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的革兰氏阴性植物病原菌寄生的中心事件似乎是HRP(类型III蛋白分泌系统)介导的‘AVR’效应蛋白转位到植物细胞中。AVR蛋白之所以这样命名，是因为它们在菌株中的存在使病原体在携带同源R(抗性)基因的潜在宿主中无毒。植物细胞内的AVR-R蛋白相互作用会触发超敏反应(HR)，这是一种与防御相关的程序性细胞死亡。虽然AVR蛋白可能会在非寄主或抗性植物中背叛寄生虫进行防御监视，但它们似乎是寄主植物中寄生所共同需要的。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蛋白的传递需要HRP(HR和致病性)和HRC(HR和保守)基因。HRC基因编码的核心成分在所有III型分泌系统中都是保守的，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，它们似乎指导蛋白质跨细菌的内膜和外膜转运。HRP基因编码Harpin(HrpZ)、HRP菌毛亚基(HrpA)、调节蛋白和一些未知功能的蛋白，这些蛋白被认为在与宿主细胞接触时促进该途径的充分激活和形成细胞外易位复合体。确定对这两个过程作出贡献的HRP组件是本提案的一个特别重点。目前AVR蛋白转移到植物细胞的证据是间接的，直到最近还没有观察到AVR蛋白在培养物中分泌。然而，目前已证实克隆在菊花欧文氏菌粘粒pCPP2156上的HRP/HRC基因簇在大肠杆菌中具有分泌已被广泛研究的丁香欧文氏菌AVR蛋白的功能，如AvrB、AvrPto和AvrRpt2。此外，尽管在培养中不分泌AvrB，但根据免疫印迹分析，已发现在培养中它能分泌AvrPto和AvrRpt2。AvrPto在大肠杆菌(PCPP2156)和丁香假单胞菌(无论表达AvrPto的病原体或菌株)中的分泌尤其强劲，这支持了AVR靶向信号的探索。耶尔森氏菌转位到宿主细胞的Yop效应蛋白通过Yop mRNA的前15个密码子携带的信号靶向于III型途径(一些YOP还带有伴侣依赖的靶向结构域)。在动植物病原菌的转位效应蛋白中，靶向信号普遍存在，小肠结肠炎耶尔森菌和大肠杆菌(PCPP2156)可互换地分泌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，足以将NPT报告蛋白靶向III型途径。紫丁香菌在培养过程中分泌AvrPto和AvrPto1-15-NPT，为HRP组分功能的遗传研究提供了新的分子表型。这个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(分泌HrpZ和AvrPto所必需的)不同的表观分泌行为，以及控制AVR转位到植物中的因素。紫丁香菌hrp/hrc基因的研究紫丁香61克隆在粘粒PHIR11上，使荧光假单胞菌和大肠杆菌等非致病细菌能够在携带同源R基因的受试植物上诱导依赖AVR的HR。这个HRP/HRC簇已经完全测序，位于HRP致病岛的中心，两侧是编码HRP分泌的额外底物的基因。该簇中的所有26个HRP/HRC基因都存在非极性突变。紫丁香疫霉HRP/HRC基因在最小但不复杂的培养基中表达，并通过在培养基中加入植物细胞额外诱导20倍。这种诱导需要活的植物细胞和一个功能正常的HRP分泌系统，它为鉴定感受植物细胞的HRP组分提供了一个表型。本项目的具体目标是：1.确定影响HRP途径蛋白质差异分泌行为的靶向信号，并建立检测HRP介导的蛋白质转运到植物细胞的改良方法。2.通过体外生化分析，确定丁香假单胞菌HRP分泌系统的所有胞外成分，使其产量最大化的条件，以及这些蛋白质在超分子复合体中的潜在相互作用。3.确定HRP系统的每个成分对一组分子表型的贡献(以及该成分的适当定位)，这些分子表型旨在剖析HRP介导的AVR蛋白的分泌和易位。

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.