茶树炭疽菌毒性效应蛋白的鉴定及其功能研究

Anthracnose is one of the main foliar diseases of tea plant affecting plant growth and quality of tea. Our previous study confirmed that Colletotrichum camelliae is a dominant pathogen causing tea plant anthracnose in China; effector proteins secreted by Colletotrichum camelliae could interact with disease resistance proteins in tea plant, followed by the activation of MAPK signaling pathway and multiple downstream defense responses thereby causing plant immunity. However, the key effectors of C. camelliae are still unclear. In this study, in order to identify key virulence effectors from the 36 candidate effector proteins screened in the previous research, the PVX virus expression vector and tobacco transient expression system will be used to identify their virulence and subcellular localization, and the yeast signal peptide screening experiment will be used to verify their secretion function; through overexpression and knockout of effector genes, the localization of the effector proteins in C. camelliae organization and the effect of key virulence functional domains on their pathogenicity will be further clarified. The results will provide a theoretical basis for exploring the interactive resistance protein in the tea plant and their regulation mechanism in disease resistance.

炭疽病是影响茶树生长与茶叶品质的主要病害之一。申请人前期研究证实，炭疽菌Colletotrichum camelliae是我国茶树炭疽病优势病原菌；炭疽菌分泌的效应蛋白与茶树抗病蛋白发生互作，激活寄主MAPK信号通路并调控下游防御反应产生免疫。但炭疽菌关键效应蛋白却不清楚。本研究利用PVX病毒表达载体和烟草瞬时表达系统，对前期筛选的36个炭疽菌候选效应蛋白进行毒性鉴定及其亚细胞定位分析，结合酵母信号肽筛选实验验证效应蛋白的分泌功能，明确关键毒性效应蛋白；通过过表达和敲除效应子基因等方法，进一步明确炭疽菌效应蛋白的组织定位、关键毒性功能域对致病性的影响。研究结果将为今后探索茶树互作抗病蛋白及其调控的抗病机制提供理论依据。

炭疽病是影响茶叶生产的主要因素之一，山茶炭疽菌是茶树炭疽病的优势病原菌，但对于病原菌的致病机理却仍不清楚。本研究对山茶炭疽菌进行基因组测序，发现该菌基因组大小为67.56 Mb，注释获得15343个基因，GC含量49.02%，组装成20个Scaffold，最长Scaffold为8.43 Mb。诱导发现，C. camelliae发育形成孢子（Con）、芽管（GT）、附着胞（App）、初级菌丝（CIH）、次级菌丝（IL）结构。基于不同结构转录组测序，共筛选出12820条差异表达基因，对C. camelliae不同组织差异基因的表达模式进行分析及其GO和KEGG富集。GO富集结果表明，在活体营养阶段（GT、App、CIH）差异基因主要富集到生物过程和细胞组分，而在死体营养阶段（IL）差异基因主要富集到分子功能。KEGG富集结果表明，除GT阶段差异基因主要富集于遗传信息处理途径外，其余阶段主要富集于代谢途径。对在附着胞黑化过程中相关基因和碳水化合物活性酶（CAZys）的表达模式进行分析后，发现cAMP和MAPK信号通路相关基因与附着胞黑化有关，并且该类基因均在App、CIH、IL阶段上调表达，黑色素合成途径关键基因在App阶段上调表达；筛选了278个CAZys差异表达基因，基因表达谱表明这些基因在App、CIH、IL阶段显著上调表达，其中多糖裂解酶基因（CcPOL）仅在IL阶段特异性显著表达。对基因组和转录组联合分析，发现该菌果胶裂解酶（CcPL）在死体营养阶段发挥主要作用。建立了PEG介导的山茶炭疽菌遗传转化体系，并对候选的7条CcPLs基因进行功能鉴定，结合烟草瞬时过表达、茶树致病性测试、分泌蛋白验证试验，证明CcPLs可以影响山茶炭疽菌形态发育变化，其中CcPL16可以降低病原菌的致病力。研究结果对阐明茶树免疫机理提供了坚实的科学依据。