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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: We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus.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.

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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Phytoalexin accumulation in W53SD-OX rice plants.A, Expression analysis of momilactone biosynthetic genes. qRT-PCR analysis was performed using total RNA isolated from uninfected rice leaves. Values indicate relative mRNA levels normalized to the expression of the UBQ gene (n = 3); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. B, Accumulation of momilactones in uninfected W53SD-OX rice plants. Momilactone levels in the rice leaves were determined by LC-MS/MS. The results are the average of at least 3 independent experiments. Bars indicate the standard error of the mean. NT, non-transformed control; OX, OsWRKY53-overexpressing rice plants; SD-OX; OsWRKY53SD-overexpressing rice plants. N.D.: not detected.
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pone-0098737-g006: Phytoalexin accumulation in W53SD-OX rice plants.A, Expression analysis of momilactone biosynthetic genes. qRT-PCR analysis was performed using total RNA isolated from uninfected rice leaves. Values indicate relative mRNA levels normalized to the expression of the UBQ gene (n = 3); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. B, Accumulation of momilactones in uninfected W53SD-OX rice plants. Momilactone levels in the rice leaves were determined by LC-MS/MS. The results are the average of at least 3 independent experiments. Bars indicate the standard error of the mean. NT, non-transformed control; OX, OsWRKY53-overexpressing rice plants; SD-OX; OsWRKY53SD-overexpressing rice plants. N.D.: not detected.

Mentions: Based on the results described above, it appears that a subset of defense-related genes is further activated in W53SD-OX rice plants compared to W53-OX plants. Thus, the 151 genes upregulated in W53SD-OX were organized into molecular function Gene Ontology (GO) categories based on their primary functions. Most of the genes grouped into the following categories: binding (GO:0005488), catalytic activity (GO:0003824), nucleic acid binding transcription factor activity (GO:0001071), and transporter activity (GO:0005215). But some of these genes grouped into the following 2 non-GO categories: defense-related genes and molecular function unknown (Table S5). Besides, almost all of the defense-related genes (22 out of 23 genes) among the 151 upregulated genes fell into the group I, including several PR protein genes such as β-1,3-glucanase, chitinase, and PR-5 genes (Table 1). Given that W53SD-OX rice plants showed further-enhanced resistance to the virulence rice blast fungus, we finally focused on the group I genes, especially those relevant to defense response. To further validate the results of the microarray analysis, we performed qRT-PCR analysis using NT and each of 2 independent W53-OX and W53SD-OX rice plants. As shown in Fig. 5, the expression of most of these genes was significantly upregulated in W53SD-OX plants compared to NT plants, and some of these genes' transcripts were also accumulated in W53-OX plants compared to NT. We also noticed that OsCPS4 and CYP99A2, which encoded biosynthetic enzymes of rice diterpenoid phytoalexins (momilactones), were included in the above defense-related genes (Table 1). Momilactones are synthesized from geranylgeranyl diphosphate (GGDP) through 2 cyclization and multiple oxidation steps [46], [47]. We therefore examined the expression levels of these above 2 genes (OsCPS4 and CYP99A2) and 3 additional genes (OsKSL4, OsMAS and CYP99A3) for momilactone biosynthesis by qRT-PCR analysis. We found that transcript levels of these momilactone-biosynthetic genes except OsKSL4 were increased only in the W53SD-OX rice plants, a result consistent with the basal accumulation of momilactones in these plants (Fig. 6). Taken together, these results suggest that the potentiated induction of these defense-related genes at steady state provides further enhanced disease resistance of W53SD-OX rice plants against rice blast fungus at the time of infection compared to W53-OX 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)

Phytoalexin accumulation in W53SD-OX rice plants.A, Expression analysis of momilactone biosynthetic genes. qRT-PCR analysis was performed using total RNA isolated from uninfected rice leaves. Values indicate relative mRNA levels normalized to the expression of the UBQ gene (n = 3); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. B, Accumulation of momilactones in uninfected W53SD-OX rice plants. Momilactone levels in the rice leaves were determined by LC-MS/MS. The results are the average of at least 3 independent experiments. Bars indicate the standard error of the mean. NT, non-transformed control; OX, OsWRKY53-overexpressing rice plants; SD-OX; OsWRKY53SD-overexpressing rice plants. N.D.: not detected.
© Copyright Policy
Related In: Results  -  Collection

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

pone-0098737-g006: Phytoalexin accumulation in W53SD-OX rice plants.A, Expression analysis of momilactone biosynthetic genes. qRT-PCR analysis was performed using total RNA isolated from uninfected rice leaves. Values indicate relative mRNA levels normalized to the expression of the UBQ gene (n = 3); bars indicate the standard error of the mean. Three independent experiments were performed, and a representative result is shown. B, Accumulation of momilactones in uninfected W53SD-OX rice plants. Momilactone levels in the rice leaves were determined by LC-MS/MS. The results are the average of at least 3 independent experiments. Bars indicate the standard error of the mean. NT, non-transformed control; OX, OsWRKY53-overexpressing rice plants; SD-OX; OsWRKY53SD-overexpressing rice plants. N.D.: not detected.
Mentions: Based on the results described above, it appears that a subset of defense-related genes is further activated in W53SD-OX rice plants compared to W53-OX plants. Thus, the 151 genes upregulated in W53SD-OX were organized into molecular function Gene Ontology (GO) categories based on their primary functions. Most of the genes grouped into the following categories: binding (GO:0005488), catalytic activity (GO:0003824), nucleic acid binding transcription factor activity (GO:0001071), and transporter activity (GO:0005215). But some of these genes grouped into the following 2 non-GO categories: defense-related genes and molecular function unknown (Table S5). Besides, almost all of the defense-related genes (22 out of 23 genes) among the 151 upregulated genes fell into the group I, including several PR protein genes such as β-1,3-glucanase, chitinase, and PR-5 genes (Table 1). Given that W53SD-OX rice plants showed further-enhanced resistance to the virulence rice blast fungus, we finally focused on the group I genes, especially those relevant to defense response. To further validate the results of the microarray analysis, we performed qRT-PCR analysis using NT and each of 2 independent W53-OX and W53SD-OX rice plants. As shown in Fig. 5, the expression of most of these genes was significantly upregulated in W53SD-OX plants compared to NT plants, and some of these genes' transcripts were also accumulated in W53-OX plants compared to NT. We also noticed that OsCPS4 and CYP99A2, which encoded biosynthetic enzymes of rice diterpenoid phytoalexins (momilactones), were included in the above defense-related genes (Table 1). Momilactones are synthesized from geranylgeranyl diphosphate (GGDP) through 2 cyclization and multiple oxidation steps [46], [47]. We therefore examined the expression levels of these above 2 genes (OsCPS4 and CYP99A2) and 3 additional genes (OsKSL4, OsMAS and CYP99A3) for momilactone biosynthesis by qRT-PCR analysis. We found that transcript levels of these momilactone-biosynthetic genes except OsKSL4 were increased only in the W53SD-OX rice plants, a result consistent with the basal accumulation of momilactones in these plants (Fig. 6). Taken together, these results suggest that the potentiated induction of these defense-related genes at steady state provides further enhanced disease resistance of W53SD-OX rice plants against rice blast fungus at the time of infection compared to W53-OX rice plants.

Bottom Line: We previously reported that OsWRKY53-overexpressing rice plants showed enhanced resistance to the rice blast fungus.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.

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