Delivery of Effector Proteins to Plant Cells via the Hrp Type III Protein Secretion System of Pseudomonas Syringae

丁香假单胞菌和其他常见革兰氏阴性植物病原体寄生的中心事件似乎是Hrp （III型蛋白分泌系统）介导的“Avr”效应蛋白易位进入植物细胞。Avr蛋白之所以如此命名，是因为它们在菌株中的存在使病原体对携带同源R（抗性）基因的潜在宿主无毒。植物细胞内Avr-R蛋白相互作用引发超敏反应（hypersensitive response， HR）；防御相关的程序性细胞死亡尽管在非寄主或抗性植物中，Avr蛋白可能背叛寄生虫的防御监视，但它们似乎是寄主植物寄生所必需的。因此，了解Avr蛋白的传递机制是了解细菌植物致病性的关键，并可能产生新的治疗方法。Avr蛋白的递送需要hrp （HR和致病性）和hrc （HR和保守性）基因。hrc基因编码在所有III型分泌系统中保守的核心成分，包括人类致病性耶尔森氏菌、志贺氏菌和沙门氏菌的分泌系统，它们似乎指导细菌内外膜的蛋白质易位。hrp基因编码一个harpin (HrpZ)，一个hrp毛囊亚基（HrpA），调节蛋白和一些功能未知的蛋白，这些蛋白被认为在与宿主细胞接触时促进通路的完全激活和细胞外易位复合物的形成。确定促进这两个进程的Hrp组成部分是本提案的特别重点。目前Avr蛋白易位到植物细胞的证据是间接的，直到最近还没有观察到在培养物中分泌Avr蛋白。然而，现在已经确定，从菊花Erwinia的cosmid pCPP2156上克隆的hrp/hrc基因簇在大肠杆菌中发挥功能，分泌丁香假单胞菌Avr蛋白，如AvrB， AvrPto和AvrRpt2。此外，尽管P. syringae在培养物中不分泌AvrB，但根据免疫印迹分析，在培养物中发现它分泌AvrPto和AvrRpt2。AvrPto在大肠杆菌（pCPP2156）和丁香假单胞菌中（无论其表达的病原体或菌株）的分泌特别强劲，这支持了对Avr靶向信号的探索。耶尔森菌易位到宿主细胞中的Yop效应蛋白通过Yop mRNA前15个密码子携带的信号靶向III型途径（一些Yops还携带伴侣依赖的靶向结构域）。mRNA靶向信号在动植物致病菌易位效应蛋白中具有普遍性，小肠结肠炎Y.小肠结肠炎和大肠杆菌（pCPP2156）可互换分泌AvrB、AvrPto、YopE和YopQ。与YopQ一样，AvrPto的前15个密码子是分泌天然蛋白所必需的，并且足以靶向III型途径的Npt报告蛋白。丁香P. syringae在培养中同时分泌AvrPto和AvrPto1-15-Npt，为Hrp成分功能的遗传探索提供了新的分子表型。本项目特别感兴趣的是AvrB、AvrPto、HrpZ（被认为是针对植物细胞外部的）和HrpA （HrpZ和AvrPto的分泌所必需的）不同的表观分泌行为以及控制Avr转运到植物中的因素。丁香假体的hrp/hrc基因。在cosmid pHIR11上克隆的丁香61使非致病性细菌，如荧光P.和大肠杆菌，在携带同源r基因的受试植物上引发avr依赖性HR。hrp/hrc基因簇已被完全测序，位于hrp致病岛的中心，其两侧是编码hrp分泌底物的基因。在该基因簇中，所有26个hrp/hrc基因均存在非极性突变。丁香假单胞杆菌hrp/hrc基因在最小但不复杂的培养基中表达，并通过在培养基中添加植物细胞而被诱导20倍。这种诱导需要活的植物细胞和功能正常的Hrp分泌系统，它提供了一种表型，用于鉴定感应植物细胞的Hrp成分。本项目的具体目标是：1。确定影响Hrp通路中蛋白质的不同分泌行为的靶向信号，并开发改进的检测Hrp介导的蛋白质转运到植物细胞的方法。2. 鉴别紫丁香辣根过辣素分泌系统的所有细胞外成分，最大化其产生的条件，以及这些蛋白质在超分子复合物中的潜在相互作用，通过体外生化分析确定。3. 确定Hrp系统的每个组成部分（以及该组成部分的适当定位）对旨在解剖Hrp介导的Avr蛋白分泌和易位的分子表型的贡献。

The central event in the parasitism of Pseudomonas syringae and other common gram-negative plant pathogens appears to be the Hrp (type III protein secretion system)-mediated translocation of 'Avr' effector proteins into plant cells. Avr proteins are so named because their presence in a strain renders a pathogen avirulent in a potential host that carries a cognate R (resistance) gene. Avr-R protein interactions inside plant cells trigger the hypersensitive response (HR); a defense-associated programmed cell death. Although Avr proteins may betray the parasite to defense surveillance in nonhost or resistant plants, they appear collectively required for parasitism in host plants. Thus, an understanding of Avr protein delivery mechanisms is key to understanding bacterial plant pathogenicity and is likely to yield novel therapies. The delivery of Avr proteins requires hrp (HR and pathogenicity) and hrc (HR and conserved) genes. hrc genes encode core components that are conserved in all type III secretion systems, including those of human pathogenic Yersinia, Shigella, and Salmonella, and they appear to direct protein translocation across the inner and outer membranes of bacteria. hrp genes encode a harpin (HrpZ), a Hrp pilus subunit (HrpA), regulatory proteins, and several proteins of unknown function that are postulated to promote full activation of the pathway and formation of an extracellular translocation complex upon contact with host cells. Identifying Hrp components that contribute to these two processes is a particular focus of this proposal. The current evidence for Avr protein translocation into plant cells is indirect, and until recently Avr proteins have not been observed to be secreted in culture. However, it has now been established that a hrp/hrc gene cluster cloned on cosmid pCPP2156 from Erwinia chrysanthemi functions in Escherichia coli to secrete well-studied P. syringae Avr proteins, such as AvrB, AvrPto, and AvrRpt2. Furthermore, although P. syringae does not secrete AvrB in culture, it has been found to secrete AvrPto and AvrRpt2 in culture, as based on immunoblot analyses. The secretion of AvrPto is particularly robust in E. coli(pCPP2156) and P. syringae (regardless of the pathovar or strain in which it is expressed), that has supported an exploration of Avr targeting, signals. The Yop effector proteins that Yersinia translocates into host cells are targeted to the type III pathway by a signal carried in the first 15 codons of Yop mRNA (some Yops also carry chaperone-dependent targeting domains). The mRNA targeting signal appears universal among the translocated effector proteins of plant and animal pathogens, and Y. enterocolitica and E. coli(pCPP2156) secrete AvrB, AvrPto, YopE and YopQ interchangeably. As with YopQ, the first 15 codons for AvrPto are necessary for secretion of the native protein and are sufficient for targeting an Npt reporter protein to the type III pathway. The secretion of both AvrPto and AvrPto1-15-Npt by P. syringae in culture provides new molecular phenotypes for genetic exploration of Hrp component functions. Of particular interest for this project are the different apparent secretion behaviors of AvrB, AvrPto, HrpZ (thought to be targeted to the exterior of the plant cell), and HrpA (required for the secretion of HrpZ and AvrPto) and the factors controlling Avr translocation into plants. The hrp/hrc genes of P. syringae pv. syringae 61, cloned on cosmid pHIR11, enable nonpathogenic bacteria, like P. fluorescens and E. coli, to elicit an Avr-dependent HR on test plants that carry cognate R-genes. This hrp/hrc cluster has been completely sequenced and is at the center of a Hrp pathogenicity island flanked by genes encoding additional substrates for Hrp secretion. Nonpolar mutations have been constructed in all 26 of the hrp/hrc genes in the cluster. P. syringae hrp/hrc genes are expressed in minimal, but not complex, media and are induced an additional 20-fold by the addition of plant cells to the medium. This induction requires living plant cells and a functional Hrp secretion system, and it provides a phenotype for identifying Hrp components that sense plant cells. The specific objectives of this project are: 1. Identify targeting signals affecting the differential secretion behaviors of proteins traveling the Hrp pathway, and develop improved assays for detecting Hrp-mediated protein translocation into plant cells. 2. Identify all extracellular components of the P. syringae Hrp secretion system, the conditions maximizing their production, and the potential interaction of these proteins in a supramolecular complex as determined by in vitro biochemical assays. 3. Determine the contribution of each component of the Hrp system (and proper localization of the component) to a panel of molecular phenotypes aimed at dissecting Hrp-mediated Avr protein secretion and translocation.