芸薹黄化病毒（BrYV）P0蛋白在本生烟上诱发坏死反应的机制研究

Numerous studies have focused on the molecular mechanisms of viral pathogenicity and host resistance. The poleroviruses, which are worldwide distributed, can infect many crops of economic importance and cause severe plant diseases. Brassica yellows virus (BrYV) is a newly identified member of Polerovirus which induces a hypersensitive response (HR)-like reaction in Nicotiana benthamiana. It was then proved that P0 protein, the RNA silencing suppressor of BrYV, is responsible for induction of the HR-like reaction in N. benthamiana. However, the molecular mechanism underlying HR-like reaction induced by P0 protein is not clear. Since the study of plant resistance to poleroviruses is lack now, this project will focus on the P0 protein to investigate the mechanism of this HR-like reaction, based on the previous studies. Global mutagenesis of BrYV P0 will find the key amino acids that function in inducing HR-like reaction. Yeast two-hybridization and co-immunoprecipitation techniques will be used to screen the host proteins that differ in interaction with P0 and its mutant. In order to elucidate the pathways and genes involved in P0-induced HR-like reaction, RNA-Seq will be used to analyze the differentially expressed genes and biological processes between N. benthamiana plants transiently expressing P0 or its mutant. Reverse genetic, biochemical, and biological analysis will be used to identify functions of candidate host factors. This study will not only provide new insights in understanding the molecular mechanism by which the plant viruses induce HR reaction, but also offer theoretical guidance on controlling the viral disease caused by poleroviruses in the future.

植物病毒致病性及寄主抗病性的分子机制一直是研究的热点。马铃薯卷叶病毒属病毒能引起多种经济作物的严重病害，该属的芸薹黄化病毒（BrYV）能在特定寄主上引起类似过敏性坏死反应（hypersensitive response, HR）的现象，并且其决定因子被鉴定为其编码的RNA沉默抑制子P0蛋白。但是，P0诱导坏死反应的分子机制还不清楚。考虑到该属病毒抗性机制的研究目前较为缺乏，本项目拟在前期研究基础上，以BrYV P0诱发类似HR坏死反应的关键位点进行精细定位；利用酵母双杂交、免疫共沉淀等技术筛选与P0及其代表性突变体在互作上有差异的寄主蛋白；对瞬时表达P0及其代表性突变体的本生烟进行转录组测序，综合运用生物信息学结合反向遗传学验证、生物学测定、生物化学等手段，解析P0蛋白在本生烟上诱发坏死反应的分子机制。研究结果将为深入理解病毒和寄主植物的分子互作以及制定病毒病害的防控策略提供参考和指导。

植物病毒致病性及寄主抗病性的分子机制一直是研究的热点。马铃薯卷叶病毒属病毒可侵染十多科植物，引起多种重要作物的严重病害，因此对该属病毒抗病相关基因的挖掘非常重要。该属的芸薹黄化病毒（BrYV）能在特定寄主上引起类似过敏性坏死反应（hypersensitive response, HR）的现象，并且其决定因子被鉴定为其编码的RNA沉默抑制子P0蛋白。但是，P0诱导坏死反应的分子机制还不清楚。本项目拟在前期研究基础上，对BrYV三个基因型的P0蛋白的生物学活性进行分析发现P0BrA比P0BrB和P0BrC的致病性要强，并寻找到了影响P0致病性的关键氨基酸位点，为进一步研究病毒致病性的分子机理提供了线索。进一步筛选了与P0互作的病毒蛋白和寄主因子，发现P0能够与长距离运动相关蛋白P3a互作，同时与P3a互作At3a-251、Nb3a-251、AtGTR1、AtNRT1.7、AtPUP14蛋白的过量表达虽然降低了BrYV的积累量，但是却促进了BrYV在本生烟接种叶上产生的坏死反应，进而发现这些寄主蛋白与P0共表达时可以促进由P0诱导产生的坏死表型；结合3a-251蛋白能够与P0、P3a共定位于细胞核周围，猜测3a-251与P0、P3a三者形成复合体从而共同发挥抗病毒的作用。P0不表达及丧失抑制子功能的BrYV突变体不能系统侵染本生烟，在豌豆耳突花叶病毒2号（Pea enation mosaic virus 2, PEMV 2）的帮助下可以系统侵染本生烟，然而却没有出现协生的症状，初步认为P0蛋白是BrYV与PEMV 2协生所引起坏死症状的主要决定因子。本项目关于P0作用机制的研究为阐明病毒致病性的分子机制提供了理论依据，并为基于基因编辑技术创制抗病毒作物新种质提供参考。