Source: UNIV OF WISCONSIN submitted to
COLONIZATION OF LEGUME SPROUTS BY SALMONELLA ENTERICA
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
Reporting Frequency
Annual
Accession No.
0232562
Grant No.
(N/A)
Project No.
WIS01694
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Oct 1, 2013
Project End Date
Sep 30, 2017
Grant Year
(N/A)
Project Director
ANE, JE, M.
Recipient Organization
UNIV OF WISCONSIN
21 N PARK ST STE 6401
MADISON,WI 53715-1218
Performing Department
Bacteriology
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.
Animal Health Component
0%
Research Effort Categories
Basic
80%
Applied
20%
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7121419103050%
7121419110050%
Goals / Objectives
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.
Project Methods
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.

Progress 10/01/13 to 09/30/17

Outputs
Target Audience:researchers working on plant-microbe interactions, food safety, human enteric pathogens, nitrogen fixation, and synthetic biology are the primary target audiences for this work are the primary target audiences. Researchers working on plant-microbe interactions will be interested in this work because it demonstrates an interaction between plants and a bacterial species that has traditionally been considered a mammalian pathogen. This project shows an interesting plant-microbe relationship that could suggest the possibility of an alternative lifestyle for human enteric pathogens within the rhizosphere and endosphere of plants. Food safety researchers are interested in this work due to the significant toll of Salmonella enterica contamination of food products has on both human health and the economy. Legume sprouts are considered to be a food that is a very high risk for contamination with human enteric pathogens, and specifically Salmonella enterica, due to their production methods and consumer preference to consume raw sprouts. Identifying bacterial factors that enable Salmonella enterica to grow in the rhizosphere and determining plant factors that hinder this growth is essential to designing and maintaining safe food production systems. Researchers in the human enteric pathogen field will be interested in this research because it demonstrates a unique ecological niche in which enteric pathogens can thrive outside of their tradition mammalian hosts. Understanding how Salmonella enterica and other enteric pathogens cycle through environments is essential for understanding their overall lifestyle, their role in natural habitats, and their prevalence in human food production systems. Researchers interested in nitrogen fixation will be interested in this work because it demonstrates that the nif genes can be expressed in a heterologous system and that this will result in a functioning nitrogenase. This is a step forward towards engineering a synthetic nitrogen-fixing symbiosis in cereals, the primary source of calories for humans. Synthetic biologists will be interested in this work because it involves reconstructing the biosynthetic circuit for producing nitrogenase and transferring it to a naïve organism (Salmonella enterica). This requires 20 genes organized into seven operons, which is much larger than any de novo genetic circuit published to date. Our collaborators have constructed hundreds of variant combinations of the refactored nif genes and tested hundreds of them for the ability to produce functional nitrogenase in Salmonella enterica. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The postdoctoral fellow benefitted in several ways from working on this project and acquired several new technical skills while working on this project. He was trained to safely handle BSL-2 organisms and work in a BSL-2 workspace. He learned how to conduct 15N and analyze experiments. He has accepted a tenure-track position at another university and plans to continue studying nitrogen-fixing organisms, so this skill will help him in his future research endeavors. The postdoctoral fellow also worked on his teaching skills by helping his PI to develop and teach a course on the microbiome. Specifically, he was involved with the development of two lectures (which he subsequently taught) and he further helped write questions for in-class formative assessments, midterms, and the final. As part of the lectures he taught, he was introduced to Top Hat, a software platform that facilitates in-class formative assessments. The postdoctoral fellow also improved his skills as a mentor by mentoring three undergraduate students during this funding period. Helping undergraduate students to learn lab etiquette, the scientific method, how to perform experiments, etc. prepared him to teach and mentor undergraduates in his new position as an assistant professor. How have the results been disseminated to communities of interest?Results from the current funding period have only been shared with other members of the lab and collaborators. These results will be in a manuscript that is already in preparation and intended for submission to the Molecular Plan-Microbe Interactions (MPMI) journal. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? During the last year of this project, we demonstrated that Salmonella can not only colonize legume sprouts but also the sprouts of several cereal crops like maize and rice. This has some interesting public health implications that will need to be explored further, but also potentially represents a new tool to manipulate interactions between cereals and bacteria. Therefore, we tested the ability ofSalmonella entericaexpressing refactorednifgenes to fix nitrogen in association with plant hosts. Our collaborators in the Voigt lab at the MIT provided us with two modified strains of Salmonella enterica ATCC 14028. One strain was transformed with a plasmid containing refactored nif genes and a low-expression controller and one transformed with a plasmid containing refactored nif genes and a high-expression controller. In a pilot experiment, we checked the ability of the strain with the high-expression controller to fix nitrogen as measured by a 15N incorporation experiment. Zea mays B73 seeds were sterilized and germinated, then grown in sterile Turface® for two weeks. When seedlings were 4-6 inches tall, they were inoculated with 1 ml each of the high-controller strain of Salmonella enterica ATCC 14028 at an OD600 = 0.1 and then incubated for a further 8-10 days. The seedlings were then transferred to 2-L gas sampling bags then sealed and injected with 10 ml 15N and incubated for 3-5 days. Following incubation, the seedlings were removed from the gas sampling bags, autoclaved, and dried down in an oven for 2-3 days. The dried samples were then ground to a powder with glass beads and a mixer mill and submitted for isotope ratio mass spectrometry by Dr. Harry Read in the Department of Soil Science. Unfortunately, our first assays did not show any evidence of nitrogen fixation for these strains on maize.

Publications


    Progress 10/01/15 to 09/30/16

    Outputs
    Target Audience:The primary target audiences for this work are researchers in the plant/microbe interaction field, the food safety field, and the human enteric pathogen field. Researchers in the plant/microbe field are interested in this work because it demonstrates an interesting plant/microbe relationship that could suggest the possibility of an alternative lifestyle for human enteric pathogens within the rhizosphere of plants. Food safety researchers are interested in this work due to the significant toll that Salmonella contamination of food products has on both human health and the economy. Researchers in the human enteric pathogen field are interested in this research because it demonstrates a unique ecological niche in which enteric pathogens can thrive outside of their tradition mammalian hosts. Understanding how Salmonella and other enteric pathogens cycle through environments is important in understanding their overall lifestyle, their role in natural environments, and their prevalence in human food production systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The graduate student continued developing skills in several areas. The graduate student continued to improve mentoring skills, research skills, analysis skills, and developed bioinformatics skills that advanced this research. How have the results been disseminated to communities of interest?Results have been shared with the broader research community through collaborative discussions with researchers at numerous other institutions. This work will be further disseminated shortly through publication. What do you plan to do during the next reporting period to accomplish the goals?One goal for the next reporting period is to address Objective 2 and try identifying Salmonella-specific markers, which may allow detecting infected or contaminated plants at different levels of the food chain. Given the outstanding ability of Salmonella to colonize a wide range of leguminous and non-leguminous crops, another goal will be to investigate the possibility of using Salmonella as a chassis to engineer nitrogen-fixing associations in non-leguminous crops.

    Impacts
    What was accomplished under these goals? Objective 1: With the goal of understanding the contribution that the general stress regulon plays in Salmonella colonization of sprouts, we performed colonization experiments using an attenuated strain of Salmonella that has lower general stress tolerance than virulent Salmonella. We demonstrated that the attenuated strain had a lower tolerance to reactive oxygen species when grown in legume root exudates, as compared to a virulent strain. Further, the attenuated strain displayed lower overall growth on sprouts, as compared to a virulent strain. Objective 3: With the goal of better understanding plant genes that influence Salmonella colonization, the graduate student worked to identify the role that the plant FLS2 receptor, which recognizes bacterial flagellin and stimulates an immune response, plays in influencing Salmonella growth on Medicago truncatula. The graduate student determined that FLS2 triggered plant defenses influence the growth of attenuated Salmonella, but did not influence the growth of virulent Salmonella. This result suggests that virulent Salmonella is capable of overcoming plant produced reactive oxygen species. The production of reactive oxygen species through stimulation of FLS2 could prime Salmonella growing on sprouts to more readily neutralize the oxidative stress encountered in macrophages during human infection. This work is being prepared for publication.

    Publications


      Progress 10/01/14 to 09/30/15

      Outputs
      Target Audience:The primary target audiences for this work are researchers in the plant/microbe interaction field, the food safety field, and the human enteric pathogen field. Researchers in the plant/microbe field are interested by this work because it demonstrates an interaction between plants and a bacterial species that has traditionally been considered a mammalian pathogen. This project demonstrates an interesting plant/microbe relationship that could suggest the possibility of an alternative lifestyle for human enteric pathogens within the rhizosphere of plants. Food safety researchers are interested in this work due to the significant toll that Salmonella contamination of food products has on both human health and the economy. Legume sprouts are considered to be a food that is very high risk for contamination with human enteric pathogens, and specifically Salmonella, due to their production methods and consumer preference to consume sprouts raw. Identifying bacterial factors that enable Salmonella to grow in the rhizosphere and determining plant factors that hinder this growth is important in understanding how to design and maintain safe food production systems. Researchers in the human enteric pathogen field are interested in this research because it demonstrates a unique ecological niche in which enteric pathogens can thrive outside of their tradition mammalian hosts. Understanding how Salmonella and other enteric pathogens cycle through environments is important in understanding their overall lifestyle, their role in natural environments, and their prevalence in human food production systems. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?The graduate student has benefited in several ways from working on this project. He has demonstrably improved his technical and analytical skills, teaching and mentorship skills, as well as his communication skills. The graduate student has gained significant technical skills while working on this project. He has learned how to design and execute large-scale transcriptional analysis projects, as well as large-scale genetic screens of bacteria. These skills will be highly transferrable in his future research. He has also gained significant data analysis skills. Through both coursework and the mentorship of his PD, he has become proficient in the use of the statistical analysis program R. Developing this skill set has allowed him to become a more efficient and effective researcher. Further, developing the ability to both analyze and graphically represent his data in new and powerful ways has made him a more effective communicator when sharing his results with the scientific community. The graduate student also had the opportunity to develop his teaching skills by participating in a teaching practicum. Participating in this practicum enabled him to participate in the teaching of upper level undergraduate students. Additionally, he gained valuable insights into the successful design and management of an undergraduate course. He has continued to develop his skills as a mentor through the mentorship of three undergraduate students during this funding period. He has utilized numerous campus resources to ensure that he is providing the best mentorship that he can to his students. He has also implemented the use of Individual Develop Plans for his undergraduates to ensure that he is providing the most appropriate mentorship. Developing these mentorship skills will help him in future work on this project, and will also be highly transferrable to future positions. The graduate student has also used his position on this project to develop skills that are outside of the classical scientific skill set. He has participated in events and courses at the University of Wisconsin School of Business to develop his abilities in communicating effectively with members of the public that are outside of the scientific community. Participating in these events has allowed him to gain insights into the concerns and priorities of the business community, and understand how they differ from the concerns and priorities of the scientific community. Having an understanding of these different communities has allowed him to develop communication skills and perspectives that will allow him to communicate effectively to both communities. Having this skill set will be useful for his future professional success and will have benefits for both the scientific community and the business community. How have the results been disseminated to communities of interest?Results from the project have been disseminated to the University of Wisconsin - Madison community by the graduate student through both a seminar presentation on his research as well as many discussions with fellow researchers. Results been shared with the broader research community through collaborative discussions with researchers at numerous other institutions. This work will be further disseminated through publications that are anticipated during the next reporting period. Additionally, the graduate student will present his results at a future conference during the next funding period. What do you plan to do during the next reporting period to accomplish the goals?Goal 1: Identify Salmonella genes that contribute to the survival of these bacteria in the environments and to the colonization of legume sprouts During the current reporting period, strategies to perform transcriptomic analysis and genetic screening to identify Salmonella genes that contribute to the survival of Salmonella in the rhizosphere of legumes have been developed and optimized. During the next reporting period, these strategies will be implemented to generate a target list of Salmonella genes that contribute to survival in the legume rhizosphere. Following the initial identification of these Salmonella genes, Salmonella strains will be developed that either lack or overexpress identified genes. These strains will then be characterized in their ability to colonize Medicago truncatula to elucidate the role that the genes play in colonization. Further characterization will be done on these strains to identify the precise mechanism through which their ability to colonize Medciago truncatula has been influenced. Goal 2. Characterize plant genes regulated by Salmonella colonization. During the next reporting period, we will focus our efforts on work related to goals one and two. Goal 3. Identify genetic loci of Medicago truncatula that control the colonization by Salmonella Work on this goal will continue to focus on characterizing the role that Medicago truncatula bacterial recognition receptors have on Salmonella colonization. The recognition of bacteria through these receptors causes a cascade of downstream effects in plants. We will continue to characterize how recognition of Salmonella through bacterial recognition receptors influences Salmonella colonization by determining which downstream effect plays the most significant role in decreasing Salmonella colonization. This will be accomplished using genetic strategies from both Salmonella and Medicago truncatula.

      Impacts
      What was accomplished under these goals? Goal 1: Identify Salmonella genes that contribute to the survival of these bacteria in the environments and to the colonization of legume sprouts Significant work has been performed to accomplish this goal during the current reorting period. Three separate strategies have been pursued to achieve the identification of Salmonella genes that contribute to its survival and colonization in the legume rhizosphere. First, a strategy of transcriptional analysis using RNA sequencing has been developed. A protocol has been developed and optimized to purify Salmonella mRNA from Salmonella populations that have colonized Medicago truncatula seedlings. This was a significant challenge, as the Salmonella population of interest is found in the interior of the plant and must be purified from this tissue rapidly and without degradation. Further, the RNA population purified contains both plant and bacterial RNA and steps were developed to address this. Using this protocol, as well as unique control conditions that have been developed, we will be able to identify Salmonella genes that are preferentially regulated when Salmonella is colonizing the rhizosphere and internal tissues of Medicago truncatula. Second, a genetic screening approach is being used. In collaboration with Professor Joel Griffitts of Brigham Young University, a transposon library screening approach is being used to identify Salmonella genes that are required for Salmonella to colonize both the rhizosphere and the internal tissues of Medicago truncatula. This approach is complementary to the RNAseq strategy and will aid in the rapid identification of Salmonella genes that should be investigated with further approaches. By combining both transcriptional and genetic approaches, we will vastly increase our ability to successfully choose targets to follow with more in depth experimental analysis. The development of these strategies has been a significant mark of progress for this project. We are excited to have developed these tools, and we are confident that they will yield meaningful results. Lastly, we are using an approach to determine the significance of Salmonella genes that we predict to be important for the colonization of Medicago truncatula. We have selected multiple Salmonella genes that we predict to be involved in the colonization of Medicago truncatula based on preliminary results we have obtained as well as published observations from the field. We are currently testing Salmonella strains that lack these genes to identify Medicago truncatula colonization phenotypes. Using both untargeted as well as targeted approaches, our work on this objective is making steady progress towards the identification of Salmonella genes that play a role in the colonization of Medicago truncatula. Goal 2. Characterize plant genes regulated by Salmonella colonization. Upon detailed analysis of previous experiments done on this objective, as well as, results from other aspects of this project, we have determined that it is not likely that plants have specific genes that are regulated in response to Salmonella. Rather, the plant response to Salmonella is that of a general response to the presence of bacteria. Due to the lack of specificity of this response, we are focusing our research efforts on the first and second objectives of this project. Goal 3. Identify genetic loci of Medicago truncatula that control the colonization by Salmonella Several strategies have been used to determine genetic loci of Medicago truncatula that influence Salmonella colonization. The initial strategy that was undertaken was to screen polymorphic lines of Medicago truncatula to identify lines that exhibited differences in Salmonella colonization. This work did not reveal any statistically significant differences in colonization. A secondary strategy was focused on looking for differences in Salmonella colonization between different species of legumes. This work also failed to identify any differences that warranted further investigation. A third approach to this goal has focused on using untargeted metabolomics to identify metabolites in root exudates that influence Salmonella growth. This work has also not been successful in identifying any factors that have an influence on Salmonella growth. Following these experiments, we have come to the conclusion that Salmonella is successful as a plant colonizer due to its diverse metabolic and lifestyle capabilities. This makes it challenging to identify plant host factors that have a significant impact on Salmonella colonization. The strategies that we have primarily utilized would reveal factors that are modulated between plant lines, as opposed to factors that are either present or not present. The intermediary nature of these potential differences between lines has likely played a role in our difficultly in identifying plant loci that influence Salmonella colonization. Due to this, we have modified our strategy and are now focused on using Medicago truncatula lines that have been genetically modified to lack certain receptors that recognize bacteria. This switch in strategy has been successful, and we have identified a Medicago truncatula gene that has a statistically significant impact on Salmonella colonization when it is knocked out. We will continue to focus on using this more targeted approach to characterize genetic loci that influence Salmonella colonization in Medicago truncatula.

      Publications


        Progress 10/01/13 to 09/30/14

        Outputs
        Target Audience: p { margin-bottom: 0.1in; direction: ltr; line-height: 120%; text-align: left; widows: 2; orphans: 2; } This project is currently in a basic science phase and its target audience is the scientific community. Studying how Salmonella, a human enteric pathogen, is able to colonize, survive, and grow on plants will provide insights into the lifestyle and lifecycle of Salmonella that will be useful in developing control and detection methods. This work will also be of general interest to food producers (especially sprout growers), the food safety community, and the general public, as it focuses on a significant food borne human pathogen. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided? Working on this project has enabled Taylor Wahlig to advance his knowledge of the field as well as his analytic and experimental skills through independent study and coursework. Taylor Wahlig has also been able to improve his mentorship skills by providing research mentorship to three undergraduate students involved in undergraduate research programs at UW-Madison. Further, Taylor Wahlig is performing community outreach through the mentorship of two middle school science students who are participating in a local science fair. This project has also provided support to Brianna Bixler, an undergraduate at UW-Madison. This project has allowed Brianna to develop her analytical, experimental, and communication skills as she prepares to enter a career in science. How have the results been disseminated to communities of interest? We have published a paper in the journal PLoS One describing the relationship between Medicago truncatula and Salmonella. This paper details the impact that inoculation size has on Salmonella colonization of Medicago truncatula and also describes how Medicago truncatula gene expression changes in response to Salmonella colonization. Additionally, initial results of this project have been disseminated to the UW-Madison community through an undergraduate mentored research poster session. Further, the Ané lab frequently participates in outreach events sponsored by the University of Wisconsin, such as Family Science Night, Family Garden Day, Darwin Day, and the Madison Middle School Science Symposium. The research done on this project has been disseminated to the general public during these outreach events. What do you plan to do during the next reporting period to accomplish the goals? Objective 1: Work on this objective will continue with a focus on nutritional environmental factors that influence Salmonella colonization. Focus will be directed on Salmonella growth and survival in rhizospheric environments of different legume species. Additional host factors, such as root exudate composition, that determine Salmonella growth will be considered. Objective 2: qRT-PCR primers will be optimized and primers for additional genes will be designed. Work will be initiated to identify specific plant genes that influence Salmonella colonization utilizing existing Medicago truncatula mutants and RNA interference lines. Additional legume species, such as alfalfa (Medicago sativa), and soybean (Glycine max) will be screened for Salmonella colonization phenotypes. Species that display differential colonization will be further characterized. Seedlings from EMS mutagenized Medicago truncatula populations will be screened for Salmonella colonization phenotypes. Objective 3: Based on the results of Objective 2, promoters will be selected and work will be initiated on this objective.

        Impacts
        What was accomplished under these goals? Objective 1: Salmonella colonization studies on polymorphic lines of Medicago truncatula have been completed. No significant colonization differences were detected between these polymorphic lines. This work is now focused on identifying colonization differences between different legume species, such as Medicago truncatula, alfalfa (Medicago sativa), and soybean (Glycine max). The role that root exudate composition plays in Salmonella colonization differences between these species is also being investigated. Objective 2: Several strategies have been initiated to identify and characterize Medicago truncatula genes that are involved in Salmonella colonization. Based on previously performed microarray analysis, qRT-PCR primers were designed for an initial set of 6 genes that were significantly upregulated during Salmonella colonization. Testing and optimization of these primers is in progress. Additionally, ethyl methanesulfonate (EMS) mutagenized populations of Medicago truncatula have been produced to screen for novel genes associated with Salmonella colonization. Further, we are addressing this objective using a nontargeted metabolomics approach to identify metabolites in root exudates that may be essential for Salmonella growth in the rhizosphere of legume sprouts. Objective 3: Work on this objective will begin once candidate genes have been identified and validated in Objective 2.

        Publications

        • Type: Journal Articles Status: Published Year Published: 2014 Citation: Jayaraman, D., Vald�s-L�pez, O., Kaspar, C. W. & An�, J.-M. Response of Medicago truncatula seedlings to colonization by Salmonella enterica and Escherichia coli O157:H7. PLoS One 9, e87970 (2014).