水稻黑条矮缩病毒P6蛋白与水稻的分子互作机制

Rice black-streaked dwarf virus (RBSDV) is the most important virus that harms rice and corn production in China. Given that there are no any reported references about the molecular mechanism of RBSDV-host interaction and the virus-host interaction is very important for virus to infect host, this project will use RBSDV P6-rice interaction as a breakthrough, screen and identify host interaction factors, identify interaction domains and key amino acid sites of RBSDV P6 and host factors, analyze subcellular localizations and expression levels of P6 and host factors, study function of host factors on infection and symptom development, explore biochemical mechanism of RBSDV P6-host interaction, reveal the molecular mechanism of interaction between RBSDV P6 and rice with techniques such as conventional and split-ubiquitin yeast two-hybrid assays, Co-IP, mass spectrometry, bimolecular fluorescence complementation, GST pull-down, gene silencing and editing, biochemical assays and bioinformatics. Thereby, results of this project will lay a foundation for elucidating molecular mechanism of RBSDV-rice interaction and provide new theory bases for preventing and controlling this viral disease.

水稻黑条矮缩病毒（RBSDV）是危害我国水稻和玉米生产的最重要病毒。鉴于目前未见有关RBSDV病毒与水稻寄主互作分子机理的报道以及病毒与寄主互作对于病毒侵染水稻的重要性，本项目以RBSDV编码的P6蛋白与水稻互作作为突破口，拟通过常规和分裂泛素化酵母双杂交技术、免疫共沉淀、蛋白质谱、双分子荧光互补实验、GST pull-down、基因沉默和编辑、生化实验、生物信息学等技术筛选鉴定与RBSDV P6蛋白互作的水稻寄主因子，鉴定P6与寄主因子的互作结构域及关键氨基酸位点，明确P6和寄主因子的亚细胞定位及共表达时互作蛋白的表达水平，分析寄主因子在病毒侵染和症状形成中的作用，探索P6蛋白与寄主因子互作的生化机制，解析RBSDV P6蛋白与寄主水稻的分子互作机制，从而为阐明RBSDV与水稻的互作机制打下基础，并为防控该水稻病毒病提供新的理论依据。

水稻黑条矮缩病毒（RBSDV）是危害我国水稻和玉米的最重要病毒，解析RBSDV与水稻寄主的互作机制是制定该病毒病防控策略的关键。本项目构建了水稻的常规和分裂泛素化酵母cDNA文库，制备了RBSDV P6的单克隆抗体，利用酵母双杂交实验、免疫共沉淀、蛋白质谱、双分子荧光互补实验、pull-down、基因沉默和编辑、生化实验、生物信息学等技术筛选鉴定到与RBSDV P6互作的OsSCE1、OsSKL、OsTFIID、OsZMPL、OsJAZ6、OsNAP、OsDdRP、OsPCDL1、OsPGK、OsRP和OsACL等14个水稻寄主因子，鉴定了OsSCE1、OsPCDL1、OsSKL等8个寄主因子与RBSDV P6的互作结构域及关键氨基酸，分析了RBSDV P6和寄主因子的亚细胞定位，明确了OsSCE1、OsTFIID、OsZMPL、OsJAZ6和OsPCDL1五个水稻互作寄主因子在病毒侵染中的生物学功能，发现OsPCDL1Os、TFIID和OsZMPL正调控病毒的侵染，OsSCE1和OsJAZ6负调控病毒的侵染；探索了RBSDV P6与寄主因子互作的生化机制，发现RBSDV P6能抑制OsSCE1的类泛素E2连接酶功能而削弱植物的抗病毒免疫反应，RBSDV P6蛋白抑制OsPCDL1的核酸酶活性并改变其与P-body的定位，且P6蛋白也能抑制OsSKL的叶绿体定位。上述研究结果解析了RBSDV P6蛋白与寄主水稻的分子互作机制，从而为全面阐明RBSDV与水稻的互作机制奠定基础，并为防控该水稻病毒病提供新的理论依据。