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

高等植物的超敏反应（hypersensitive response， HR）是一种与抵抗不相容病原体（包括植物致病菌）有关的快速细胞自杀。丁香假单胞菌（Pseudomonas syringae）的hrp基因是其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基因在坏死性革兰氏阴性植物病原体中广泛存在（也许是普遍存在），聚集在细菌基因组或巨质粒上，并编码III型分泌途径的成分，该途径被植物和动物病原体用于分泌毒力蛋白，并且似乎专门用于高等真核生物的细菌发病机制。因此，了解这一途径和运输它的蛋白质似乎是理解细菌植物致病性的关键。丁香辣根hrp基因功能簇的研究。syringae 61克隆产生cosmid pHIR11，为探索III型途径，特别是Hrp系统提供了一个特别有用的工具。Cosmid pHIR11能使腐生菌，如荧光假单胞菌和大肠杆菌，在烟草和许多其他植物的叶片中诱导HR，从而证明荧光假单胞菌（pHIR11）的25 kb区域，而不是P.s. syringae 61和其他模型丁香假单胞菌菌株，诱导HR。影响在植物和动物病原体中高度保守的含有III型成分的四个hrp操纵子的突变产生了免疫印迹检测的HrpZ亚细胞定位的不同模式，这表明这四个操纵子对细菌内外膜易位的控制存在差异。avr基因与植物中相应的抗性基因以基因对基因的方式相互作用，在种-品种水平上控制着丁香属植物的寄主特异性。荧光假单胞菌（pHIR11）在大豆中不诱导HR，但将表达avrB（与RPG1相互作用）反式的荧光假单胞菌（pHIR11）接种于携带RPG1抗性基因的品种后，可诱导HR。然而，如果hrpZ或编码III型途径成分的hrp基因发生突变，则不会引发HR。hrmA基因位于紫丁香P.s. 61的hrp簇旁边，是荧光P.s. （pHIR11）诱导烟草HR的必需基因，而不是紫丁香P.s. 61。令人惊讶的是，发现荧光假单胞菌（pHIR11） hrmA突变体分泌野生型HrpZ。像一个典型的avr基因一样，hrmA在许多丁香假单胞菌菌株中缺失，包括烟草病原体烟草假单胞菌，当它在该细菌中异源表达时，它具有不亲和性和HR活性。HrmA蛋白在烟叶中浸润时不引起HR，但在生物转化的烟草悬浮培养细胞中短暂表达时，会产生明显的细胞死亡。这些观察结果和已知的III型途径在耶尔森氏菌中直接将抗宿主蛋白传递到动物细胞的能力导致了以下模型，该模型是该建议的基础。harpin样蛋白“混杂”地分泌到外质体中，而avr样蛋白则在接收到某些信号后直接传递到植物细胞中。因此，有两类蛋白质在控制其分泌和作用部位方面有所不同，III型途径是这些蛋白质协调传递的关键。拟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种表达时是否会引起细胞死亡和活性氧产生或其他易于测定的反应，这些蛋白质是否以hrp依赖的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适当的hrp突变体或植物信号培养中观察到。2. 确定细菌诱导HR明显需要HrpZ和Avr蛋白的基础，并进一步确定HrpZ在发病机制中的作用。3. 鉴定Hrp通路上的其他蛋白质。4. 在pHIR11 hrp簇中构建hrpJ、hrpA和hrcU的非杨树突变，并确定它们在Avr信号的部署、HrpZ跨内外膜分泌以及其他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. ***