Limits...
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.

Show MeSH

Related in: MedlinePlus

Overexpression of a phosphorylation-mimic mutant of OsWRKY53 enhances the rice blast resistance of rice plants.A, Representative lesions of rice blast disease observed at 5 days post inoculation (dpi). Bars indicate 1 mm. B, Ratio of the classes of lesions in transgenic rice leaves infected with rice blast fungus Magnaporthe oryzae Ina86–137. Washed conidia of the blast fungus were suspended in 1 mM MES-NaOH (pH 5.7) and then were inoculated on leaves of W53-OX and W53SD-OX transgenic rice plants. The lesions were counted according to the classifications shown in A. Bars represent the ratio of lesions of each class to the total number of counted lesions in 3 or 4 individual leaf blades. Three independent experiments were performed, and a representative result is shown. Asterisks and daggers denote significant differences compared to W53-OX plants (*p<0.05; **p<0.01; ***p<0.001 vs. W53-OX #10; †p<0.05; †††p<0.001 vs. W53-OX #60, by One-way ANOVA with Tukey post hoc test) C, Development of blast disease in leaf blades evaluated by quantitating M. oryzae genomic DNA. The amount of M. oryzae 28S rDNA relative to rice genomic eEF1α DNA was determined by quantitative PCR analysis. Values are represented as mean values ±SE for 6 leaf blades. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test on log-transformed data). NT, non-transformant control rice plants; W53-OX, native OsWRKY53-overexpressing rice plants; W53SD-OX, phosphorylation-mimic mutant of OsWRKY53-overexpressing rice plants.
© Copyright Policy
Related In: Results  -  Collection

License
getmorefigures.php?uid=PMC4043820&req=5

pone-0098737-g004: Overexpression of a phosphorylation-mimic mutant of OsWRKY53 enhances the rice blast resistance of rice plants.A, Representative lesions of rice blast disease observed at 5 days post inoculation (dpi). Bars indicate 1 mm. B, Ratio of the classes of lesions in transgenic rice leaves infected with rice blast fungus Magnaporthe oryzae Ina86–137. Washed conidia of the blast fungus were suspended in 1 mM MES-NaOH (pH 5.7) and then were inoculated on leaves of W53-OX and W53SD-OX transgenic rice plants. The lesions were counted according to the classifications shown in A. Bars represent the ratio of lesions of each class to the total number of counted lesions in 3 or 4 individual leaf blades. Three independent experiments were performed, and a representative result is shown. Asterisks and daggers denote significant differences compared to W53-OX plants (*p<0.05; **p<0.01; ***p<0.001 vs. W53-OX #10; †p<0.05; †††p<0.001 vs. W53-OX #60, by One-way ANOVA with Tukey post hoc test) C, Development of blast disease in leaf blades evaluated by quantitating M. oryzae genomic DNA. The amount of M. oryzae 28S rDNA relative to rice genomic eEF1α DNA was determined by quantitative PCR analysis. Values are represented as mean values ±SE for 6 leaf blades. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test on log-transformed data). NT, non-transformant control rice plants; W53-OX, native OsWRKY53-overexpressing rice plants; W53SD-OX, phosphorylation-mimic mutant of OsWRKY53-overexpressing rice plants.

Mentions: Next, we tested the transformants for resistance to the blast fungus. The transgenic rice plants were grown in a growth chamber, inoculated with conidia of the blast fungus, and disease symptoms were characterized 5 days later. We confirmed as previously reported that W53-OX rice plants showed enhanced resistance against the blast fungus compared to non-transformed control (NT) rice plants (Fig. S3). Thus, we compared resistance to the blast fungus between W53-OX and W53SD-OX rice plants directly. We did not see statistically-significant differences in average numbers of lesions in the total infected area between 2 independent W53-OX and 2 independent W53SD-OX samples (Fig. S4). Therefore, we categorized the disease lesions into 3 classes (Fig. 4A) and compared the ratio of lesions between W53-OX and W53SD-OX rice plants. Interestingly, the ratio of necrotic spot lesions in leaves of the W53SD-OX plants was significantly increased relative to that of W53-OX plants (Fig. 4B). Moreover, the ratio of susceptible lesions in W53SD-OX plants was significantly decreased relative to W53-OX plants (Fig. 4B). We also quantified fungal biomass in leaves of NT, W53-OX and W53SD-OX rice plants. Quantitative genomic PCR analysis demonstrated that significantly reduced levels of fungal DNA were detected from W53SD-OX plants compared with W53-OX plants. It was also indicated that fungal biomass levels tend to be decreased in W53-OX plants compared with NT plants (Fig. 4C). These results strongly suggest that overexpression of a phospho-mimic mutant of OsWRKY53 further enhances the basal defense response against the virulence rice blast fungus compared to native OsWRKY53-overexpressing rice plants.


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)

Overexpression of a phosphorylation-mimic mutant of OsWRKY53 enhances the rice blast resistance of rice plants.A, Representative lesions of rice blast disease observed at 5 days post inoculation (dpi). Bars indicate 1 mm. B, Ratio of the classes of lesions in transgenic rice leaves infected with rice blast fungus Magnaporthe oryzae Ina86–137. Washed conidia of the blast fungus were suspended in 1 mM MES-NaOH (pH 5.7) and then were inoculated on leaves of W53-OX and W53SD-OX transgenic rice plants. The lesions were counted according to the classifications shown in A. Bars represent the ratio of lesions of each class to the total number of counted lesions in 3 or 4 individual leaf blades. Three independent experiments were performed, and a representative result is shown. Asterisks and daggers denote significant differences compared to W53-OX plants (*p<0.05; **p<0.01; ***p<0.001 vs. W53-OX #10; †p<0.05; †††p<0.001 vs. W53-OX #60, by One-way ANOVA with Tukey post hoc test) C, Development of blast disease in leaf blades evaluated by quantitating M. oryzae genomic DNA. The amount of M. oryzae 28S rDNA relative to rice genomic eEF1α DNA was determined by quantitative PCR analysis. Values are represented as mean values ±SE for 6 leaf blades. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test on log-transformed data). NT, non-transformant control rice plants; W53-OX, native OsWRKY53-overexpressing rice plants; W53SD-OX, phosphorylation-mimic mutant of OsWRKY53-overexpressing rice plants.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098737-g004: Overexpression of a phosphorylation-mimic mutant of OsWRKY53 enhances the rice blast resistance of rice plants.A, Representative lesions of rice blast disease observed at 5 days post inoculation (dpi). Bars indicate 1 mm. B, Ratio of the classes of lesions in transgenic rice leaves infected with rice blast fungus Magnaporthe oryzae Ina86–137. Washed conidia of the blast fungus were suspended in 1 mM MES-NaOH (pH 5.7) and then were inoculated on leaves of W53-OX and W53SD-OX transgenic rice plants. The lesions were counted according to the classifications shown in A. Bars represent the ratio of lesions of each class to the total number of counted lesions in 3 or 4 individual leaf blades. Three independent experiments were performed, and a representative result is shown. Asterisks and daggers denote significant differences compared to W53-OX plants (*p<0.05; **p<0.01; ***p<0.001 vs. W53-OX #10; †p<0.05; †††p<0.001 vs. W53-OX #60, by One-way ANOVA with Tukey post hoc test) C, Development of blast disease in leaf blades evaluated by quantitating M. oryzae genomic DNA. The amount of M. oryzae 28S rDNA relative to rice genomic eEF1α DNA was determined by quantitative PCR analysis. Values are represented as mean values ±SE for 6 leaf blades. Statistically different data groups are indicated using different letters (p<0.05 by One-way ANOVA with Tukey post hoc test on log-transformed data). NT, non-transformant control rice plants; W53-OX, native OsWRKY53-overexpressing rice plants; W53SD-OX, phosphorylation-mimic mutant of OsWRKY53-overexpressing rice plants.
Mentions: Next, we tested the transformants for resistance to the blast fungus. The transgenic rice plants were grown in a growth chamber, inoculated with conidia of the blast fungus, and disease symptoms were characterized 5 days later. We confirmed as previously reported that W53-OX rice plants showed enhanced resistance against the blast fungus compared to non-transformed control (NT) rice plants (Fig. S3). Thus, we compared resistance to the blast fungus between W53-OX and W53SD-OX rice plants directly. We did not see statistically-significant differences in average numbers of lesions in the total infected area between 2 independent W53-OX and 2 independent W53SD-OX samples (Fig. S4). Therefore, we categorized the disease lesions into 3 classes (Fig. 4A) and compared the ratio of lesions between W53-OX and W53SD-OX rice plants. Interestingly, the ratio of necrotic spot lesions in leaves of the W53SD-OX plants was significantly increased relative to that of W53-OX plants (Fig. 4B). Moreover, the ratio of susceptible lesions in W53SD-OX plants was significantly decreased relative to W53-OX plants (Fig. 4B). We also quantified fungal biomass in leaves of NT, W53-OX and W53SD-OX rice plants. Quantitative genomic PCR analysis demonstrated that significantly reduced levels of fungal DNA were detected from W53SD-OX plants compared with W53-OX plants. It was also indicated that fungal biomass levels tend to be decreased in W53-OX plants compared with NT plants (Fig. 4C). These results strongly suggest that overexpression of a phospho-mimic mutant of OsWRKY53 further enhances the basal defense response against the virulence rice blast fungus compared to native OsWRKY53-overexpressing rice plants.

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