Characterization of the hrp Cluster from Pseudomonas syringae pv. syringae 61 and TnphoA Tagging of Genes Encoding Exported or Membrane-Spanning Hrp Proteins. Hsiou-Chen Huang. Department of Plant Pathology, Cornell University, Ithaca, NY 14853-5908. Steven W. Hutcheson(2), and Alan Collmer(1). (1)Department of Plant Pathology, Cornell University, Ithaca, NY 14853-5908; (2)Department of Botany, University of Maryland, College Park 20742.. MPMI 4:469-476. Accepted 31 May 1991. Copyright 1991 The American Phytopathological Society.

The cosmid pHIR11, obtained from Pseudomonas syringae pv. syringae 61, enables P. fluorescens to elicit the hypersensitive response (HR) in tobacco leaves. To locate functional loci, pHIR11 was mutagenized with TnphoA in Escherichia coli, and transposon insertion derivatives were mobilized into P. fluorescens and screened for their HR phenotype. Forty-one unique insertions that affected HR elicitation were identified. HR‾ pHIR11::TnphoA derivatives were transferred into the P. s. pv. syringae genome by marker exchange. Thirteen complementation groups were then defined by analysis of merodiploids containing all combinations of TnphoA insertions in the chromosome and in pHIR11. P. s. pv. syringae mutants in 12 of the complementation groups (II-XIII) exhibited Hrp‾ phenotypes, including loss of the ability to cause disease or to multiply in bean, to elicit the HR in tobacco leaves, or to cause HR-associated ionic responses in suspension-cultured tobacco cells. P. s. pv. syringae mutations in complementation group I were phenotypically distinct from those in the hrp region, producing a delayed HR (necrosis appeared after 24-48 hr) in tobacco leaves and retaining virulence in bean leaves. Two genes, in complementation groups IV and X, respectively, were determined to encode exported or membrane-spanning proteins by the PhoA⁺ (alkaline phosphatase) phenotype of TnphoA insertions. Analyses of Hrp-PhoA hybrid proteins immunostained with anti-PhoA antibodies and assays of PhoA activity in mutant cultures indicated that the expression of both genes was derepressed in a minimal medium known to derepress other hrp genes. Various pairs of HR‾ P. s. pv. syringae mutants co-inoculated into tobacco leaves failed to elicit the HR despite the use of high levels of inoculum. The core region of the hrp cluster (complementation groups III-XII) was homologous with the hrp cluster from P. s. pv. phaseolicola NPS3121 but not with hrpM from P. s. pv. syringae PS9020, as determined by Southern blot hybridizations. These results suggest that 12 of the 13 complementation groups analyzed are involved in the production of a single factor or activity that is necessary for bacterial multiplication in planta and for elicitation of both the HR and disease symptoms. Production or deployment of this factor requires two Hrp proteins that are either exported or have periplasmic domains