广谱抗病蛋白RPW8.1介导稻瘟病抗性的分子机理研究

The molecular mechanism of rice blast resistance is a long-lasting active area of investigation. Most of the functionally characterized rice blast resistance genes encode NB-LRR (nucleotide-binding-site-leucine-rich-repeat) family proteins that mediate race-specific resistance. Race-specific resistance is easily lost due to the fast variation of the dominant pathogen strains. Thus, it is necessary to diversify the resistant resource for rice breeding, rice production and investigation of molecular mechanism of the rice blast resistance. RPW8.1 was isolated from the model plant Arabidopsis. In our previous studies, we found that the expression of RPW8.1 was able to enhance resistance against multiple filamentous pathogens such as powdery mildew and downy mildew. Intriguingly, the transgenic rice plants expressing RPW8.1 driven by a pathogen-inducible promoter exhibited resistance to rice blast. These preliminary results imply that there is a significant strategy that we can enrich rice blast resistant resource by introducing broad-spectrum genes from other family of plants into rice. To understand how RPW8.1 initiates defense responses and confers rice blast resistance, we propose the following experiments. First, the resistant spectrum, the cellular characteristics of resistance, the production of reactive oxygen species, the accumulation of callose, and the expression of defense-related genes will be investigated in the transgenic rice plants expressing RPW8.1. Second, comparative transcriptome analysis will be performed on the RPW8.1 transgenic (blast-resistant) plants and the non-transgenic (blast-susceptible) plants, so as to reveal the signal transduction pathways manipulated by RPW8.1. Third, RPW8.1-interacting proteins will be screened via yeast-two-hybridization (Y-2-H) from a library constructed from Magnaporthe oryzae-infected rice plants. Finally, the function of the key genes and RPW8.1-interactors identified in transcriptome analysis and Y-2-H, respectively, will be characterized through reverse genetic approaches. Data from this proposed research will significantly contribute to our knowledge on the cross-species transfer of broad-spectrum resistance genes.

稻瘟病抗性的分子机理一直是一个重要的研究热点。目前分离出的稻瘟病抗性基因主要编码NBS-LRR类蛋白，多属小种特异性抗性，基因类型单一，抗性难以持久。因此，有必要增加抗病基因类型和抗性基因资源。RPW8.1是从模式植物拟南芥中分离出的一个广谱抗病基因，我们前期研究发现，它通过提高拟南芥的基础抗性产生对白粉病菌和霜霉病菌等丝状病原菌的抗病性；而且，利用稻瘟病菌侵染诱导性启动子在水稻中表达RPW8.1，获得的转基因水稻具有稻瘟病抗性。本项目拟在此基础上，通过抗谱测定、抗病细胞学分析、超氧化物迸发与胼胝质积累、防御相关基因的表达分析几个方面来剖析RPW8.1转基因水稻对稻瘟病的抗病性，同时利用差异转录组分析、互作蛋白的筛选鉴定和功能研究来系统阐明RPW8.1如何调控水稻的防御系统产生对稻瘟病的抗病性，从而明确在水稻中表达异源广谱抗病基因产生抗病性的分子机理，为跨物种转移广谱抗病基因提供理论指导。

稻瘟病抗性机理一直是植物病理学的一个研究热点，RPW8.1在拟南芥中通过增强基础抗性产生对白粉病和霜霉病的抗性。用稻瘟病菌侵染诱导性启动子在水稻中表达RPW8.1，可明显提高对稻瘟病菌的抗性。通过抗病细胞学分析，发现对稻瘟病抗性表现为抗扩展。通过差异转录组分析发现乙烯信号在RPW8.1介导的细胞死亡和抗病性中起重要作用。通过筛选鉴定RPW8.1介导的细胞死亡和抗病性的增强子和抑制子突变体，发现调控叶片发育的基因AS1和调控生物钟及小RNA合成的基因XCT正调控RPW8.1介导的细胞死亡和抗病性，而膜联蛋白基因ANN8负调控RPW8.1介导的细胞死亡和抗病性。而且，筛选RPW8.1的互作蛋白鉴定到一个控制乙烯信号的关键基因，RPW8.1一方面可能通过激活乙烯信号通路来增强PTI；另一方面乙烯信号通路又反馈抑制RPW8.1的表达。虽有报道，调控免疫相关的基因可以通过跨物种转移并在受体物种中发挥作用，已经报道的跨物种基因转移包括从双子叶植物到单子叶植物、从单子叶植物到双子叶植物以及在不同种双子叶植物间，但还没有从双子叶植物到单子叶植物转移抗病基因的报道。本项目实现了从模式双子叶植物拟南芥向模式单子叶植物水稻转移抗病基因，为跨物种转移抗病基因提供了新的成功案例，也为在农作物中利用模式植物的有利基因提供依据。RPW8.1可以通过对乙烯信号和增强PTI信号来调控植物免疫力，因此，可以利用RPW8.1这种功能，通过转基因技术在不同作物中定向、定点、定位表达RPW8.1来构筑抗病性，创制抗病新种质和培育抗病新品种。