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Overexpression of phosphomimic mutated OsWRKY53 leads to enhanced blast resistance in rice.

Chujo T, Miyamoto K, Ogawa S, Masuda Y, Shimizu T, Kishi-Kaboshi M, Takahashi A, Nishizawa Y, Minami E, Nojiri H, Yamane H, Okada K - PLoS ONE (2014)

Bottom Line: WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses.An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster).When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

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Phosphorylation of OsWRKY53 does not alter its W-box binding ability.A, W-box-specific DNA-binding activity of OsWRKY53. GMSA assay was performed using purified recombinant OsWRKY53 protein and 32P-labeled W-box probe containing the W-box cis-elements in the OsWRKY53 promoter. The specificity of the W-box binding activity was demonstrated by competition assay using 125-fold excess amount of unlabeled W-box probe (W) and mutated W-box probe (mW). B, Phosphorylation of OsWRKY53 does not enhance its W-box binding activity. Purified recombinant OsWRKY53 protein was phosphorylated using the OsMPK6 activated by OsMKK4DD. GMSA was performed as in A. C, W-box binding activity of OsWRKY53 variant proteins. Purified recombinant OsWRKY53, OsWRKY53SA and OsWRKY53SD proteins were subjected to GMSA. GMSA was performed as in A. WT, native His-OsWRKY53; SA, His-OsWRKY53SA; SD, His-OsWRKY53SD.
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pone-0098737-g002: Phosphorylation of OsWRKY53 does not alter its W-box binding ability.A, W-box-specific DNA-binding activity of OsWRKY53. GMSA assay was performed using purified recombinant OsWRKY53 protein and 32P-labeled W-box probe containing the W-box cis-elements in the OsWRKY53 promoter. The specificity of the W-box binding activity was demonstrated by competition assay using 125-fold excess amount of unlabeled W-box probe (W) and mutated W-box probe (mW). B, Phosphorylation of OsWRKY53 does not enhance its W-box binding activity. Purified recombinant OsWRKY53 protein was phosphorylated using the OsMPK6 activated by OsMKK4DD. GMSA was performed as in A. C, W-box binding activity of OsWRKY53 variant proteins. Purified recombinant OsWRKY53, OsWRKY53SA and OsWRKY53SD proteins were subjected to GMSA. GMSA was performed as in A. WT, native His-OsWRKY53; SA, His-OsWRKY53SA; SD, His-OsWRKY53SD.

Mentions: It has been reported that phosphorylation of the group I WRKY proteins NtWRKY1 and NbWRKY8 by MAP kinase enhanced their W-box specific DNA-binding activity [29], [34]. To investigate whether phosphorylation of OsWRKY53 by OsMPK3/OsMPK6 enhanced W-box binding activity, we performed a gel mobility shift assay (GMSA). First, we analyzed the W-box-specific DNA binding activity of OsWRKY53 by using a W-box probe derived from the native promoter region of this gene. When incubated with the OsWRKY53 recombinant protein, a retarded band was observed, and this retardation of the labeled probe was abolished by competition using an unlabeled probe. In contrast, an unlabeled W-box mutated probe could not compete with the labeled probe (Fig. 2A).


Overexpression of phosphomimic mutated OsWRKY53 leads to enhanced blast resistance in rice.

Chujo T, Miyamoto K, Ogawa S, Masuda Y, Shimizu T, Kishi-Kaboshi M, Takahashi A, Nishizawa Y, Minami E, Nojiri H, Yamane H, Okada K - PLoS ONE (2014)

Phosphorylation of OsWRKY53 does not alter its W-box binding ability.A, W-box-specific DNA-binding activity of OsWRKY53. GMSA assay was performed using purified recombinant OsWRKY53 protein and 32P-labeled W-box probe containing the W-box cis-elements in the OsWRKY53 promoter. The specificity of the W-box binding activity was demonstrated by competition assay using 125-fold excess amount of unlabeled W-box probe (W) and mutated W-box probe (mW). B, Phosphorylation of OsWRKY53 does not enhance its W-box binding activity. Purified recombinant OsWRKY53 protein was phosphorylated using the OsMPK6 activated by OsMKK4DD. GMSA was performed as in A. C, W-box binding activity of OsWRKY53 variant proteins. Purified recombinant OsWRKY53, OsWRKY53SA and OsWRKY53SD proteins were subjected to GMSA. GMSA was performed as in A. WT, native His-OsWRKY53; SA, His-OsWRKY53SA; SD, His-OsWRKY53SD.
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Related In: Results  -  Collection

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Show All Figures
getmorefigures.php?uid=PMC4043820&req=5

pone-0098737-g002: Phosphorylation of OsWRKY53 does not alter its W-box binding ability.A, W-box-specific DNA-binding activity of OsWRKY53. GMSA assay was performed using purified recombinant OsWRKY53 protein and 32P-labeled W-box probe containing the W-box cis-elements in the OsWRKY53 promoter. The specificity of the W-box binding activity was demonstrated by competition assay using 125-fold excess amount of unlabeled W-box probe (W) and mutated W-box probe (mW). B, Phosphorylation of OsWRKY53 does not enhance its W-box binding activity. Purified recombinant OsWRKY53 protein was phosphorylated using the OsMPK6 activated by OsMKK4DD. GMSA was performed as in A. C, W-box binding activity of OsWRKY53 variant proteins. Purified recombinant OsWRKY53, OsWRKY53SA and OsWRKY53SD proteins were subjected to GMSA. GMSA was performed as in A. WT, native His-OsWRKY53; SA, His-OsWRKY53SA; SD, His-OsWRKY53SD.
Mentions: It has been reported that phosphorylation of the group I WRKY proteins NtWRKY1 and NbWRKY8 by MAP kinase enhanced their W-box specific DNA-binding activity [29], [34]. To investigate whether phosphorylation of OsWRKY53 by OsMPK3/OsMPK6 enhanced W-box binding activity, we performed a gel mobility shift assay (GMSA). First, we analyzed the W-box-specific DNA binding activity of OsWRKY53 by using a W-box probe derived from the native promoter region of this gene. When incubated with the OsWRKY53 recombinant protein, a retarded band was observed, and this retardation of the labeled probe was abolished by competition using an unlabeled probe. In contrast, an unlabeled W-box mutated probe could not compete with the labeled probe (Fig. 2A).

Bottom Line: WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses.An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster).When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster.

View Article: PubMed Central - PubMed

Affiliation: Biotechnology Research Center, The University of Tokyo, Bunkyo-ku, Tokyo, Japan.

ABSTRACT
WRKY transcription factors and mitogen-activated protein kinase (MAPK) cascades have been shown to play pivotal roles in the regulation of plant defense responses. We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus. In this study, we identified OsWRKY53 as a substrate of OsMPK3/OsMPK6, components of a fungal PAMP-responsive MAPK cascade in rice, and analyzed the effect of OsWRKY53 phosphorylation on the regulation of basal defense responses to a virulence race of rice blast fungus Magnaporthe oryzae strain Ina86-137. An in vitro phosphorylation assay revealed that the OsMPK3/OsMPK6 activated by OsMKK4 phosphorylated OsWRKY53 recombinant protein at its multiple clustered serine-proline residues (SP cluster). When OsWRKY53 was coexpressed with a constitutively active mutant of OsMKK4 in a transient reporter gene assay, the enhanced transactivation activity of OsWRKY53 was found to be dependent on phosphorylation of the SP cluster. Transgenic rice plants overexpressing a phospho-mimic mutant of OsWRKY53 (OsWRKY53SD) showed further-enhanced disease resistance to the blast fungus compared to native OsWRKY53-overexpressing rice plants, and a substantial number of defense-related genes, including pathogenesis-related protein genes, were more upregulated in the OsWRKY53SD-overexpressing plants compared to the OsWRKY53-overexpressing plants. These results strongly suggest that the OsMKK4-OsMPK3/OsMPK6 cascade regulates transactivation activity of OsWRKY53, and overexpression of the phospho-mimic mutant of OsWRKY53 results in a major change to the rice transcriptome at steady state that leads to activation of a defense response against the blast fungus in rice plants.

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Related in: MedlinePlus