诱发植物坏死效应蛋白VmE02在腐烂病菌侵染定殖苹果中的作用机理

Apple Valsa canker, caused by the weakly parasitic ascomycete Valsa mali (Vm), resulting in severe necrosis on tree bark and even death to trees, has always been key problem in apple production. However, lacking of deep understanding of the mechanism of pathogenesis of canker on tree bark, no efficient control measures developed so far. Thus, it is very urgent to deeply understand the interaction process between this necrotrophic fungus and its host. The understanding will be very helpful for development of more efficacious control measures in future. Effectors secreted by pathogens play critical roles in microbe - plant interactions. In our recent studies, an unannotated hypothetical effector protein VmE02 was found, which could induce cell death on Arabidopsis thaliana and other plants. qRT-PCR showed that this gene was significantly up-regulated during early infection stage (6-24 h after inoculation) and the knockout mutant of this effector gene showed no significant difference in pathogenicity on apple twigs compared with Vm wild type 03-8 strain. The hypothesis is that VmE02, inducing host cell death, plays an important role in the Vm infection and colonization of apple bark. Then, what is the mechanism of this effector protein during V. mali - apple interaction? Therefore, this project will focus on effector VmE02 to find its function and uncover the related pathway. We plan to a) reveal plant responses activity to this effector protein through histological, cytological and biochemical techniques in different aspects; b) identify the interaction target via Y2H and Co-IP; c) verify the function of the target and analyze the function by VIGS, RNAi and gene-knockout. The results could reveal the mechanism of this protein regulating cell death on model plants and host plant and lay a scientific foundation on getting new control strategies for apple Valsa canker.

弱寄生黑腐皮壳引起的苹果树腐烂病造成皮烂枝枯树死，经济损失巨大，一直是苹果生产上的重点防治对象。由于对病菌特异性侵染定殖树皮引致坏死溃烂的机理缺乏深入认识，导致高效防治技术匮乏。因此，解析该死体营养型真菌与苹果互作机理成为亟待解决的关键问题。研究发现效应蛋白在病原菌-植物互作中发挥重要作用。项目前期筛选获得一个诱导拟南芥等多种植物细胞坏死效应蛋白VmE02。该基因在病菌定殖初期显著上调表达，但其缺失突变体致病力无显著变化。由此推测该蛋白诱导的细胞坏死在病菌侵染定殖初期发挥重要作用。为此，本项目拟从组织细胞学等不同层面揭示植物对该蛋白应答反应特征及机理，通过Co-IP等技术筛选鉴定互作靶标，利用VIGS、RNAi及基因敲除等技术分析靶标功能，从而揭示其导致细胞坏死的机理及其在病菌定殖中的作用机理。研究结果有助于深入揭示病菌特异性定殖树皮的分子机理，进而为腐烂病针对性防控策略制定提供科学依据。

由黑腐皮壳属真菌引起的苹果树腐烂病造成皮烂、枝枯、树死，经济损失巨大。由于对该病菌侵染定殖树皮引致坏死溃烂的机理缺乏深入认识，对其与寄主互作关系知之甚少，因而，解析该病菌Valsa mali（Vm）与苹果（Malus domestica）互作的机理成为亟待解决的关键问题。课题组前期从强致病力菌株03-8中筛选到一个引起植物坏死的效应基因VmE02，发现其在病菌侵染初期显著上调表达，过表达突变体致病力提升。为揭示其在Vm侵染定殖苹果中的作用，本课题利用原核表达、组织细胞学、遗传学、免疫共沉淀（Co-IP）、Pull-down及微量热泳动（MST）等技术，首先获得了具有活性的VmE02纯化蛋白，明确了植物的应答反应特征：VmE02分子能够激发植物一系列免疫反应，包括明显的活性氧迸发、胼胝质沉积、免疫标志基因HIN1、HSR203J、PR1、PR2、PR4、LOX上调表达、抗氧胁迫酶CAT、POD、SOD活性升高，且通过VIGS技术沉默植物受体激酶BAK1和SOBIR1后，VmE02不能诱导植物细胞坏死，从而揭示了VmE02为病原相关模式分子（PAMP）。进而，我们研究了VmE02分子与植物的互作机理：首先在本氏烟（Nicotiana benthamiana）中鉴定到受体蛋白RE02，两者在植物体内和体外均互作，结合常数为1.77 ± 0.67 μM；沉默本氏烟RE02后，核盘菌（Sclerotinia sclerotiorum）和辣椒疫霉（Phytophthora capsici）侵染程度分别增加了54.1%和75.3%；再通过序列比对在苹果中找出一个RE02的同源蛋白MdRE02，沉默苹果MdRE02后，Vm侵染程度增加了45.7%，说明RE02及MdRE02正调控植物免疫。进一步的研究发现RE02能够与BAK1和SOBIR1直接互作，且沉默BAK1和SOBIR1能显著降低VmE02激发的植物免疫反应，说明VmE02分子通过受体蛋白RE02、受体激酶BAK1和SOBIR1调控植物免疫性。本项目明确了腐烂病菌03-8菌株VmE02是一个激发子，且深入揭示了其在病菌侵染定殖苹果过程中的作用机理，为免疫诱抗剂开发和广谱抗病育种等绿色防控技术提供了理论依据。以上研究结果已经在New Phytologist上发表论文2篇，完成了课题既定任务和目标。