Molecular Basis of Phytopathogenicity of Pseudomonas solanacearum

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Pseudomonas solanacearum is an ecologically and economically important phytopathogen causing a lethal wilt disease of over 200 solanaceous plants worldwide. The research proposed investigates biochemical and genetic basis of its pathogenicity, focusing on major extracellular macromolecules. A greater than 10-kb segment (Region I) of the P. solanacearum genome in which insertions simultaneously cause a major decrease in virulence, production of extracellular polysaccharide slime (EPS), and synthesis of two major extracellular proteins (EXPs) has been identified. One objective of the proposed research is to characterize Region I gene products and investigate their involvement in virulence and synthesis of extracellular virulence molecules. This will be accomplished by E. coli maxicell analysis of Region I subclones, TnphoA mutagenesis, and in vitro biochemical and in planta studies of nonpolar isertion mutants of Region I. The gene encoding a major 28-kDa EXP, whose level correlates with EPS levels, will be cloned and similarly analyzed. For the biochemical studies we will first develop methods to detect accumulation of intermediates in EPS synthesis. Expression of Region I depends on two distinct, unlinked loci that encode exported (membrane) proteins, and a locus located 6 kb downstream of Region I; other studies suggest that all these loci control other virulence genes unlinked to Region I. Additional project objectives are characterization of the regulatory proteins by DNA sequence and gene product analysis, exploring crosstalk between them by analysis of Region I expression in multiple mutant background, and investigation of signals affecting virulence gene regulation.