稻瘟病菌雷帕霉素靶蛋白TOR调控细胞自噬的分子机制研究

Autophagy is essential for the rice blast fungus Magnaporthe oryzae to form functional appressorium leading to infection in the host rice plant. However, its molecular mechanism is poorly known. Target of rapamycin (TOR) controls cell growth and metabolism in response to environmental cues, and negatively regulates autophagy in several eukaryotic species such as yeast Saccharomyces cerevisiae, plant Arabidopsis and mammals. Our previous study found that TOR associates with the complex of rapamycin/FKBP12 in M. oryzae. The autophagy process was significantly reduced when TOR kinase activity was inhibited or TOR expression was down-regulated during appressorium development in M. oryzae. These results suggest that TOR positively regulates autophagy in M. oryzae, which is different from the mechanism that observed in yeast, Arabidopsis or mammals. In this study, we will examine the gene expression and subcellular localization of TOR, determine the kinase activity of TOR protein and its potential relationship with autophagy progression, dissect the TOR associated signal transduction of autophagy in M. oryzae. Through performing this study, we will elucidate the molecular regulation of TOR on autophagy in M. oryzae. Our study will provide the implicit knowledge for controlling rice blast disease effectively.

细胞自噬在稻瘟病菌侵染水稻的致病过程中发挥必需作用，但稻瘟病菌的自噬调控机理并不清楚。雷帕霉素靶蛋白TOR是酵母、拟南芥和哺乳动物等真核生物自噬过程的负调控因子，并在细胞生长和分化过程中起重要作用。申请人前期研究发现，雷帕霉素与稻瘟病菌蛋白FKBP12形成复合物后，靶向结合蛋白TOR并抑制其活性。当基因TOR表达量或蛋白TOR活性受抑制时，稻瘟病菌侵染相关的自噬性凋亡受到显著抑制，表明蛋白TOR对稻瘟病菌自噬性凋亡起着正调控作用，这不同于酵母等真核生物中蛋白TOR对自噬的负调控作用。本项目拟采用分子生物学、细胞生物学、生物化学及转录组学等各学科相关技术方法，分析基因TOR的表达模式和蛋白TOR的亚细胞定位；蛋白TOR对自噬的影响，及TOR激酶活性与自噬的关系；TOR所参与的信号调控网络和互作因子。本项目将阐明TOR调控稻瘟病菌自噬的分子机制，为鉴定稻瘟病防治靶标奠定理论和应用基础。

自噬在病原真菌的致病过程中发挥必需作用，但病原真菌的自噬调控机理仍不清楚。本项目以水稻病原真菌稻瘟病菌为模式，综合使用细胞生物学、遗传学、生物化学、植物病理学等多学科技术手段，解析了稻瘟病菌雷帕霉素靶蛋白MoTOR对自噬的分子调控机制，取得了以下主要研究成果：蛋白MoTOR在细胞质中发挥调控功能，并在生长发育各阶段均会表达；MoTOR对附着胞发育阶段的自噬性凋亡及营养物质动员起着重要调控作用，从而影响稻瘟病菌致病过程；MoTOR的激酶活性是稻瘟病菌生长发育及致病所必需的；MoTOR正调控表观因子MoSNT2的转录表达，两者协同调控生长发育及致病；MoSNT2作用于MoTOR信号途径下游，发挥染色质组蛋白表观调控功能，负责识别组蛋白H3乙酰化修饰并招募组蛋白去乙酰化酶，参与调节组蛋白H3的去乙酰化过程，从而控制与致病、生长、产孢等生理过程相关的大量基因的转录表达。此外，本项目还针对稻瘟病菌与水稻的亲和及非亲和相互作用，开展了细胞生物学的比较分析，明确了亲和与非亲和互作过程之间的差异主要集中于侵染钉形成及侵染菌丝的增殖。本项目揭示了MoTOR调控稻瘟病菌自噬的分子机制，丰富了对稻瘟病菌与水稻互作机理的认识，可为稻瘟病防治提供新的理论和应用基础。