植物细菌病原体毒性的激活机制研究

Plant diseases caused by bacterial pathogens pose an important threat to food security worldwide. Ralstonia solanacearum is one of the most destructive bacterial plant pathogens, and is able to infect important crops, including potato, tomato, tobacco, banana, pepper and eggplant, among others. Like many other bacterial pathogens of plants and animals, R. solanacearum is able to manipulate host cells by using a type III secretion system (T3SS), which is a molecular syringe that injects bacterial proteins directly inside host cells. These proteins, called type III effectors (T3Es), target important cellular functions, such as immunity, in order to enable bacterial infection and cause disease. Deciphering T3E mode of action will be essential for our understanding of fundamental aspects of bacterial diseases in plants and the engineering of efficient plant resistance against them..In recent years, research from several groups has been directed towards understanding T3E targets and virulence activities. However, an important question remains unanswered, which is how T3Es get activated once they are delivered inside plant cells. Several T3Es display toxic enzymatic activities that need to be specifically activated in host cells, but, most importantly, T3Es need to travel through the T3SS in an unfolded state, requiring host enzymes or other host factors to achieve their active conformation. Several T3Es from plant and animal pathogens have been shown to undergo phosphorylation inside host cells, but the mechanistic insights of their phosphorylation-based activation and the plant proteins involved in the process remain unknown. .In this proposal, we describe a novel approach to characterize T3E activation inside the plant cell based on post-translational modifications (PTMs) by host enzymes. We will take advantage of the large repertoire of T3Es injected by R. solanacearum to identify T3Es that undergo PTMs, and will follow innovative approaches to identify the plant enzymes involved in the process of activation of T3Es. This approach will also allow us to design unprecedented PTM-based T3Es translocation markers, to identify different T3Es that are injected inside plant cells at different stages of the infection process. Our research will uncover fundamental aspects of the infection by bacterial pathogens, which is an essential step towards the generation of crop varieties more resistant to bacterial infections.

细菌病原体引起的植物疾病对全球粮食安全造成重大威胁。青枯菌破坏性极强，能感染土豆、番茄等重要农作物。它通过Ⅲ型分泌系统(T3SS)将Ⅲ型效应子蛋白(T3E)直接注入宿主细胞，被注入的T3E可干扰植物多种细胞功能，最终导致植物青枯病。因此破译T3E的作用模式对理解植物细菌性疾病，提高植物抗病能力至关重要。近年的研究直指T3E的靶标及其毒力，但T3E在宿主细胞内的激活过程仍待解答。一些T3E在宿主细胞内被磷酸化而激活，但其详细激活过程及参与的蛋白仍未知。我们采用新方法，利用青枯菌向植物宿主细胞注入许多不同T3E的优势来鉴定需要翻译后修饰的T3E以及植物中参与T3E激活的酶。该手段有助于我们详细阐述T3E在植物细胞内的活化过程，设计翻译后修饰依赖的T3E易位标记，以识别病原体在侵染的不同阶段注入植物内的不同T3E。本研究将揭示细菌病原体侵染植物的基本层面，是培育有更强抗病力农作物品种的必要一步。

细菌性病原体引起的植物病害是威胁世界粮食安全的重要因素。青枯菌(Ralstonia solanacearum)破坏性极强，能感染土豆、番茄、烟草等重要农作物。它通过Ⅲ型分泌系统(T3SS)将Ⅲ型效应子蛋白(T3E)直接注入宿主细胞，被注入的T3E可干扰植物多种细胞功能，最终导致植物青枯病。因此破译T3E的作用模式对理解植物细菌性疾病，提高植物抗病能力至关重要。. 在本项目中，我们采用了一种新的方法，通过识别植物靶标和宿主酶的翻译后修饰(PTMs)来表征植物细胞内的T3E活性。利用青枯菌向植物宿主细胞注入大量不同T3E这一优势，采用创新的方法识别植物中参与T3E激活过程的酶。目前，我们已经确定了许多在植物细胞内进行PTMs的T3Es，以及一些T3Es的靶点。RipAY在植物细胞中被磷酸化，这种磷酸化是激活其GGCT活性所必需的。RipAY利用这种GGCT活性来降解植物细胞中的谷胱甘肽，从而导致免疫反应的抑制。我们还对T3E RipE1进行了深入的描述，它通过植物免疫受体的识别在植物细胞中诱导免疫。然而，我们也发现RipAY利用其GGCT活性抑制RipE1识别下游谷胱甘肽的积累，消除其免疫诱导活性，并通过毒力因子的进化反映出一种细菌毒力策略。此外，还鉴定了其他几种T3Es及其植物靶标，以及对这些T3Es的分子活性至关重要的磷酸化事件。我们的研究揭示了细菌性病原体感染的基本方面，是向培育更能抵抗细菌感染的作物品种迈出的重要一步。