Characterization of the Synergistic Interaction Between Salmonella Enterica and Zanthomonas Vesicatoria in the Tomato Phyllosphere

NON-TECHNICAL SUMMARY: The incidence of salmonellosis caused by the consumption of fresh produce has surpassed outbreaks associated with the consumption of animal products and continues to rise. Tomato is one of the most likely produce associated with Salmonellosis outbreaks from consumption of contaminated produce. Salmoenlla fails to grow on tomato plants or fruit in the field. However, in the presence of the plant pathogen Xanthomonas vesicatoria, Salmonella can grow, even in the absence of plant disease. A growing Salmonella population on the tomato plant leads to a higher incidence of contaminated fruit. Experiments will be preformed that identify the mechanisms that allows growth of Salmonella with X. vesicatoria. Also, the specificity of this interaction will be tested by examining another foliar pathogen of tomato and determining whether Salmonella can grow during co-colonization of it. Finally, the genes that have previously been identified as important for Salmonella colonization of seedlings will be characterized for their function. To determine the function of these genes, biofilm, plant, and swarm assays and protein sequence analysis will be completed. Completion of this work will confirm whether Salmonella and X. vesicatoria form a dual-genera biofilm on the leaf surface; confirm the specificity of this interaction; and expand our knowledge of the genetic mechanisms that allow Salmonella to colonize plants. Understanding the interaction of Salmonella with other bacteria on plants is a first step toward solving the food safety crisis of contaminated produce. <P>APPROACH: Methods: Objective 1. We propose to use the X. vesicatoria and S. enterica strains carrying the constitutive DsRed and gfp plasmids and examine their spatial distribution on the tomato leaf surface by confocal microscopy. We propose to inoculate tomato leaves simultaneously with the two genera and in sequence, X. vesicatoria followed by S. enterica.We will examine the Xv-Se interaction with confocal microscopy. We will also employ site-directed mutagenesis to create a gumB mutant, that is reduced in biofilm formation in Xv, and test the effect of such mutation on the S. enterica - X. vesicatoria interaction. From available data in the literature, it is unclear whether X. vesicatoria biofilm mutants that are reduced in virulence are thus, due to poor initial plant colonization or some other mechanism. Therefore, we will enumerate phyllosphere populations of both X. vesicatoria and S. enterica. Objective 2. We will use plant experiments with Psuedomonas syringae pv. tomato to determine if the other foliar plant pathogen of tomato helps Salmonella to grow. We will examine whether S. enterica can grow in the co-colonized tomato phyllosphere with P. syringae pv. tomato. Plants co-colonized with S. enterica - P. syringae pv. tomato will be examined by confocal microscopy to determine the extent of S. enterica internalization. Objective 3. We will characterize the functions of FUN genes by biofilm, swarming, plant assays, and sequence analysis. Our approach to characterize the FUN genes is multifaceted with the goal of revealing gene function and the specific role in plant colonization. These genes were originally identified on alfalfa seedlings on which S. enterica prefers to colonize the roots. Mutants will be examined for tomato phyllosphere attachment and continued colonization capacity. Swarm assays will be performed and if FUN gene mutants have differential phenotypes from the wild type, swarm cells will be examined for differentiation characteristics - hyperflagellation, antibiotic resistance, and surfactant or osmotic agent production. Since water is probably one of the most important routes of pre-harvest contamination, we will examine whether our FUN gene mutants of interest have a phenotype in water, especially those that had differentially gene expression in S. enterica cells in alfalfa irrigation water. Once phenotypes for the FUN gene mutants have been complemented, comparative genomics between plant and animal pathogens will be conducted by BLASTP analysis and maximum parsimony.