甘蓝枯萎病菌1号生理小种（Foc1）效应子鉴定及免疫调控机制研究

Fusarium oxysporum f. sp conglutinans (Foc), a major soil-bornefungal pathogen, is one of the most destructive diseases of cabbage worldwide. Foc race 1(Foc1) has spread over major cabbage producing areas in north China, causing huge losses to cabbage industry. Identification of the effectors of Foc1 and analysis of their molecular mechanism in immune suppression are significant important to understand the molecular mechanisms for Foc1 pathogenicity. In this study, we plan to identify the pathogenic effectors of Foc1 by combining the omics methods, including genomics, resequencing, transcriptomics and proteomics, with molecular genetic experiments. The candidate effectors will subject to stringent validation experimentally, including gene deletion, complement, and hypersensitive responses experiment. We will further analyze the function of the effectors through a series of molecular experiments, such as subcellular localization of the effectors, constructing the deletions and identification of targets genes for the effectors by co-immunoprecipitation. Our goal is to identify pathogenic effectors for Foc1 and elucidate the molecular mechanism of immune regulation, and the results will provide theoretical guidance for establishing innovative control methods and breeding strategy of resistant cabbages.

甘蓝枯萎病是一种毁灭性的土传病害,其中1 号生理小种（Foc1）是危害我国甘蓝生产的优势病原菌。鉴定Foc1的毒性及非毒性效应子，并分析其调控寄主免疫反应的分子机制对于解析甘蓝枯萎病菌的致病机理具有重要意义。本研究拟通过结合基因组、重测序、转录组、蛋白组等组学方法和分子遗传实验对Foc1的效应子进行鉴定。在此基础上，通过构建GFP 融合蛋白对目标基因进行亚细胞定位、构建目的基因的删除片段鉴定其功能域、通过免疫共沉淀(Co-IP)鉴定效应子的互作蛋白等方法系统解析效应子的功能及其调控寄主免疫反应的分子机制。该研究对于丰富尖孢镰刀菌的致病机理具有重要的意义，并可为甘蓝枯萎病控制及育种策略提供理论支持。

甘蓝枯萎病是一种毁灭性的土传病害,其中1 号生理小种（Foc1）严重制约着我国北方甘蓝产业发展。鉴定Foc1的致病效应子，并分析其抑制寄主免疫反应的分子机制对于解析甘蓝枯萎病菌的致病机理和规律有着重要的意义。本研究利用三代Pacbio测序技术完成了Foc1 和2号（Foc2）的基因组测序及组装，与二代测序组装的基因组相比，基因组的大小分别提升了9.7Mb和7.4Mb；contig的N50分别达到了1.58Mb和1.28Mb,比二代测序技术组装分别提高了0.72Mb和0.64Mb。通过Foc1和Foc2的比较基因组分析，分别鉴定了90个Foc1特异基因和441个Foc2特异基因。通过蛋甘蓝枯萎病侵染甘蓝后的伤流液的蛋白组的分析，鉴定了可能参与Foc1致病的4个效应子蛋白，并构建了4个效应子蛋白的突变菌株。我们进一步对候选效应子的功能进行了研究，，对基因的敲除突变菌株致病力以及部分生物学特性进行测定，敲除突变体与野生型相比，发现4个效应子中只有FOCA1-16510的致病力下降，而FOCA1-11385缺失突变体致病力上升。而生长速率与产孢量的测定显示，只有 ∆FOCA1-01935、∆ FOCA1-13811的生长速率略缓慢，且产孢量差异均不显著。这表明FOCA1-16510和FOCA1-11385参与了Foc1的侵染和致病过程，而FOCA1-01935和∆FOCA1-13811只参与Foc1的生长发育过程。为了进一步深入了解Foc的致病机制及Foc1和Foc2致病性差异的原因，我们对Foc2特异的效应子g177基因进行敲除，发现g177影响Foc2的生长发育及致病力。上述的研究表明，Foc的致病机制是一个多效应子参与的复杂机制，对Foc效应子致病机制的研究，对于理解Foc的致病机制及抗病育种均具有重要的意义。