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

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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.