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Colonization of Legume Sprouts by Salmonella Enterica

Kaspar, Charles; Ane, Jean-Michel
University of Wisconsin - Madison
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Numerous disease outbreaks due to the consumption of raw produce contaminated with Salmonella enterica, a human enteric bacterial pathogen, have been reported. The goal of our research is to gain a better understanding in plant responses to human enteric pathogens in order to reduce the incidence of food borne illnesses and improve the contribution of agriculture to the welfare of consumers.

We propose the following objectives: 1. Define the contributions of the Salmonella general stress regulon and environmental factors on the colonization of legume sprouts. 2. Characterize plant genes induced by Salmonella colonization. 3. Identify genetic loci of Medicago that influence Salmonella colonization.

By the end of this research project, we expect to generate the following outputs: 1. Better knowledge on plant responses to Salmonella and on the role of stress responses in Salmonella during plant colonization; 2. Identification of Salmonella-specific markers, which may allow detecting infected or contaminated plants at different levels of the food chain; 3. Genetic resources, specifically transgenic lines of Medicago truncatula and mutants, that will allow a better understanding of plant colonization by human enteric pathogens.

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Non-Technical Summary:
The demand for fresh produce is greater than ever due to changes in eating preferences of consumers in industrialized nations. Increased consumption of raw fruits, salads and sprouts led to an increased number of reported illnesses due to contaminated produce. In the United States, bacterial pathogens are the major contributors to food-borne illness caused by fresh produce. In outbreaks caused by bacterial pathogens, Salmonella enterica is most frequently involved, accounting for nearly half of the outbreaks. To date, very little is known about the plant determinants that allow or facilitate the surface or internal colonization of crops. The goal of our research is to gain a better understanding on the plant responses to human enteric pathogens. To address these issues, we propose to use Medicago truncatula as a model plant and alfalfa and soybean as target crops. Our specific aims are to: 1. Identify Salmonella genes that contribute to the survival of these bacteria in the environments and to the colonization of legume sprouts. 2. Characterize plant genes regulated by Salmonella colonization. 3. Identify genetic loci of Medicago truncatula that control the colonization by Salmonella. Medicago, alfalfa and soybean plants will be infected with Salmonella and the colonization level will be evaluated. Likewise, the survival of Salmonella strains will be monitored under different environmental conditions. We will assess the effect of different plant compounds on Salmonella colonization. These experiments will allow us to identify genetic and environmental factors that affect the colonization of crop tissues by these human pathogens. For objective 2, we will identify plant genes that are regulated specifically by Salmonella colonization. To understand the biological role of these genes, their expression will be decreased or increased in Medicago plants. Medicago plants with modified expression of these genes will be infected with Salmonella and the colonization levels will be tested as described previously. With these experiments, we are expecting to better understand how legume sprouts respond to Salmonella colonization. For objective 3, based on the genes identified in objective 2, we will develop reporter constructs that get activated specifically under colonization by Salmonella. This will allow us to detect more easily the presence of Salmonella in plant tissues for genetic research but also for practical purposes. Transgenic plants expressing these reporters will be mutagenized and mutants will be analyzed by classical genetics to identify plant loci that control Salmonella colonization. We expect to generate the following outputs: 1. Better knowledge on the response of crops to Salmonella colonization and on the role of stress responses in Salmonella during plant colonization; 2. Identification of plant reporters, which will allow detecting more easily and more specifically plants infected or contaminated with Salmonella; 3. Genetic resources (reporter genes and mutants) that will allow an improved understanding of plant colonization by human enteric pathogens.

Objective 1. Plants from polymorphic lines of Medicago truncatula, alfalfa (Medicago sativa), and soybeans (Glycine max) will be grown in CYG seed growth pouches. Plants will be inoculated at the root level using individual Salmonella serovars and cocktails. After 4, 6, 8 and 10 days of germination, the total amount of viable Salmonella will be assessed by plate counts. Internal colonization will be evaluated by surface sterilization and plate counts, but also by microscopy using a confocal microscope. These experiments will be conducted under different environmental conditions. The survival of different Salmonella strains and rpoS expression will be monitored in various environmental stress conditions by colony counting. The growth inhibition and antimicrobial activity of plant isoflavonoids and triterpene saponins will be tested in Luria Broth. Induction of the RpoS stress protection system will be monitored by Western blot. Induction of morphological changes will be monitored by microscopy.

Objective 2. The expression of 25 Salmonella-responsive genes identified by microarrays will be confirmed by quantitative RT-PCR using individual Salmonella serovars. To test the response specificity, gene expression will be also be monitored after inoculation with bacterial symbionts and a few plant pathogens. Expression levels will be monitored in various genotypes and environmental conditions as in objective 1 and correlated with the colonization patterns. To test the role of regulated genes in the colonization process, expression of genes specific for the response to Salmonella will be silenced by RNA interference or artificial miRNAs or over-expressed using strong and constitutive promoters. Constructs will be introduced into Agrobacterium rhizogenes MSU440 and used to generate transgenic roots. Transgenic roots will be challenged with various Salmonella serovars and colonization evaluated as described in objective 1. Insertion- and deletion mutants will be obtained for genes found to influence colonization by RNA interference. Homozygous mutants will be selected and tested for their colonization by Salmonella as described previously.

Objective 3. Ten promoters will be cloned to develop promoter-GUS fusions. These constructs will be introduced into Medicago truncatula roots by Agrobacterium rhizogenes transformation to confirm GUS induction in the presence of Salmonella. Based on these results, we will select two constructs to generate stable transgenic lines of Medicago truncatula Jemalong A17. The line showing the strongest and most specific expression in response to Salmonella will be used for fast neutron bombardment mutagenesis. A M2 population will be developed and screened using a non-lethal GUS assay. Putative mutants will be grown in pots and allowed to self-pollinate. The phenotype will be checked at the M3 generation. Confirmed mutants will be back-crossed to non-mutagenized parents and crossed with each other for allelism tests. In-depth characterization of resulting phenotypes will be conducted as described in objective 1 and expression of plant genes induced by Salmonella, identified in objective 2, will be examined.

Funding Source
Nat'l. Inst. of Food and Agriculture
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Bacterial Pathogens