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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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In vitro phosphorylation of OsWRKY53 by chitin-responsive OsMPKs.A, Putative MAPK phosphorylation sites in the SP cluster region of OsWRKY53, the loss-of-phosphorylation OsWRKY53 mutant with all 6 Ser substituted to Ala (OsWRKY53SA), and the phospho-mimicking OsWRKY53 mutant with all 6 Ser substituted to Asp (OsWRKY53SD). B, In vitro phosphorylation of OsWRKY53 by OsMPK3 and OsMPK6. Recombinant His-OsWRKY53 and His-OsWRKY53SA proteins were used as the substrate for rice mitogen-activated protein kinases (OsMPKs) activated by a constitutively active form of the rice MAP kinase OsMKK4DD. Proteins separated by SDS-PAGE were blotted on membrane and probed with a Phostag-biotin antibody (top panel). The arrowhead indicates the position of phosphorylated OsWRKY53 (P-OsWRKY53). The membranes were reprobed followed by probing with an anti-His antibody to detect added substrate OsWRKY53 and OsWRKY53SA proteins (middle panel). OsMPKs in the reaction mixtures were also detected by immunoblot analysis with anti-OsMPK3 and anti-OsMPK6 antiserum (bottom panel). WT, native His-OsWRKY53; SA, His-OsWRKY53SA.
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pone-0098737-g001: In vitro phosphorylation of OsWRKY53 by chitin-responsive OsMPKs.A, Putative MAPK phosphorylation sites in the SP cluster region of OsWRKY53, the loss-of-phosphorylation OsWRKY53 mutant with all 6 Ser substituted to Ala (OsWRKY53SA), and the phospho-mimicking OsWRKY53 mutant with all 6 Ser substituted to Asp (OsWRKY53SD). B, In vitro phosphorylation of OsWRKY53 by OsMPK3 and OsMPK6. Recombinant His-OsWRKY53 and His-OsWRKY53SA proteins were used as the substrate for rice mitogen-activated protein kinases (OsMPKs) activated by a constitutively active form of the rice MAP kinase OsMKK4DD. Proteins separated by SDS-PAGE were blotted on membrane and probed with a Phostag-biotin antibody (top panel). The arrowhead indicates the position of phosphorylated OsWRKY53 (P-OsWRKY53). The membranes were reprobed followed by probing with an anti-His antibody to detect added substrate OsWRKY53 and OsWRKY53SA proteins (middle panel). OsMPKs in the reaction mixtures were also detected by immunoblot analysis with anti-OsMPK3 and anti-OsMPK6 antiserum (bottom panel). WT, native His-OsWRKY53; SA, His-OsWRKY53SA.

Mentions: First, we focused on potential MAPK phosphorylation sites of OsWRKY53, especially Ser or Thr residues followed by Pro, which is a minimal consensus motif for MAPK phosphorylation [27], [28]. The deduced amino acid sequence of OsWRKY53 possessed 6 SP and 2 TP motifs, 6 of which were concentrated in the N-terminal region as the SP cluster conserved among several group I WRKY proteins (Fig. 1A and Fig. S1). To investigate whether OsWRKY53 was phosphorylated by rice MAPKs, OsMPK3, OsMPK6 and a Thioredoxin-His-tagged OsWRKY53 recombinant proteins were expressed and purified from E. coli, and subjected to in vitro MAPK phosphorylation assay. As shown in Fig. 1B OsWRKY53 was phosphorylated by recombinant OsMPK3 and OsMPK6 activated by a constitutively active form of OsMKK4, OsMKK4DD. We also confirmed this phosphorylation did not occur without OsMPK3 and OsMPK6.


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)

In vitro phosphorylation of OsWRKY53 by chitin-responsive OsMPKs.A, Putative MAPK phosphorylation sites in the SP cluster region of OsWRKY53, the loss-of-phosphorylation OsWRKY53 mutant with all 6 Ser substituted to Ala (OsWRKY53SA), and the phospho-mimicking OsWRKY53 mutant with all 6 Ser substituted to Asp (OsWRKY53SD). B, In vitro phosphorylation of OsWRKY53 by OsMPK3 and OsMPK6. Recombinant His-OsWRKY53 and His-OsWRKY53SA proteins were used as the substrate for rice mitogen-activated protein kinases (OsMPKs) activated by a constitutively active form of the rice MAP kinase OsMKK4DD. Proteins separated by SDS-PAGE were blotted on membrane and probed with a Phostag-biotin antibody (top panel). The arrowhead indicates the position of phosphorylated OsWRKY53 (P-OsWRKY53). The membranes were reprobed followed by probing with an anti-His antibody to detect added substrate OsWRKY53 and OsWRKY53SA proteins (middle panel). OsMPKs in the reaction mixtures were also detected by immunoblot analysis with anti-OsMPK3 and anti-OsMPK6 antiserum (bottom panel). WT, native His-OsWRKY53; SA, His-OsWRKY53SA.
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Related In: Results  -  Collection

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

pone-0098737-g001: In vitro phosphorylation of OsWRKY53 by chitin-responsive OsMPKs.A, Putative MAPK phosphorylation sites in the SP cluster region of OsWRKY53, the loss-of-phosphorylation OsWRKY53 mutant with all 6 Ser substituted to Ala (OsWRKY53SA), and the phospho-mimicking OsWRKY53 mutant with all 6 Ser substituted to Asp (OsWRKY53SD). B, In vitro phosphorylation of OsWRKY53 by OsMPK3 and OsMPK6. Recombinant His-OsWRKY53 and His-OsWRKY53SA proteins were used as the substrate for rice mitogen-activated protein kinases (OsMPKs) activated by a constitutively active form of the rice MAP kinase OsMKK4DD. Proteins separated by SDS-PAGE were blotted on membrane and probed with a Phostag-biotin antibody (top panel). The arrowhead indicates the position of phosphorylated OsWRKY53 (P-OsWRKY53). The membranes were reprobed followed by probing with an anti-His antibody to detect added substrate OsWRKY53 and OsWRKY53SA proteins (middle panel). OsMPKs in the reaction mixtures were also detected by immunoblot analysis with anti-OsMPK3 and anti-OsMPK6 antiserum (bottom panel). WT, native His-OsWRKY53; SA, His-OsWRKY53SA.
Mentions: First, we focused on potential MAPK phosphorylation sites of OsWRKY53, especially Ser or Thr residues followed by Pro, which is a minimal consensus motif for MAPK phosphorylation [27], [28]. The deduced amino acid sequence of OsWRKY53 possessed 6 SP and 2 TP motifs, 6 of which were concentrated in the N-terminal region as the SP cluster conserved among several group I WRKY proteins (Fig. 1A and Fig. S1). To investigate whether OsWRKY53 was phosphorylated by rice MAPKs, OsMPK3, OsMPK6 and a Thioredoxin-His-tagged OsWRKY53 recombinant proteins were expressed and purified from E. coli, and subjected to in vitro MAPK phosphorylation assay. As shown in Fig. 1B OsWRKY53 was phosphorylated by recombinant OsMPK3 and OsMPK6 activated by a constitutively active form of OsMKK4, OsMKK4DD. We also confirmed this phosphorylation did not occur without OsMPK3 and OsMPK6.

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.

Show MeSH
Related in: MedlinePlus