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

通过丁香假单胞菌的 HRP III 型蛋白分泌系统将效应蛋白递送至植物细胞

• 批准号: 9982646
• 负责人: Alan Collmer
• 金额: --
• 依托单位: Cornell Univ - State: AWDS MADE PRIOR MAY 2010
• 依托单位国家: 美国
• 项目类别: Continuing grant
• 财政年份: 2000
• 资助国家: 美国
• 起止时间: 2000-05-15 至 2004-04-30
• 项目状态: 已结题
• 来源: https://www.nsf.gov/awardsearch/showAward?AWD_ID=9982646&HistoricalAwards=false
• 关键词: Delivery; Effector; Proteins; Plant; Cells

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.

假单胞菌和其他常见的革兰氏阴性植物病原体的寄生中的中心事件似乎是Hrp（III型蛋白分泌系统）介导的“Avr”效应蛋白到植物细胞中的易位。Avr蛋白如此命名是因为它们在菌株中的存在使得病原体在携带同源R（抗性）基因的潜在宿主中无毒。Avr-R蛋白在植物细胞内的相互作用触发过敏反应（HR）;一种防御相关的程序性细胞死亡。虽然Avr蛋白可能背叛寄生虫防御监视在非宿主或抗性植物，它们出现集体需要寄生在宿主植物。因此，理解Avr蛋白的传递机制是理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组分，包括人类致病性耶尔森氏菌、志贺氏菌和沙门氏菌的那些，并且它们似乎指导蛋白质易位穿过细菌的内膜和外膜。hrp基因编码harpin（HrpZ）、Hrp菌毛亚基（HrpA）、调节蛋白和几种功能未知的蛋白质，这些蛋白质被认为在与宿主细胞接触时促进途径的完全激活和细胞外易位复合物的形成。确定有助于这两个进程的人力资源规划组成部分是本建议的一个特别重点。目前的证据Avr蛋白易位到植物细胞是间接的，直到最近还没有观察到Avr蛋白分泌的文化。然而，现在已经确定，克隆在来自欧文氏菌的粘粒pCPP 2156上的hrp/hrc基因簇在大肠杆菌中发挥功能，以分泌充分研究的P. lingae Avr蛋白，如AvrB、AvrPto和AvrRpt 2。此外，尽管P. lingae在培养物中不分泌AvrB，但基于免疫印迹分析，已发现其在培养物中分泌AvrPto和AvrRpt 2。AvrPto的分泌在E. coli（pCPP 2156）和P. lingae（无论其在其中表达的致病变种或菌株），这支持了Avr靶向信号的探索。耶尔森氏菌易位到宿主细胞中的Yop效应蛋白通过Yop mRNA的前15个密码子中携带的信号靶向III型途径（一些Yop还携带分子伴侣依赖性靶向结构域）。mRNA靶向信号在植物和动物病原体的易位效应蛋白中具有普遍性，并且Y. enterocolitica和E. coli（pCPP 2156）可互换地分泌AvrB、AvrPto、YopE和YopQ。与YopQ一样，AvrPto的前15个密码子对于天然蛋白的分泌是必需的，并且足以将Npt报告蛋白靶向III型途径。在培养物中，P. lingae分泌的AvrPto和AvrPto 1 -15-Npt为Hrp组分功能的遗传探索提供了新的分子表型。该项目特别感兴趣的是AvrB、AvrPto、HrpZ（被认为靶向植物细胞外部）和HrpA（HrpZ和AvrPto的分泌所需）的不同表观分泌行为以及控制Avr易位到植物中的因素。将烟草疫霉致病变种的hrp/hrc基因克隆到大肠杆菌BL 21（DE 3）中。克隆在粘粒pHIR 11上的pH 1261能使荧光假单胞菌和大肠杆菌等非致病性细菌。大肠杆菌，以引发一个Avr依赖的HR测试植物携带同源R-基因。该hrp/hrc簇已被完全测序，位于Hrp致病岛的中心，两侧是编码Hrp分泌额外底物的基因。非极性突变已经在簇中的所有26个hrp/hrc基因中构建。P. lingae hrp/hrc基因在最小但不复杂的培养基中表达，并通过向培养基中添加植物细胞诱导额外的20倍。这种诱导需要活的植物细胞和功能性的Hrp分泌系统，并且它提供了用于鉴定感测植物细胞的Hrp组分的表型。本项目的具体目标是：1.识别影响Hrp途径蛋白质差异分泌行为的靶向信号，并开发用于检测Hrp介导的蛋白质易位到植物细胞中的改进的检测方法。2.确定所有的细胞外组分的P. erichingae Hrp分泌系统，最大限度地提高其生产的条件下，这些蛋白质在超分子复合物的潜在相互作用，如体外生化测定确定。3.确定Hrp系统的每个组分（以及组分的适当定位）对一组分子表型的贡献，旨在剖析Hrp介导的Avr蛋白分泌和易位。