辣椒CMPG1在高温高湿下调节MLO1对青枯病抗性的作用机理研究

Bacterial wilt, caused by Ralstonia solanacearum, which is more serious in pepper plants under high temperature and high humidity (HTHH) than room temperature, which is one of the serious problems in pepper production. However, the mechanism underlying bacterial wilt resistance of pepper remains poorly understood. We previously found that MLO1 act as a negative regulator in pepper response to R. solanacearum under room temperature, but act as a positive regulator to R. solanacearum under high temperature and humidity. In addition, it was predicted and verified through BiFC that MLO1 might interact with CMPG1, an E3 ligase that mediate ubiquitination or monoubiquitination of MLO1, which might associate with the functional conversion of MLO1 under two conditions. To affirm this hypothesis, the relationship between MLO1 and the genes including GST3 and PRP1 that specifically respond to R. solanacearum under high temperature and humidity will be assayed by transient overexpression of MLO1 and virus induced MLO1 silencing in pepper plants. The CMPG1/MLO1 interaction will be confirmed by co-IP and MST based on the results of co-expression and subcellular co-localization assay of CMPG1 and MLO1.The function of CMPG1 in the resistance to R. solanacearum under high temperature and humidity will be assayed using CMPG1 silencing pepper plants and its overexpressing Nicotiana tabacum T2 plants. The activity of E3 ligase will also be detected and the possible ubiquitination or monoubiquitination of MLO1 mediated by CMPG1 and its effect on the function of MLO1 in pepper resistance to R. solanacearum under high temperature and humidity will also be assayed. The results will help to further elucidate the molecular mechanism of pepper resistance to bacterial wilt under high temperature and high humidity, and provide a basis for its genetic improvement.

高温高湿下辣椒青枯病发生异常严重，但辣椒在高温高湿下的抗病机制却鲜见报道。项目前期发现辣椒MLO1在常温下负调节抗青枯病，但在高温高湿下转变为正调节抗青枯病，且预测并通过BiFC验证MLO1可能与E3连接酶CMPG1互作。我们推测CMPG1介导的MLO1泛素化特别是单泛素化可能与MLO1在两种条件下的功能转化有关。为了验证这一假设，本项目拟进一步分析瞬间超表达和病毒诱导沉默MLO1对GST3等高温高湿下青枯菌侵染特异性应答PR基因表达的影响；运用双分子荧光互作、免疫共沉淀和微量热泳动验证CMPG1与MLO1的互作。利用病毒诱导沉默和转基因烟草分析CMPG1高温高湿下抗青枯病中的作用，解析CMPG1在高温高湿下青枯菌侵染下对MLO1泛素化或单泛素化，确定其位点，分析单泛素对MLO1在高温高湿下抗青枯病中的作用。研究结果有利于阐明辣椒在高温高湿下抗青枯病机制，为辣椒抗病遗传改良提供依据。

高温高湿(HTHH)条件下植物病害比常温条件下更为严重，因此高温高湿条件下的抗病性是植物生存的瓶颈。然而，植物在HTHH下如何应对病原菌仍然知之甚少。本文以辣椒-青枯病病原系统为例，通过表达谱分析、病毒诱导基因沉默和过表达的功能丧失和功能获得测定以及蛋白质-蛋白质相互作用测定，比较了MLO1和PUB21在室温/高湿(RTHH)或HTHH条件下辣椒对青枯病免疫的功能特征。结果表明，在RTHH处理下，MLO1在辣椒应答青枯菌的免疫中起负调节作用，而在HTHH处理下起正调节作用。与此相反，PUB21在RTHH处理下应答青枯菌具有正调节作用，而在HTHH处理下则具有负调节作用。重要的是，PUB21在所有测试条件下都与MLO1相互作用，但只有PUB21-MLO1在HTHH和RSHT条件下导致CaMLO1降解，从而关闭对RSHT的不适当免疫。这些结果表明，MLO1和PUB21在辣椒对青枯菌的免疫中具有明显的环境依赖性作用，并相互作用，导致MLO1在RSHT时特异性降解，从而关闭由MLO1介导的不适当免疫，对于将来进一步研究辣椒在高温高湿环境下的抗病分子机制具有重要的理论指导意义。