Overexpression of phosphomimic mutated OsWRKY53 leads to enhanced blast resistance in rice.
Overexpression of phosphomimic mutated OsWRKY53 leads to enhanced blast resistance in rice.
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DOI：
10.1371/journal.pone.0098737
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发表时间：
2014
期刊：
影响因子：
3.7
通讯作者：
Okada K
中科院分区：
文献类型：
--
作者：
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
WRKY转录因子和丝裂原活化蛋白激酶（MAPK）级联在植物防御反应的调控中起着关键作用。我们之前报道过OsWRKY 53过表达的水稻植株对稻瘟病真菌表现出增强的抗性。在本研究中，我们鉴定了OsWRKY 53作为水稻中真菌PAMP响应性MAPK级联的组分OsMPK 3/OsMPK 6的底物，并分析了OsWRKY 53磷酸化对稻瘟病菌Ina 86 -137菌株毒力小种的基础防御反应的调节作用。体外磷酸化实验表明，OsMKK 4激活的OsMPK 3/OsMPK 6在OsWRKY 53重组蛋白的多簇丝氨酸-脯氨酸残基（SP簇）处发生磷酸化。当OsWRKY 53与OsMKK 4的组成型活性突变体在瞬时报告基因分析中共表达时，发现OsWRKY 53的增强的反式激活活性依赖于SP簇的磷酸化。过量表达OsWRKY 53的磷酸化模拟突变体（OsWRKY 53 SD）的转基因水稻与过量表达OsWRKY 53的天然水稻相比，表现出对稻瘟病菌的进一步增强的抗病性，并且与过量表达OsWRKY 53的水稻相比，大量的防御相关基因（包括病程相关蛋白基因）在过量表达OsWRKY 53 SD的水稻中被更多地上调。这些结果有力地表明OsMKK 4-OsMPK 3/OsMPK 6级联调节OsWRKY 53的反式激活活性，并且OsWRKY 53的磷酸模拟突变体的过表达导致水稻转录组在稳态下发生重大变化，从而激活水稻植物对稻瘟病菌的防御反应。
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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