木薯细菌性枯萎病菌III型效应蛋白基因的筛选及功能鉴定

Cassava bacterial blight (CBB), incited by Xanthomonas axonopodis pv. manihotis (Xam), is the most important bacterial disease of cassava, a staple food source for millions of people in developing countries. Crop improvement efforts have aimed at attaining CBB resistance; however, these efforts have been largely unsuccessful, most likely due to genetic diversity among Xam strains and the pathogenic mechanism. Bacterial phytopathogen effectors proteins were critical in infecting its host plants, colonization and the occurrence of disease. However, the study on its effectors proteins was extremely limited. Two different pathogenic strains of Xam were separated in our previous study, and a weak virulence strains of Xanthomonas genome has been sequenced, and the candidate effectors genes were predicted .In this project, comparative genomic analysis of two different pathogenic strains have be adopted, and the effectors protein genes of the strong virulence strains of Xam would be identified via comparative genomics analysis and the deletion mutant of the candidate genes would been obtained through the insertion vector. The biological characteristics and pathogenicity of the mutant would be carried out. The related regulatory pathways and biochemical mechanisms of the candidate effectors protein genes would be further analyzed with cassava protoplast transient expression system and yeast two hybrid. The project was carried out to clarify the function of candidate effector genes and regulatory pathways, and provide experimental evidence for the interaction mechanism between Xam and cassava. The results will provide a theoretical basis for the development of new disease prevention and control strategies.

地毯草黄单胞菌引起的木薯细菌性枯萎病是生产上的重要病害，严重影响了木薯产业的健康发展。病原菌的III型效应蛋白对其在寄主植物的侵染（识别）、定殖和病害发生都至关重要，然而地毯草黄单胞菌对木薯的致病机理还不清楚，对其效应蛋白的研究也极其有限。我们的前期研究分离了2个致病性不同的菌株，并对一个弱致病力黄单胞菌菌株基因组进行了测序，预测了候选的III型效应蛋白基因。本项目在此基础上，拟通过致病性强弱不同的2个地毯草黄单胞菌菌株的比较基因组学分析，候选强致病性菌株可能的效应蛋白基因，并通过缺失突变鉴定候选效应蛋白基因对病原菌生物学特性、致病性的影响；借助酵母双杂、木薯原生质体瞬时表达系统进一步对候选效应蛋白的相关调控途径及生化机制进行分析，初步阐明候选III型效应蛋白的功能及调控通路。项目的开展为阐明地毯草黄单胞菌与木薯的互作机理提供了基因资源和实验依据，同时也为新型病害防控策略的制定提供理论。

地毯草黄单胞菌引起的木薯细菌性枯萎病是生产上的重要病害，严重影响了木薯产业的健康发展。病原菌的III型效应蛋白对其在寄主植物的侵染、定殖和病害发生都至关重要，然而地毯草黄单胞菌对木薯的致病机理还不清楚，对其效应蛋白的研究也极其有限。.我们的前期研究分离了2个致病性不同的菌株，本项目在此基础上，在对致病性有差异的Xam1和Xam11两个菌株的致病相关生理生化指标检测，明确了2种病原菌在菌落形态、生长速度、胞外酶等生理生化表现上存在差异；进一步采用illumina测序平台对2个菌株的基因组进行了测序，通过比较分析候选了30个效应蛋白基因进行了功能鉴定，其中XopA、XopAG、XopC2、XopAU、XopQ等基因的敲除明显降低了突变体的致病性。采用酵母双杂交对XopC2、XopAU互作蛋白进行了筛选，获得互作蛋白20个，推测XopC2可能通过泛素蛋白酶体的竞争性结合调控致病性，XopAU可能通过调控MAPK通路调节植株对病原菌的抗性。.为明确XopC2的作用机制，我们采用互作转录组测序的方法，对Xam1、Xam11、Xam11△XopC2菌株浸染木薯后不同时间点的转录情况进行了分析，证实了我们的推测，该效应蛋白通过蛋白翻译后的修饰和降解调控病原菌的致病性。进一步的结果正在验证中。.项目的开展为阐明地毯草黄单胞菌与木薯的互作机理提供了基因资源和实验依据，同时也为新型病害防控策略的制定提供理论。项目实施期间，项目主持人获得省部级荣誉6次，获批国家木薯产业体系专项经费1项、吸纳博士1名、晋升职称1人、获得国家自然基金2项，培养研究生3名，本科生4名，发表论文14篇，其中中文核心期刊10篇，SCI4篇，标注资助5篇，参加国内外相关学术会议8次。