A Combined Genomic and In Vivo Imaging Platform to Understanding Salmonella-Tomato Plant Host Interactions

<p>NON-TECHNICAL SUMMARY:<br/> Large-scale gastroenteritis outbreaks related to consumption of Salmonella-contaminated tomatoes have caused widespread economic losses. Salmonella can survive and grow in a wide variety of produce including tomatoes, and is not removed from fresh produce by washing or current disinfectants. Improving eradication requires a better understanding of the microbiological, plant, and environmental factors involved in Salmonella contamination and how they interact. These studies will illuminate both how contamination occurs and how pathogens survive or proliferate in plant tissues. Here we plan to identify conditions and pathogen pathways that alter Salmonella fitness and which represent vulnerabilities that can be exploited to ameliorate contamination. For this we will use novel strategies that combines; 1) a sensitive, real-time assay that
illuminates plant-foodborne pathogen interactions during mechanical damage, environmental stresses and phytopathogens infection, and 2) genome-wide fitness assays of molecularly barcoded Salmonella mutants to uncover those environmental factors. In Aim 1, we will determine the influence of physical and environmental factors and phytopathogen infection on the interaction of S. Typhimurium with tomato plants; In Aim 2, we will identify, using high throughput genomic screening, those environmental conditions and pathogen pathways required for S. Typhimurium survival in tomato plants; and In Aim 3 we will evaluate selected novel compounds for their effect on Salmonella persistence in tomato. Our innovative studies will identify novel Salmonella-plant interaction mechanisms and lay the groundwork for development of effective control products to reduce Salmonella burden in fresh produce,
improve product safety, and thus promote sustainable and safe tomato production.
<p>APPROACH:<br/> APPROACH Aim1a: Determine the influence of temperature and humidity on survival and internalization of S. Typhimurium in tomato seedlings and fruit. Tomato seedlings OH9242 will be inoculated with bioluminescent S. Typhimurium by a) spray inoculation of leaves, and b) through wound by clipping the cotyledons. Inoculated tomato seedlings will be maintained in growth chambers with four different temperature and relative humidity (RH) regimes. Tomato seedlings will be imaged in real-time to monitor the growth and movement of S. Typhimurium using an in vivo imaging system weekly during the first three weeks. After sacrificing three seedlings for recovery of S. Typhimurium at each time point, the remaining 11 seeding will be transplanted to large pots and kept in the same growth chambers to harvest fruit for analysis (4-8 weeks). Tomato fruits will be sampled
at early fruit formation, green mature and ripening stages for biophotonic imaging. Hypocotyls, cotyledons, stems and roots will be collected in whirl-pack sample bags. Suspended in peptone water, and the supernatant will be plated on XLT-4 agar plates. To isolate internalized Salmonella, the plant tissue will be surface sterilized by dipping in 70% ethanol followed by 0.6 % sodium hypochlorite Aim 1b: Role of grafting in Salmonella transmission in tomato seedlings. The grafting tool, will be exposed to different concentrations of S. Typhimurium Xen33. For rootstock inoculation, 21-day-old rootstock seedlings will be decapitated with one horizontal cut 5 mm below the cotyledons with a blade exposed to bioluminescent S. Typhimurium and 18-day-old scion seedlings will be de-rooted with a single cut with a sterile blade exposed to sterile water. For scion inoculation, rootstock seedlings
will be decapitated and scion seedlings will be de-rooted with a single cut with a blade exposed to a bioluminescent S. Typhimurium suspension. Grafted plants will be placed in a growth chamber at 22°C in darkness under 80-90 % relative humidity for 7 days. The movement of the bioluminescent bacteria up and down the stem from the graft union will be determined Aim1c: Determine the influence of phytopathogens on S. Typhimurium internalization and survival in tomato plants. Tomato plants will be inoculated at the seedling and flowering stage. Cmm strain C290 will be inoculated to 3-week-old seedlings. Xe strain 110C will be inoculated by spraying the bacterial suspension on the foliage. One week later, seedlings will be challenged with bioluminescent Salmonella. After inoculation, seedlings will be kept in a growth chamber and biophotonic images will be taken weekly for 3 weeks and
bacterial population will also be determined. To inoculate plants at the flowering stage, stems will be inoculated in the middle by cutting the petioles of two leaves. Xe 110C will be inoculated by spraying foliage. One week later, Salmonella will be inoculated. Samples of flower, leaves, stems, and fruit from three plants will be taken for biophotonic imaging weekly until the fruits are ripe. Bacterial CFU data will be log transformed and analyzed using SAS statistical software. Aim 2. Determine, using genomic screening, those environmental conditions and pathogen pathways required for S. Typhimurium survival in tomato plants. We will introduce a DNA-Tag, (a unique, mutant-specific 25bp molecular barcode) to track the abundance of each individual mutant in the pool of mutants during the competitive growth under stress. The tags are used as a proxy to measure strain abundance, for
example, if the number of "counts" of any barcode changes (increases or decreases) from the beginning to the end of the experiment, we infer that the gene marked by that barcode is required for sensitivity or resistance to that condition. Briefly, the 90,000 barcoded S. Typhimurium-Tn5 transposons will be added to S. Typhimurium genomic DNA and transposed in vitro as we described for C. albicans. Transposed DNA will then be transformed into competent S. Typhimurium by electroporation. The high density transposon mutant pool will initially be screened in parallel by subjecting it to chronic, sublethal environmental stresses that mimic those that might be encountered as a consequence of tomato harvest, collection and post-harvest processing. Specifically, we will test the pools in the following stresses (high and low temperature, extremes of humidity, extremes of salt, a titration of
sodium hydroxide, bleach and co-culture with the phytopathogens Cmm, Xanthomonas. In addition to these environmental stresses, the most potent S. Typhimurium -active compounds described in our preliminary data will be screened against this pool in competitive liquid culture. To best explore the effects of these compounds, and samples collected at 5 time points. For environmental conditions, the robotic system will be modified such that the growth chamber can accommodate a range of temperatures and humidity. For experiments in which the Salmonella transposon library is challenged with phytopathogens, we will perform these experiments as co-cultures in media that allows for proliferation of both organisms. Following genomic DNA extraction the transposon mutants will be selected by the barcode PCR reaction which is specific for the mutant genomic DNA. After the screens have been completed,
genomic DNA will be extracted, barcodes amplified and sequenced. Following bar-seq, data will be analyzed by first binning each read to its experimental bin, and then each unique, strain-specific barcode will be matched to a list of all barcode sequences. Normalized mapped reads based on total counts are expressed as the log2 ratio of normalized counts in control vs. experimental. The greater the log2 ratio, the more sensitive that particular strain is to the drug/condition tested against. Determine the importance of S. Typhimurium genes for survival in tomato. Suspensions of S. Typhimurium mutants (n=5) will be inoculated in tomato seedlings by clipping cotyledons and plants at the flowing stage by spraying as described in Aim 1c. Thirty seedlings and 10 plants will be inoculated for each strain and controls. The bacterial growth and internalization will be evaluated by isolation of S.
Typhimurium on the surface of tissue and internal tissue as described in Aim 1a. Ten fruits will be randomly sampled weekly after flower inoculation and the bacterial populations will be assessed. Data analysis and expected results. The chemogenomic screens will be analyzed using our custom sequencing analysis software that transforms raw sequence reads into a measure of the sensitivity of each transposon mutant in the pool of mutants. These data will comprise two datasets: 1) a compendium of genome-wide sensitivity profiles containing the relative sensitivity for each gene in the Salmonella genome and 2) a ranked list of putative protein targets and target pathways. Aim3a: Evaluate the selected compounds for their effects on Salmonella survival and internalization in tomato seedlings. Tomato seedlings will be inoculated with bioluminescent Salmonella strain by clip inoculation and will
be kept under favorable temperature and humidity conditions. Selected compounds will be sprayed a day before inoculation and then applied to seedlings on a weekly. The efficacy of selected compounds on inactivating Salmonella will be determined by biophotonic imaging. Aim3b: Evaluate the selected compounds for their effects on inactivating Salmonella on tomato fruit. Freshly harvested ripened and unripened tomatoes will be incubated for 24 hour at 32°C. Fruits will be submerged in bacterial suspension for 30 min. Fruits will be air dried in a biosafety hood and exposed to the small molecules by fogging. The treated tomato will be stored at 90% RH and 20 °C for 5 days before analysis. External as well as internal Salmonella will be evaluated.