Suppression of innate immunity by an ADP-ribosyltransferase type III effector

描述（由申请人提供）：真核先天免疫系统是病原体需要绕过的重要屏障，以引起疾病。该系统的几个组成部分在真核生物中是保守的。最近，通过III型蛋白分泌系统（TTSSs）注入宿主细胞的细菌病原体效应物已被证明能够抑制真核生物的先天免疫。植物病原菌丁香假单胞菌依赖TTSS对植物致病。p.s. pv。番茄DC3000效应基因hopU1类似于ADP核糖基转移酶（ADP- rts）基因。这些基因编码动物细菌性病原体中一些最容易理解的毒素（例如霍乱毒素）。本研究的初步数据表明，HopU1是一种活性ADP-RT，它能使几种植物蛋白发生adp核苷化。质谱分析表明，叶绿体和富含甘氨酸的rna结合蛋白是HopU1的体外底物。这些都是adp - rt的新型底物。此外，HopU1能够抑制植物先天免疫系统的几种反应，这种反应依赖于它的ADP-RT活性位点。缺乏HopU1底物AtGRP7的拟南芥突变体对丁香假单胞菌的易感性增强，这表明它是先天免疫的一个组成部分。AtGRP7是一种富含甘氨酸的RNA结合蛋白，这表明该病原体靶向参与RNA代谢的蛋白来抑制先天免疫。所提出的实验的中心假设是AtGRP7和HopU1 ADP-RT III型效应物的其他靶点可能是先天免疫的组成部分。一些实验试图利用生化和分子生物学方法阐明AtGRP7在先天免疫中的作用。由于资源丰富、真核生物天然免疫系统的相似性以及研究成本高，丁香拟南芥病理系统是研究天然免疫系统的一个很好的模型。这些实验将有助于对细菌发病和先天免疫的分子机制有一个基本的了解。

DESCRIPTION (provided by applicant): The eukaryotic innate immune system represents an important barrier that pathogens need to circumvent in order to cause disease. Several components of this system are conserved in eukaryotes. Recently, bacterial pathogen effectors that are injected into host cells by type III protein secretion systems (TTSSs) have been shown to be capable of suppressing innate immunity in eukaryotes. The bacterial plant pathogen Pseudomonas syringae is dependent on a TTSS to cause disease on plants. The P. s. pv. tomato DC3000 effector gene hopU1 resembles ADP ribosyltransferases (ADP-RTs) genes. These genes encode some of the best understood toxins in bacterial pathogens of animals (e. g., cholera toxin). Preliminary data within this proposal show that HopU1 is an active ADP-RT and that it ADP-riboslylates several plant proteins. Mass spectrometry determined that chloroplast and glycine-rich RNA-binding proteins acted as in vitro substrates for HopU1. These are novel substrates for ADP-RTs. Moreover, HopU1 has the ability to suppress several responses of the plant innate immune system in a manner that is dependent on its ADP-RT active site. An Arabidopsis mutant lacking one HopU1 substrate, AtGRP7, displayed enhanced susceptibility to P. syringae suggesting that it is a component of innate immunity. AtGRP7 is a glycine-rich RNA-binding protein, which suggests this pathogen targets proteins involved in RNA metabolism to suppress innate immunity. The central hypothesis of the proposed experiments is that AtGRP7 and perhaps other targets of the HopU1 ADP-RT type III effector are components of innate immunity. Several of the experiments seek to elucidate the role AtGRP7 plays in innate immunity using biochemical and molecular biological approaches. The P. syringae-Arabidopsis pathosystem is an excellent model to study the innate immune system because of the resources available, the similarities between innate immune systems between eukaryotes, and the cost efficient research. These experiments will contribute to a fundamental understanding of the molecular mechanism of bacterial pathogenesis and innate immunity. The Specific Aims are the following: (1) Determine the molecular consequence of ADP- ribosylation on the function of AtGRP7 and elucidate the role this protein plays in innate immunity; (2) Identify additional substrates of HopU1 and verify their involvement in innate immunity; (3) Analyze the affect that HopU1 has on host-microbe interactions. Project Narrative: Identifying the eukaryotic targets for the P. syringae HopU1 ADP-ribosyltransferase will contribute to our understanding of bacterial pathogenesis and will likely reveal important components of the innate immune system. One HopU1 target belongs to a large group of proteins called glycine-rich RNA binding proteins, which are not well understood, and this research will likely increase our understanding of these proteins. Because there are considerable similarities between the innate immune systems in plants and mammals we expect that our findings will be relevant to the mission of the NIH and be broadly interesting to researchers studying molecular mechanisms of bacterial pathogenesis and innate immunity.