苹果树腐烂病菌效应蛋白VmEP1抑制植物免疫性的机理研究

The weakly parasitic pathogen Valsa mali, causing destructive rot in phloem and xylem of apple tree, and even tree death, has been the key objects of prevention and treatment in apple production. However, little information about the substances involved in infection, colonization and pathogenic process is still the primary limitation for disease management. In the recent studies, a total of 413 effector protein candidates was predicted based on the V. mali genome. And the effector protein VmEP1 annotated as hypothetical protein, which led to significant suppression of BAX-induced cell death in Nicotiana benthamiana, was identified. Moreover, its gene knockout mutant showed much lower virulence on apple, which implies effector protein VmEP1 contributed to the pathogen infection and colonization in apple. Therefore, this project further plan to a) analyze the gene features about signal peptide and domain; b) confirm the subcellular localization of VmEP1 in N. benthamiana; c) identify the interaction target in N. benthamiana through yeast two hybrid and co-immunoprecipitation, and then verify the target function by virus induced gene silencing and overexpression; d) explore the homologous interaction target protein in apple; e) finally, observe the phenotypes of mutant ΔVmEP1 by cytological methods. These results may reveal the mechanism of suppressing plant cell death by VmEP1, which will help establish sustainable disease management strategies for apple valsa canker.

由弱寄生性苹果黑腐皮壳侵染树干引起的苹果树腐烂病极易造成韧皮部和木质部溃烂甚至整树死亡，导致严重经济损失，一直是生产上的重点防治对象。但对病菌哪些物质参与侵染、定殖和致病过程却知之甚少。课题组在前期完成了病菌全基因组测序的基础上，从预测获得的413个效应基因中筛选获得了一个能够抑制由激发子Bax引起的烟草叶片细胞坏死效应蛋白VmEP1，注释为功能未知假想蛋白，且基因敲除突变体对苹果致病性明显降低，推测为致病关键因子。因此，本项目拟首先应用异源表达体系明确信号肽，利用缺失突变和瞬时表达技术明确功能域；同时利用组织细胞学技术观察VmEP1作用部位及植物的防卫反应；用酵母双杂交和免疫共沉淀技术(Co-IP)筛选鉴定靶标蛋白，基因沉默(VIGS)分析靶标功能；进而在苹果全基因组比对其同源基因，并验证靶标蛋白，从而揭示VmEP1抑制植物免疫性的机理，为腐烂病可持续控制策略制定提供新的思路和理论依据。

由弱寄生性苹果黑腐皮壳侵染树干引起的苹果树腐烂病极易造成树皮腐解溃烂甚至整树死亡，导致严重经济损失，但对病菌侵染、定殖和致病机理却了解很少。前期从病菌全基因组预测的 413 个效应基因中筛选获得一个能够抑制由激发子 Bax 引起的烟草叶片细胞坏死效应蛋白 VmEP1，注释为功能未知假想蛋白，敲除突变体对苹果致病性明显降低，推测为致病关键因子。为此，本项目首先鉴定了VmEP1信号肽并通过酵母异源表达证明其信号肽具有外泌功能。利用亚细胞定位发现VmEP1主要定位在植物细胞核内。进行VmEP1缺失突变，借助本氏烟瞬时表达以及本实验室建立的PEG介导的遗传转化体系，发现VmEP1 蛋白340-354位氨基酸对其抑制BAX诱导的坏死及病原菌致病性起关键作用，明确了其功能域。明确了苹果对ΔVmEP1防卫反应的细胞学特征，发现VmEP1能够抑制寄主免疫应答反应并促进病菌侵染。利用酵母双杂交、免疫沉淀以及LC-MS技术共获得9个VmEP1候选互作靶标，利用双分子荧光互补技术（BiFC）和免疫共沉淀（Co-IP）技术，发现苹果病程相关蛋白PR10（MdPR10）、本氏烟同源蛋白（NbPR10）均与VmEP1存在互作关系。通过烟草瞬时表达结合定量PCR检测，发现VmEP1能够抑制本氏烟茉莉酸、水杨酸以及乙烯信号，促进核盘菌的侵染。利用病毒诱导的基因沉默（VIGS）技术，发现NbPR10被沉默后VmEP1丧失抑制茉莉酸、水杨酸以及乙烯信号通路的功能，揭示了效应蛋白VmEP1通过靶向植物 PR10蛋白而抑制植物免疫反应促进病菌侵染。本项目明确了效应蛋白VmEP1 抑制植物免疫的机理，可为植物病害可持续控制提供新的思路和理论依据。..