Merighi Massimo, Doris Majerczak and David Coplin.

Department of Plant Pathology, The Ohio State University, Columbus 43210-1087.

Pantoea stewartii is a Gram-negative bacterial pathogen of corn. Phytopathogenic bacteria are believed to inject pathogenicity proteins (effectors) directly into host cells via a specialized delivery system termed a type III secretion system. This system is encoded by hrp (hypersensitive response and pathogenicity) genes. The hrp system of P. stewartii is environmentally controlled by at least four genes that constitute a regulatory cascade that perceives metabolic/plant signals and regulates the expression of both secretion and effector genes. hrpX and hrpY encode a two component regulatory system, with HrpX functioning as a sensor and HrpY as a response regulator. HrpY may directly or indirectly upregulate hrpS, which encodes a transcriptional activator. HrpS then activates a gene for the alternate sigma factor, HrpL, which in turn enables RNAP to recognize promoters of target genes in the hrp regulon.

Null mutations of hrpX did not alter pathogenicity of P. stewartii to corn, but overexpression of hrpX reduced the virulence of a wild-type strain. Overexpression of hrpY did not affect virulence, but instead eliminated the need for preinduction of hrp genes in order for P. stewartii to elicit an HR in tobacco. Site-directed mutation of the conserved aspartate (D57) residue in HrpY, which serves as the phosphorylation site, to alanine and asparagine revealed that phosphorylation of this site is not required for activation of hrp genes. In contrast, mutation of D57 to glutamate, which should have constitutively activated the protein, depressed hrp gene transcription. Taken together, these findings suggest that the unphosphorylated form of HrpY may serve as a transcriptional activator and that HrpX may function primarily as a phosphatase. This mode of action is the opposite of that for most known bacterial two component systems.

Using GUS and unstable GFP as reporter genes, we studied the regulation the hrpS, hrpL and hrpJ promoters in bacteria that were in contact with corn cells. Preliminary data indicated that all of the fusions, especially the hrpSp-GFP fusion, were much more strongly expressed in planta than when bacteria were grown on artificial media and that this induction occurred within two hours of inoculation.