Pathogen and Host Mechanisms Involved in Salmonella Infection of Tomato

Non-Technical Summary: We seek to identify the genetic networks required by Salmonella to proliferate on and within tomatoes, a common vehicle for transmission of the bacteria to humans, and to determine the means by which the plant responds to suppress Salmonella infection. This work is important as vegetables and produce have become major sources of human salmonellosis in this country. Additionally, little is known about the bacterial constituents required for growth in produce plants. We thus hypothesize that the expression of specific Salmonella genes is required for survival and replication of the pathogen in tomatoes. These genes will be differentially regulated, so that they can be identified through their specific induction upon infection of the plant. We further hypothesize that tomato detects Salmonella, and this limits infection by the bacterium. Our goals for this proposal are therefore to define Salmonella genes and genetic pathways that are essential for survival and proliferation in tomatoes. Such pathways are potential intervention targets for preventing produce contamination. In parallel, this project will characterize the molecular responses of tomato leaves and fruits to attempted Salmonella infection and will assess whether these responses play a role in inhibiting Salmonella survival and growth in these tissues. <P> Approach: We will employ the following approaches to achieve the goals of this proposal: 1. We will use the recombinase-based system to identify Salmonella genes that are differentially expressed when bacteria are exposed to specific environmental conditions, such that transient expression causes a permanent and selectable change in the bacterial phenotype that remains even after gene expression has ceased. Fusion of promoters active only when exposed to the ripe fruit of the tomato will induce bacterial conversion to sucrose resistance only after the bacteria have been exposed to that environment, thus selecting for differentially expressed bacterial genes. 2. We will create mutants harboring deletions of the regulated genes and test them for their ability to survive within tomato fruit in competition with the wild type strain. Ripe round tomatoes will be inoculated with a mutant and the wild type, and tomatoes will then be incubated for 7 days prior to harvesting the internalized bacteria. 3. We will make random transposon insertions in the Salmonella chromosome of strains carrying in planta-induced promoter fusions to cre and lacZ, and screen for those that inappropriately express it when grown in laboratory medium. Our first effort will be to test each of these insertions for their effects on each of the induce genes that we have identified. We will next choose mutants of regulators for their ability (or inability) to survive in plants using competition assays. 4. We will characterize how the tomato immune system responds to attempted infection by Salmonella. Initial experiments will be performed using established assays of PAMP-triggered immunity (PTI) in whole plants, isolated leaves, or leaf protoplasts. We will determine which PTI responses are induced by Salmonella and evaluate which PAMPs of the bacterium are most important for the immune response. Finally, we will use RNAi technology to silence genes known to be involved in tomato PTI against Pseudomonas syringae and assess whether these genes also play a role in inhibiting growth and survival of Salmonella in tomato tissues.