正番茄斑萎属病毒抗性基因RTSW广谱抗性鉴定及无毒基因的作用机制研究

由正番茄斑萎病毒属病毒（orthotospoviruses）造成的作物斑萎病已成为云南农作物生产的重要威胁。前期的抗病性鉴定发现来源于野生烟草抗性基因RTSW对orthotospoviruses具有广谱抗性。本项目的开展将进一步明确RTSW的抗病谱，阐明RTSW的广谱抗性由不同病毒NSm介导的过敏性反应(HR)决定。项目将通过包括：（1）鉴定NSm诱导HR反应的最小结构域和关键氨基酸位点；（2）明确NSm激发HR的细胞定位和关键氨基酸位点的蛋白翻译后修饰类型；（3）获得抗病烟草中与NSm最小结构域互作的蛋白来解析无毒基因NSm的作用机制，进而明确RTSW烟草的广谱抗性机制。项目的结果对于进一步认识广谱抗病基因的作用机制及抗病蛋白与Avr蛋白的互作机制有着重要的科学意义，同时研究成果有可能直接克隆获得RTSW抗性基因，促使基础研究成果走向应用。

Orthotospoviruses (order Bunyavirales, family Tospoviridae) are the most destructive viral pathogens of plants that threaten food security in Yunnan. Recently, a single dominant gene conferring complete resistance to TSWV (RTSW) was found in a wild tobacco, Nicotiana alata, and has been introgressed into cultivar tobacco. Our previous result showed RTSW confers broad-spectrum resistance against both American-type and Asian-type orthotospoviruses and NSm of Tomato spotted wilt virus (TSWV) is the avirulence (Avr) factor of RTSW. In this project, we will collect more orthotospoviruses and preform disease resistance test to further demonstrate broad-spectrum resistance of RTSW. In order to illustrate molecular mechanism of Avr function of NSm, 1)minimal functional domain, key amino acid sites in the NSm will be identified by sequential deletions, domain swap and/or site-directed mutagenesis. 2)Subcellular localization of minimal domain of NSm and post-translational modification of key domain/motif or amino acid sites will be investigated. 3) Co-immunoprecipitation (Co-IP) analysis will be performed to screen the host factor that interacts with NSm minimal domains. The results will provide a new example of a plant R mediating broad-spectrum resistance and shed light the molecular mechanism of NSm-induced HR in RTSW tobacco.