稻瘟病菌内质网相关蛋白质降解途径关键基因MoHrd1的作用机制研究

Endoplasmic reticulum (ER) constitutes an essential compartment required for protein folding and assembly. Only correctly folded proteins are capable of exercising their proper functions within the secretory pathway, while misfolded proteins are further subjected to re-folding or promptly degraded to prevent the accumulation of misfolded proteins and eliminate the induction of ER stress, which is detrimental to cell survival. ER-associated degradation (ERAD) is important for ER stress recovery and functions by targeting misfolded proteins and transports them out of the ER into the cytosol for subsequent ubiquitylation and degradation by cytosolic proteasomes. Rice blast is the most destructive disease of cultivated rice in China. Studying and understanding the pathogenesis of the causal agent Magnaporthe oryzae is helpful to develop sustainable strategies for breeding disease resistance cultivars and providing potential targets for producing new fungicides. M. oryzae secretes effector proteins to promote infection, previous studies found that ER stress is induced during infection. This project is intended to analyze the biological function and mechanism of MoHrd1 as a key gene in ERAD pathway, and additionally identify its target proteins and clarify their biological functions. Finally, we further aimed at illuminating the effect of MoHrd1 and its target proteins during the growth and infection-related development and further explore the mechanism of ERAD pathway in the processes required at different developmental stages.

内质网是蛋白质折叠与组装的重要场所。只有正确折叠的蛋白才能通过分泌途径行使功能，而错误折叠的蛋白需要被重新折叠或被降解，否则蛋白蓄积后将产生内质网压力，影响细胞的存活。内质网相关蛋白质降解途径（ERAD）识别错误折叠蛋白，并将其转运至细胞质中泛素化修饰后降解，是应答内质网压力的重要途径。稻瘟病是我国水稻生产上的毁灭性病害之一，从分子水平上解析稻瘟病菌致病机理有助于为抗病育种提供新的策略，同时也为新型杀菌剂的研发提供候选靶标。稻瘟病菌侵染水稻时分泌大量效应蛋白，前期研究发现病菌在侵染过程会产生内质网压力。本项目拟通过对ERAD途径关键基因MoHrd1进行生物学功能分析，并对其在ERAD中的作用机制开展研究，同时鉴定并分析其结合蛋白的生物学功能，解析MoHrd1及其结合蛋白在稻瘟病菌生长发育及致病过程中的作用，为进一步揭示ERAD途径在调控稻瘟病菌生长发育及致病过程中的分子机制奠定基础。

稻瘟病菌引起的稻瘟病严重威胁我国的水稻生产安全。ERAD途径对生物体内质网中未折叠或错误折叠蛋白的泛素化降解具有重要作用。目前，关于该途径在植物致病真菌中的功能尚不清楚。本课题利用基因敲除的方法，成功获得稻瘟病菌ERAD途径关键蛋白MoHR1和MoDER1的单基因及双基因缺失突变体。定量PCR结果发现，MoHRD1和MoDER1基因在分生孢子阶段和侵染阶段显著上调表达。亚细胞定位分析发现，MoHrd1和MoDer1均定位于内质网上。表型分析结果表明，MoHRD1和MoDER1共同参与调控病菌的菌丝生长、孢子产生及致病过程。同时缺失MoHRD1和MoDER1基因导致稻瘟病菌无性繁殖能力显著下降，致病力丧失，附着胞发育异常。进一步研究表明，MoHRD1和MoDER1基因共同参与调控错误折叠蛋白的降解、内质网压力胁迫应答、未折叠蛋白应答信号途径激活和蛋白的外泌过程。本项目研究结果表明MoHRD1和MoDER1介导的ERAD途径在稻瘟病菌菌丝生长、无性繁殖、内质网压力应答及致病过程中具有重要作用。本研究结果有助于深入阐明稻瘟病菌的致病机理，为开发以ERAD途径相关基因为靶标的新型杀菌剂提供有效参考价值。