The ability of P. syringae to elicit the hypersensitive response in nonhost plants or pathogenesis in hosts is controlled by hrp genes. The P. syringae pv. syringae 61 hrpZ gene encodes harpinPss, a 34.7 kd extracellular protein that elicits hypersensitive necrosis in tobacco and other plants. HarpinPss is heat stable, glycine rich, dissimilar in amino acid sequence to any known protein, produced only in apoplastic fluid-mimicking minimal media, and secreted in a HrpH-dependent manner. The carboxy-terminal 148 amino acid portion of harpinPss contains two directly repeated sequences of GGGLGTP and QTGT and is sufficient and necessary for elicitor activity. The necrosis elicited by harpinPss is an active response of the plant, which can be inhibited by alpha-amanitin, cycloheximide, lanthanum chloride, or sodium vanadate.

A cosmid clone isolated from a genomic library of Pseudomonas syringae pv. syringae 61 restored to all Tn5 mutants of this strain studied the ability to elicit the hypersensitive response (HR) in tobacco. Cosmid pHIR11 also enabled Escherichia coli TB1 to elicit an HR-like reaction when high levels of inoculum (109 cells per ml)were infiltrated into tobacco leaves. The cosmid, which contains a 31-kilobase DNA insert, was mobilized by triparental matings into Pseudomonasfluorescens 55 (a nonpathogen that normally causes no plant reactions), P. syringae pv. syringae 226 (a tomato pathogen that causes the HR in tobacco), and P. syringae pv. tabaci (a tobacco pathogen that causes the HR in tomato). The plant reaction phenotypes of all of the transconjugants were altered. P. fluorescens(pHIR11) caused the HR in tobacco and tomato leaves and stimulated an apparent proton influx in suspension-cultured tobacco cells that was indistinguishable from the proton influx caused by incompatible pathogenic pseudomonads. P. syringae pv. tabaci(pHIR11) and P. syringae pv. syringae 226(pHIR11) elicited the HR rather than disease symptoms on their respective hosts and were no longer pathogenic. pHIR11 was mutagenized with TnphoA (TnS ISSOL::phoA). One randomly chosen mutant, pHIR11-18, no longer conferred the HR phenotype to P. fluorescens. The mutation was marler-exchanged into the genomes of P. syringae pv. syringae strains 61 and 226. The TnphoA insertions in the two pseudomonads abolished their ability to elicit any plant reactions in all plants tested. The results indicate that a relatively small portion of the P. syringae genome is sufficient for the elicitation of plant reactions.The hypersensitive response (HR) of higher plants is characterized by the rapid, localized necrosis of tissues invaded by an incompatible pathogen (a microorganism that is pathogenic only on another host) and is associated with resistance to the pathogen (11). Elicitation of the HR by phytopathogenic bacteria is readily observed by infiltrating leaves with suspensions containing >106 bacteria per ml (10). Tobacco leaf tissue collapses within 24 h after inoculation with Pseludomonas syringae pv. syringae, for example. (5,6,16,23), and some have been cloned (14,19). These hrp genes are required for both the HR and pathogenicity and appear to be present in all P. syr-ingae pathovars (13,14,21

Bacterial surface appendages called pili often are associated with DNA and͞or protein transfer between cells. The exact function of pili in the transfer process is not understood and is a matter of considerable debate. The Hrp pilus is assembled by the Hrp type III protein secretion system of Pseudomonas syringae pv. tomato (Pst) strain DC3000. In this study, we show that the hrpA gene, which encodes the major subunit of the Hrp pilus, is required for secretion of putative virulence proteins, such as HrpW and AvrPto. In addition, the hrpA gene is required for full expression of genes that encode regulatory, secretion, and effector proteins of the type III secretion system. hrpA-mediated gene regulation apparently is through effect on the mRNA level of two previously characterized regulatory genes, hrpR and hrpS. Ectopic expression of the hrpRS gene operon restored gene expression, but not protein secretion, in the hrpA mutant. Three single amino acid mutations at the HrpA carboxyl terminus were identified that affect the secretion or regulatory function of the HrpA protein. These results define an essential role of the Hrp pilus structural gene in protein secretion and coordinate regulation of the type III secretion system in Pst DC3000.