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

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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. ***