THE HRPZ PROTEINS OF PSEUDOMONAS-SYRINGAE PVS SYRINGAE, GLYCINEA, AND TOMATO ARE ENCODED BY AN OPERON CONTAINING YERSINIA YSC HOMOLOGS AND ELICIT THE HYPERSENSITIVE RESPONSE IN TOMATO BUT NOT SOYBEAN

The Pseudomonas syringae pathovars are composed of host-specific plant pathogens that characteristically elicit the defense-associated hypersensitive response (HR) in nonhost plants. P. s. pv. syringae 61 secretes an HR elicitor, harpin(Pss), (HrpZ(Pss)), in a hrp-dependent manner. An internal fragment of the It s. pv. syringae 61 hrpZ gene was used to clone the hrpZ locus from P. s. pv. glycinea race 4 (bacterial blight of soybean) and P. s. pv. tomato DC3000 (bacterial speck of tomato). DNA sequence analysis revealed that hrpZ is the second ORF in a polycistronic operon. The amino acid sequence identities of HrpZ(Pss)/HrpZ(Psg), and HrpZ(Pss)/Hrp(Pst) were 79 and 63%, respectively, Although none of the HrpZ proteins shelved significant overall sequence similarity with other known proteins, HrpZ(Pst) contained a 24-amino acid sequence that is homologous with a region of the PopA1 elicitor protein of the tomato pathogen, Pseudomonas solanacearum GMI1000. hrpA, the upstream ORF, was highly divergent: The amino acid sequence identities of HrpA(Pss)/HrpA(Psg) and HrpA(Pss)/HrpA(Pst) were 91 and 28%, respectively, and no HrpA sequence showed similarity to known proteins. In contrast, the predicted products of the downstream ORFs in P. s. pv. syringae and P. s. pv. tomato, hrpB, hrpC, hrpD, and hrpE showed varying levels of similarity to those of yscI, yscJ, yscK, and yscL. These are colinearly arranged genes in the virC locus of Yersinia spp., which are involved in the secretion of the Yop virulence proteins via the type III pathway. The similarity of the Ysc proteins was generally stronger in comparisons with the P. s. pv. tomato Hrp proteins. The HrpZ proteins were purified by heat denaturation of contaminating proteins followed by ammonium sulfate fractionation, hydrophobic chromatography, and gel electrophoresis. All three HrpZ proteins elicited the IIR in tomato, whereas none of them elicited significant necrosis in soybean. The results indicate that HrpZ is encoded in an operon containing some of the genes involved in its own secretion and suggest that HrpZ structure does not directly determine bacterial host range.