棉花－黄萎病菌跨界RNAi及相关效应蛋白的调控机理和抗病应用研究

Verticillium dahliae is a hemibiotrophic fungus that causes vascular wilt disease in many plant species, including cotton. Cotton wilt disease is a major threat to cotton production in most cotton growing countries. We recently identified a novel penetration structure -hyphopodium , and revealed that hyphopodium-specific VdNoxB/VdPls1-dependent ROS-Ca2+ signaling is required for plant infection by V. dahliae (Zhao et al., 2016 PLoS Pathogens); we then verified that hyphopodium functions as key signaling hubs during plant infection that is the apparatus of not only breaching the host cells but also unique interfaces of secretion of fungal effectors (Zhou et al., 2017 PLoS Pathogens); we also found that the Verticillium-specific effector protein targets the host nucleus to modulate plant immunity (Zhang et al., 2017 New Phytologist); we also showed that in response to V. dahliae infection, cotton plants increased production of endogenous small RNAs (miRNAs) and exported them to the fungal hyphae for specific silencing of fungal virulent genes. Our works are the first direct experimental evidence of the mobility of RNA molecules from plants to fungal cells and inducing cross-kingdom gene silencing in fungal cells and confer disease resistance (Zhang et al., 2016 Nature Plants); and most importantly, we utilized cross-kingdom RNAi and demonstrated the effectiveness of engineering cotton cultivars by sending small RNAs to fight off V. dahliae in both laboratory and disease nursery (Zhang et al., 2016 Molecular Plant). In this project, we will identify RNAi components that would involve in cross-kingdom RNAi in V. dahliae and analyze the molecular mechanism; we will also use our T-DNA insertional mutant library and secretory protein data from liquid chromatography-mass spectrometry of V. dahliae in combination with reverse genetics approach, aiming to identify the fungal pathogen-associated molecular patterns (PAMPs) and/or secretory proteins that affect the exportation of miRNAs into fungal cells, and analyze their regulative mechanism. The research aims are to uncover novel RNAi targets to develop more effective cross-kingdom RNAi cotton plants for germplasm innovation in engineering resistance against Verticillium wilt disease.

大丽轮枝菌引起的黄萎病是一种土传维管束病害，是危害棉花最重要的病害之一，造成巨大的经济损失。我们近期鉴定了致病菌的侵染结构及其穿刺和分泌功能; 证明一些分泌蛋白进入植物细胞调控致病性；发现致病菌侵染棉花诱导一些植物miRNAs，并转运到病菌细胞中，降解病菌的致病基因，在国际上首次证明了植物-真菌跨界miRNA降解病原靶基因的抗真菌RNAi新途径；更重要的是，我们成功利用跨界RNAi技术在早熟陆地棉中提高了棉花黄萎病抗病性。基于这些前期的研究和发现，该项目拟鉴定致病菌中参与跨界RNAi的组分并解析其作用机制；并运用前期已构建的病原菌突变体库和分泌蛋白质谱数据，结合反向遗传学，鉴定影响miRNAs跨界传递和作用的病菌表面分子模式和效应蛋白，探究其调控或影响跨界RNAi的机制；旨在挖掘并整合有效的RNAi靶标，力求获得高效跨界RNAi抗病棉花，进行棉花抗黄萎病的种质创新。

大丽轮枝菌引起的棉花黄萎病，造成巨大经济损失。我们前期在国际上首次证明了植物-真菌跨界miRNA降解病原靶基因的RNAi新途径；并成功利用跨界RNAi技术在早熟陆地棉中提高棉花黄萎病抗病性。在此基础上，本项目继续探究大丽轮枝菌RNAi途径的分子机理；植物RNAi和跨界RNAi效应；大丽轮枝菌效应因子及其作用机理；完善棉花跨界RNAi抗黄萎病体系。在本项目资助下，按计划进行，并 1）鉴定了大丽轮枝菌RNAi及表观遗传组分调控致病性（Phil Trans R Soc B 2019；PLoS Pathog 2020）；2）解析了侵染过程中植物和大丽轮枝菌sRNAs、跨界RNAi的空间和时间效应（Mycology 2018；Mol Plant 2020；Plant Cell 2020；Front Plant Sci 2022；Int J Mol Sci 2022）；3) 揭示了大丽轮枝菌效应蛋白操控植物免疫等（Elife 2018；Nat Plants 2019），成果入选《2020中国农业科学重大进展》；4）获得了农业部签发的国内首个 RNAi 棉花安全评价环境释放证书，并在2021年圆满完成环境释放工作。我们还应邀对跨界RNA沉默相关研究热点撰写多篇综述及专著章节（Mol Plant 2018；Curr Opin Genet Dev 2019；Curr Opin Virol 2021；Plant Commun 2021；JIPB 2022；Methods Mol Biol 2018）。.项目执行期内，项目负责人郭惠珊研究员两次入选爱思唯尔中国高被引学者（2020/2021年度）；2021年受中央电视台《透视新科技》访谈，讲述《新型抗病棉》；获2021年第十二届大北农科技奖创新奖；并作为论坛执行主席，组织协办2022年“棉花高质量发展国际学术论坛”。