Characterization of the Pseudomonas syringae pv. syringae PSS61hrpJ and hrpU operons and analysis of their role in Avr gene function

The ability of Pseudomonas syringae to cause disease in susceptible plants and elicit the hypersensitive response (HR) in resistant genotypes resides in its hrp (hypersensitive response and pathogenicity) genes. When the 30-kb hrp gene cluster cloned from the genome of P. syringae pv. syringae Pss61 on the cosmid pHIR11 is expressed in Escherichia coli, it enables this saprophytic bacterium to elicit the HR in tobacco. To better understand the nature of hrp gene function, a 4.3-kb BglII fragment from pHIR11 containing at least three essential hrp loci was characterized. DNA sequence analysis identified five complete ORFs (designated hrpQ, hrcN, hrpO, hrpP, and {dollar}hrPQsb{lcub}A{rcub}){dollar} in this region. The predicted products of these ORFs were similar to determinants of pathogenicity and flagellar biogenesis in other bacteria. HrcN is a member of the FliI family of cytoplasmic ATPases known to be components of a novel sec-independent protein export system called type III secretion. A HrpL-dependent promoter was identified within the hrpO ORF. Complementation analysis and hrp-lacZ fusions were used to clarify the translational and transcriptional organization of this region. An in planta lysis technique demonstrated that HrcN, HrpO, and HrpQ{dollar}rmsb{lcub}A{rcub}{dollar} are required for secretion of the HrpZ protein.; Evidence that P. syringae hrp genes and avr host range determinants are co-regulated by HrpL suggested their gene products interact in some way. The nature of this interaction was examined through use of a heterologous E. coli system for phenotypic expression of P. syringae avr genes in Arabidopsis thaliana. RPM1-dependent elicitation of the HR in A. thaliana by E. coli strains expressing avrB or avrRpm1 required the secretory activities of the hrp cluster but not HrpZ, indicating that avr products or byproducts are hrp-secreted elicitors of the HR.; Demonstration of direct interaction between Avr proteins and R gene products by means of a TMV system for expression of avrB within A. thaliana cells was unsuccessful due to the apparent toxicity of AvrB to A. thaliana in the absence of RPM1. Attempts to show hrp-mediated translocation of avr products into plant cells through use of avr-reporter gene fusions and cell fractionation/immunoprecipitation techniques were not successful.