNA was ligated into the plant binary
vector pGA643 (An et al.,
1988
The cell death phenotype of tobacco leaves was photographed by using a
dissecting microscope. Autofluorescent materials and callos deposition were
detected using an UV epifluorescence microscope as described by Dietrich et
al. (1994
A bacterial pathogen (Pseudomonas syringae pv tabacci)
was cultured overnight at 30°C in Kings B medium containing 50 µg
mL–1 rifampicin and 50 µg
mL–1 kanamycin. The bacterial culture was washed
twice with 10 mM MgCl2 and resuspended in 10
mM MgCl2. Bacterial density was determined by absorbance
at OD600 nm. Bacteria were diluted to the desired concentrations in
10 mM MgCl2 for inoculation. Six-week-old plants were
inoculated by vacuum infiltration, and the inoculated plants were kept in a
greenhouse. Leaf discs were ground in 10 mM MgCl2 and
plated on appropriate Kings B plates. The number of bacteria in the leaves was
calculated by counting cfu. Infection with Phytophthora parasitica pv
nicotianae was performed as described by Mittler et al.,
(1995
A synthetic GCC box (CAT AAG AGC CGC CAC TAA AAT AAG ACC GAT CAA ATA AGA
GCC GCC AT) and GCC box mutant (CAT AAG ATC CTC CAC TAA AAT AAG ACC GAT CAA
ATA AGA TCC TCC AT; Ohme-Takagi and
Shinshi, 1995
To observe the cellular localization of BWMK1, we prepared BWMK1-smGFP or
BWMK1 KD-smGFP fusion constructs. The BamHI (1–506 amino acids)
or EcoRI (1–324 amino acids) fragment of BWMK1 was
fused to the coding region of smGFP under the control of the CaMV 35S
promoter. The fusion construct of nuclear localization signal from simian
virus 40 large T antigen and red fluorescent protein (NLS-RFP) was used as a
positive control (Lee et al.,
2001
We thank Drs. Cris Lamb and John A. Ryals for generously providing us PR protein cDNAs. We also thank Dr. Jen Sheen for providing pHBT95 plasmid and Dr. Inhwan Hwang for providing NLS-RFP plasmid. Received March 6, 2003; returned for revision April 7, 2003; accepted May 9, 2003.
Article, publication date, and citation information can be found at http://www.plantphysiol.org/cgi/doi/10.1104/pp.103.023176.
1 This work was supported by Korea Science and Engineering Foundation (grant
no. 2000–2–20900–001–1), by Crop Functional Genomic
Center (grant no. CG1512), by National Research Laboratory (grant no.
2000–N–NL–01–C–236), and by the BK21 program
from the Ministry of Education to M.J.C.
2 These authors contributed equally to the paper. * Corresponding author; e-mail mjcho{at}nongae.gsnu.ac.kr; fax 82–55–759–9363.
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TOP
ABSTRACT
RESULTS
-32P] ATP (6,000 Ci
mmol–1; Amersham). The substrate kinase activity
of GST-BWMK1 was assayed at room temperature for 20 min in a final volume of
20 µL using one of the substrates, MBP or GST-OsEREBP1, at a final protein
concentration of 0.1 mg mL–1. Reactions were
terminated by the addition of 4x SDS sample buffer. The samples were
then analyzed by 12.5% (w/v) SDS-PAGE and subsequent autoradiography. 
