Involvement of Pseudomonas syringae harpins, Avr Proteins, and the Type III (Hrp) Secretion Pathway in Plant Interactions

9631530 Collmer高等植物的过敏反应（HR）是一种快速的细胞自杀，与防御不相容的病原体有关，包括植物病原菌。该菌的hrp基因是该菌在非寄主植物中引发HR或在寄主植物中致病所必需的。已知hrp基因在致坏死的革兰氏阴性植物病原体中是广泛的（可能是通用的），聚集在细菌基因组中或巨质粒上，并编码III型分泌途径的组分，所述III型分泌途径被植物和动物病原体用于分泌毒力蛋白，并且似乎专用于高等真核生物的细菌致病。因此，了解这一途径和蛋白质的交通似乎是理解细菌植物致病性的关键。研究了烟草疫霉致病变种hrp基因的功能簇。已克隆产生粘粒pHIR 11的Escherichiagae 61为探索III型途径（一般而言）和Hrp系统（具体而言）提供了特别有用的工具。粘粒pHIR 11使嗜盐细菌如荧光假单胞菌和大肠杆菌能够在烟草和许多其它植物的叶片中引发HR，从而证明荧光假单胞菌（pHIR 11）的25 kb区域，而不是P.s.突变影响的四个hrp操纵子含有III型组件，是高度保守的植物和动物病原体产生不同的模式的免疫印迹检测HrpZ亚细胞定位，并建议这四个操纵子差异控制易位跨细菌的内膜和外膜。avr基因以基因对基因的方式与植物中相应的抗性基因相互作用，控制着小菜蛾小种-品种水平上的寄主特异性。荧光假单胞菌（pHIR 11）在大豆中不引起HR，但当用表达反式avrB（其与RPG 1相互作用）的荧光假单胞菌（pHIR 11）接种携带RPG 1抗性基因的品种时，HR被引起。然而，如果hrpZ或编码III型途径组分的hrp基因突变，则不引起HR。hrmA基因在P.s.荧光假单胞菌（pHIR 11）需要P. ingae 61，而P.s.烟草61，以引起烟草中的HR。令人惊讶的是，发现荧光假单胞菌（pHIR 11）hrmA突变体分泌野生型水平的HrpZ。像典型的avr基因一样，hrmA在许多烟草疫霉菌株中缺失，包括烟草病原体P.s.当在细菌中异源表达时，它赋予不相容性和HR活性。当hrmA蛋白渗入烟草叶片中时不引起HR，但是当hrmA在生物射弹转化的烟草悬浮培养细胞中瞬时表达时，它产生明显的细胞死亡。这些观察结果和耶尔森氏菌中III型途径的已知能力。将抗宿主蛋白直接递送到动物细胞中导致了以下模型，该提议就是基于该模型建立的。Harpin样蛋白被“混杂地”分泌到质外体中，而Avr样蛋白在接收到一些信号后被直接递送到植物细胞中。因此，有两类蛋白质，其分泌控制和作用部位不同，III型途径是这些蛋白质协同递送的关键。建议工作的具体目标是：1.确定HrmA、AvrB和/或AVRPto在适当的植物叶片中异源表达时是否可以引起细胞死亡和活性氧产生或其他容易测定的反应，蛋白质是否以hrp依赖性方式被细菌分泌到植物细胞中，以及它们的分泌是否可以在具有适当的hrp突变体或植物信号的培养物中观察到。2.确定的基础上，明显需要HrpZ和Avr蛋白在细菌诱发的HR，并进一步定义hrpZ的发病机制中的功能。3.识别Hrp途径中的其他蛋白质。4.构建pHIR 11 hrp簇中hrpJ、hrpA和hrcU的nonpoplar突变，并确定每个突变在Avr信号部署、HrpZ跨内膜和外膜分泌以及其他Hrp相关表型中的作用。***

9631530 Collmer The hypersensitive response (HR) of higher plants is a rapid cellular suicide associated with defense against incompatible pathogens, including plant pathogenic bacteria. The hrp genes of Pseudomonas syringae are necessary for the bacterium to elicit the HR in nonhost plants or pathogenesis in host plants. hrp genes are known to be widespread (perhaps universal) in necrogenic, gram-negative plant pathogens, to be clustered in the bacterial genome or on megaplasmids, and to encode components of a type III secretion pathway that is used by both plant and animal pathogens to secrete virulence proteins, and which appears dedicated to bacterial pathogenesis of higher eucaryotes. Thus, an understanding of this pathway and the proteins that traffic it appear key to understanding bacterial plant pathogenicity. The functional cluster of hrp genes from P. syringae pv. syringae 61, which was cloned to produce cosmid pHIR11, has provided a particularly useful tool for exploring the type III pathway, in general, and the Hrp system, in particular. Cosmid pHIR11 enables saprophytic bacteria, like Pseudomonas fluorescens and Escherichia coli, to elicit the HR in the leaves of tobacco and many other plants, and it thereby demonstrates that a 25 kb region of the P. fluorescens(pHIR11), but not P.s. syringae 61 and other model P. syringae stains, to elicit the HR. Mutations affecting the four hrp operons containing type III components that are highly conserved in plant and animal pathogens produced different patterns of immunoblot-detected HrpZ subcellular localization and suggest that the four operons differentially control translocation across the inner and outer membranes of the bacterium. avr genes, which interact in a gene-for-gene manner with corresponding resistance genes in plants, control host specificity at the race-cultivars level in P. syringae. P. fluorescens(pHIR11) does not elicit the HR in soybean, but when cultivars carrying the RPG1 resistance gene are inoculated with P. fluorescens(pHIR11) expressing avrB (which interacts with RPG1) in trans, the HR is elicited. However, no HR is elicited if either hrpZ or a hrp gene encoding a type III pathway component is mutated. The hrmA gene is located next to the hrp cluster in P.s. syringae 61 and is required for P. fluorescens(pHIR11), but not P.s. syringae 61, to elicit the HR in tobacco. Surprisingly, P. fluorescens(pHIR11) hrmA mutants were found to secrete wild-type levels of HrpZ. Like a typical avr gene, hrmA is missing from many P. syringae strains, including the tobacco pathogen P.s. tabaci, and it conferred incompatibility and HR activity when heterologously expressed in that bacterium. The HrmA protein does not elicit the HR when infiltrated into tobacco leaves, but when hrmA is transiently expressed in biolistically-transformed tobacco suspension-cultured cells, it produced apparent cell death. These observations and the known ability of the type III pathway in Yersinia spp. To deliver anti-host proteins directly into animal cells leads t the following model upon which this proposal is founded. Harpin-like proteins are secreted "promiscuously" into the apoplast, while Avr-like proteins are delivered directly into plant cells upon receipt of some signal. Thus, there are two classes of proteins that differ with respect to the control of their secretion and the site of their action, and the type III pathway holds the keys to the coordinated delivery of these proteins. The specific objectives of the proposed work are: 1. Determine if HrmA, AvrB, and/or AVRPto can elicit cell death and active oxygen generation or other readily assayed responses when heterologously expressed in appropriate plant leaves, if the proteins are secreted into plant cells by bacteria in a hrp dependent manner, and if their secretion can be observed in culture with appropriate hrp mutants or plant signals. 2. Determine the basis for the apparent need for both HrpZ and an Avr protein in bacterial elicitation of the HR, and further define the function of hrpZ in pathogenesis. 3. Identify additional proteins traveling the Hrp pathway. 4. Construct nonpoplar mutations in hrpJ, hrpA, and hrcU in the pHIR11 hrp cluster and determine the role of each in the deployment of Avr signals, the secretion of HrpZ across the inner and outer membranes, and other Hrp related phenotypes. ***