Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
MOLECULAR MECHANISMS OF HUMAN BACTERIAL PATHOGEN INTERNALIZATION AND SURVIVAL IN FRESH PRODUCE
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1006525
Grant No.
2015-67017-23360
Project No.
CA-D-PLS-2291-CG
Proposal No.
2014-05791
Multistate No.
(N/A)
Program Code
A1331
Project Start Date
Apr 1, 2015
Project End Date
Mar 31, 2019
Grant Year
2015
Project Director
Melotto, M.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Sciences, College Ag and Env Sciences
Non Technical Summary
The overall goal of this project is to elucidate the mechanisms underlying internalization and survival of Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium SL1344 in major fresh produces, particularly lettuce and spinach. An integrative approach involving biochemical, transcriptomic, genetic, and infection analyses as well as innovative thermo-imaging will be used in this research.This proposed research is critical for guiding the development of innovative measures to reduce pathogen load in unprocessed leafy vegetables, thereby enhancing food safety and security.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
(N/A)
Developmental
(N/A)
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
7121430110050%
2011430104050%
Knowledge Area
712 - Protect Food from Contamination by Pathogenic Microorganisms, Parasites, and Naturally Occurring Toxins; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1430 - Greens and leafy vegetables;

Field Of Science
1040 - Molecular biology; 1100 - Bacteriology;
Goals / Objectives
The goals of this project are to elucidate the mechanisms underlying the surprisingly different stomatal responses to E. coli O157:H7 and S. enterica serovar Typhimurium SL1344 and to characterize the in situ transcriptomes and survival determinants of these bacteria in fresh produce, particularly lettuce and spinach. Specific objectives include (1) characterization of differential stomatal responses to SL1344 and O157:H7 and (2) determine the molecular mechanisms of SL1344 and O157:H7 survival in the phyllosphere.
Project Methods
This research utilizes both global (transcriptomics) and targeted (specific gene mutagenesis) research approaches to address fundamental questions in plant interaction with human pathogens. The experiments described under the two specific aims provide a powerful approach to dissect the mechanism of human pathogen internalization and survival in fresh produce at the molecular level. This research will also generate genome-scale data and resources to guide future research directions in the community.A novel feature of our proposed research is that it differs from conventional studies, in which whole plants/leaves are used without distinguishing different host cell types in vivo. In particular, our proposed study takes into consideration the unique contributions of epidermis (stomate)- and apoplast-based defenses to the entry, colonization, and survival of plant and human pathogenic bacteria in the plant. As such, with the proposed research we expect to discover new phenomena and mechanisms that would likely be missed by conventional studies.In addition to making new conceptual advances, the proposed research will introduce new methodologies to the study of plant interaction with pathogenic bacteria. In particular, we have developed (i) a novel RNA-seq pipeline for determining bacterial gene expression in planta, and (ii) a non-invasive, thermo-imaging-based protocol for identification of Salmonella mutants defective in modulating stomatal defense. We anticipate that these new methodologies may have a "transformative" impact on the discovery of new human pathogen genes involved in stomatal modulation, colonization, and survival in plants.

Progress 04/01/15 to 03/31/19

Outputs
(N/A)

Impacts
What was accomplished under these goals? IMPACT: Pathogens that cause foodborne illness pose a challenge to food safety and security, as vegetable crops that are vectors for these human diseases may appear healthy, resulting in their integration into marketplaces throughout the global food supply chain. Salmonella enterica serovar Typhimurium strain SL1344 has been shown to disrupt plant innate immune signaling (including stomatal closure) and survive for long periods inside the leaf. It is crucial to understand the underlying mechanism of Salmonella persistence in leafy greens to employ science-based mitigation strategies relevant to the fresh produce industry. This NIFA grant allowed us to advance knowledge on the genetic basis of Salmonella enterica and Escherichia coli O157:H7 colonization of the lettuce phyllosphere. In particular, this research provided crucial foundational knowledge to guide the development of strategies geared toward reducing pathogen load in unprocessed leafy vegetables, thereby enhancing food safety and security. Together, our team published 8 articles in reputable journals, 12 abstracts through proceedings of scientific society meetings, and 2 dissertations. Furthermore, we currently have 6 manuscripts under review. Our major findings are highlighted below: Objective 1: The experiments were designed to determine how S. enterica is able to overcome stomatal defense, penetrate the leaf tissue, and survive in the intercellular space. By screening 337 multi-gene deletion (MGD) mutants of STm 14028s, we were able to identify 10 genomic regions that play a role in opening the stomatal pore of lettuce leaves. The major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, membrane transport, and biosynthesis of surface appendages. Interestingly, seven of these mutants showed increased population titers inside the leaf through 21 days post-inoculation, two mutants showed similar titers as the wild type bacterium, and one mutant with a large deletion, including the Salmonella pathogenicity island 2 (SPI-2) and adjacent genes, showed significantly impaired persistence in the leaf apoplast. These findings suggest that not all the genomic regions required for initiation of leaf colonization (i.e., epiphytic behavior and tissue penetration) are essential for continuing bacterial survival as an endophyte. We also observed that mutants lacking either SPI-1 (Mut3) or SPI-2 (Mut9) induce callose deposition levels comparable to those of the wild type STm 14028s; therefore, these islands do not seem to affect this lettuce defense mechanism. However, the growth of Mut9, but not of Mut3, was significantly impaired in leaf apoplastic wash fluid (AWF). This suggests that STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overall bacterial fitness in this niche, facets dependent on the gene(s) deleted in the Mut9 strain. Objective 2: To identify the molecular determinants underlying the survival of bacterial pathogens in the phyllosphere, we conducted a large RNA-seq analysis to profile plant and bacterial transcriptomes simultaneously during infection. We inoculated Arabidopsis and Lettuce with Salmonella enterica or Escherichia coli O157:H7 and extracted total RNA at 4 and 24 hours post-inoculation. In addition, we obtained RNA from the bacterial inoculum to infer the metabolic changes occurring in the bacterium when in the plant environment. We initiated a collaboration with Dr. R. Michelmore (lettuce geneticist and director of the UC Davis Genome Center) and Dr. Matthew Settles (manager of the Bioinformatics Core at UC Davis Genome Center) to analyze the plant and bacterium transcriptomic data and integrate datasets into appropriate public databases. Currently, we are finishing a comprehensive analysis of this large dataset. Thus far, our results indicate that, within 24 hours in the plant apoplast, differentially expressed genes (DEGs) in the bacterial population are associated with the type III and type IV secretion systems, motility, membrane proteins and transporters, regulation of transcription, stress resistance, as well as sugar, nitrogen, and iron metabolism. At the same time, E. coli O157:H7 induced genes in the Arabidopsis and Lettuce cells that are associated with signaling and regulatory processes, metabolism, responses to external stimuli, secondary metabolism and cell wall, and growth and development. Interestingly, STm 14028s induced similar changes in these metabolic pathways; however, the magnitude of induction (i.e., relative gene expression) and the number of genes induced in each pathway were considerably higher. These preliminary findings indicate that plants might respond differently to each human pathogen. We are in the process of writing a manuscript to soon be submitted for publication.

Publications

  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Oblessuc PR, Matiolli CC, Melotto M. Novel molecular components involved in callose-mediated defense against human pathogens. BMC Plant Biology
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Oblessuc PR, Bisneta MV, Melotto M. Common and unique Arabidopsis proteins involved in stomatal susceptibility to Salmonella enterica and Pseudomonas syringae. FEMS Microbiol Letters (in review).
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Jacob C, Melotto M. Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium 14028s colonization of lettuce leaves is dependent on the plant genotype and the level of plant defense responses. Front Plant Sci (in review).
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Rodrigues O, Melotto M. Stomata in a state of emergency, H2O2 is the locked target. New Phytologist (second round of revision).
  • Type: Journal Articles Status: Under Review Year Published: 2019 Citation: Montano J, Rossidivito G, Torreano J, Porwollik S, Sela S, McClelland M, Melotto M. Salmonella enterica serovar Typhimurium 14028s genomic regions required for colonization of lettuce leaves. Front Plant Sci (in review).
  • Type: Journal Articles Status: Other Year Published: 2020 Citation: Jacob C, Vel�squez AC, Reyes-Chin-Wo S, Han R, Joshi N, Michelmore R, Settles ML, He SY, Melotto M. Temporal and simultaneous plant and bacterial transcriptomes during Arabidopsis and Lettuce interactions with Salmonella enterica and Escherichia coli O157:H7. (final stages of preparation).
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Roy D, Melotto M. 2019. Stomatal response and human pathogen persistence in leafy greens under preharvest and postharvest environmental conditions. Postharvest Biol. Tech. 148:76-82.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Zhang L, Paasch BC, Chen J, Day B, and He SY. 2019. An important role of L-fucose biosynthesis and protein fucosylation genes in Arabidopsis immunity. New Phytologist 222: 981
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Vel�squez AC, Castroverde CDM, and He SY. 2018. Plant and pathogen warfare under changing climate conditions. Current Biology 28: R619.
  • Type: Journal Articles Status: Published Year Published: 2018 Citation: Xin XF, Kvitko B, and He SY. 2018. Pseudomonas syringae: what it takes to be a pathogen. Nature Reviews Microbiology 16: 316
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Panchal S, Melotto M. 2017. Stomate-based defense and environmental cues. Plant Signaling & Behavior 12(9): e1362517.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhang L, Zhang F, Melotto M, Yao J, He SY. 2017. Jasmonate signaling and manipulation by pathogens and insects. J. Exp. Bot. 68:1371-1385.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Melotto M, Zhang F, Oblessuc PR, He SY. 2017. Stomatal defense a decade later. Plant Physiol. 174: 531-571.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Montano J, Melotto M. 2017. Stomatal bioassay to characterize bacterial-stimulated PTI at the pre-invasion phase of infection. In: Shan L, He P (eds). Methods in Molecular Biology, vol. 1578, Plant Pattern Recognition Receptors, Springer Protocols, Chapter 19: pp. 233-241.
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Montano J. 2017. Salmonella Pathogenicity Island 2 is essential for survival in lettuce. (MSc. Thesis).
  • Type: Theses/Dissertations Status: Under Review Year Published: 2020 Citation: Jacob C. 2020. The close molecular interaction between lettuce and the two human pathogens, Salmonella enterica and Escherichia coli O157:H7. (PhD Dissertation)
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Oblessuc PR, Matiolli CC, Melotto M. 2019. Plant and human bacterial pathogens trigger similar SA-mediated defense in Arabidopsis. In: Book of Abstracts, XVIII International Congress on Molecular Plant-Microbe Interactions, Glasgow, Scotland.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2019 Citation: Jacob C, Melotto M. 2019. Differential plant defense responses of lettuce genotypes against human pathogenic bacteria. In: Book of Abstracts, XVIII International Congress on Molecular Plant-Microbe Interactions, Glasgow, Scotland.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Jacob C, Melotto M. 2018. Lettuce genotypic variation affects the interaction between the plant and the human pathogens Salmonella enterica Typhimurium 14028s and Escherichia coli O157:H7. Plant Biology Annual Meeting, American Society of Plant Biologists. July 14-18, 2018, Montreal, Canada.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2018 Citation: Oblessuc PR, Roy D, Bisneta MV, Matiolli CC, Melotto M. 2018. Genetic basis for Arabidopsis guard cell responses to human and plant pathogens. Pioneer Plant Science Symposia Series, April 16. University of California, Davis, CA. USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Melotto M, He SY, Montano J, Jacob C. 2017. Molecular mechanisms of human bacterial pathogen internalization and survival in fresh produce. USDA-NIFA Project Directors Meeting, July 8. Tampa, FL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Montano J, Jacob C, Porwollik S, McClelland M, Melotto M. 2017. Human pathogens interaction with lettuce. Plant Microbe Interaction Symposium, Joint Genome Institute (JGI), US DOE, Walnut Creek, CA. April 21, 2017.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Montano J, Porwollik S, McClelland M, Melotto M. 2016. Determining the genetic basis for stomatal aperture modulation by Salmonella enterica serovar Typhimurium strain 14028. Phytopathology 106 (12):149. American Phytopathological Society Annual Meeting. Tampa, FL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Montano J, Porwollik S, McClelland M, Melotto M. 2016. Mechanism of Salmonella enterica serovar Typhimurium 14028S invasion and persistence in lettuce. UC Davis Research Retreat on Host Microbe Interaction, Tahoe City, CA. October 27-28, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Roy D, Montano J, Jacob C, Melotto M. 2016. Differential immune response of lettuce to Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Plant Microbe Interaction Symposium, Joint Genome Institute (JGI), US DOE, Walnut Creek, CA. April 15, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Melotto M., Roy D, 2015. Towards preventing internalization and persistence of human bacterial pathogens in fresh produce. Produce Research Symposium, June 23-24. Atlanta, GA, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Melotto M. 2015. Interactions between plants and human pathogens. WIFSS-NAU Annual Symposium on One Health and Food Safety, Nanjing, China. http://www.wifss.ucdavis.edu/?p=10299.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2015 Citation: Roy D, Melotto M. 2015. Internalization and persistence of human bacterial pathogens in fresh produce. III International Conference on Fresh-cut Produce  ISHS, Davis, CA, USA.


Progress 04/01/17 to 03/31/18

Outputs
Target Audience:We have continued our efforts to present this project to undergraduate and graduates students engaged in the One Health Initiative at the UC Davis campus. This year, we have organized and hosted the "Plant Science Day" on February 6, 2018 as part of the "NAU-UC Davis Education Conference on One Health for Food Safety and Security". Students attended several seminars delivered by our research group and visited the UC Davis Student Farm where they were introduced to food safety concepts at the farm. PI Melotto has also organized and delivered a new workshop titled "Plant Immunity and Bacterial Pathogenesis: what determines the outcome of this cross-kingdom relationship?" in collaborations with researchers at Campinas State University, UNICAMP, São Paulo, Brazil. This intensive, one-week workshop was attended by graduate students from UNICAMP and The University of São Paulo (USP). Changes/Problems:During this period, we experienced some technical difficulties with our RNA-seq pipeline. However, the scope of the project remains the same and a non-cost extension of the project was approved. What opportunities for training and professional development has the project provided?One undergraduate student, three PhD students, and two post-doctoral fellow are being trained in this project. How have the results been disseminated to communities of interest?This past year, the project was highlighted at the Plant-Microbe Interaction Symposium held at the Joint Genome Institute, US DOE, Walnut Creek, CA, April 21, 2017. Among the attendees were graduate students, research scientists, technical support staff, and university professors. In June, 2017, PI Melotto was awarded a faculty summer fellowship from the Jewish National Fund and Media Watch, New York, to present this project to several research groups in Israel including Volcani Center, Tel Aviv University, Technion, Hebrew University, Arava Institute, and Tel Hai College. What do you plan to do during the next reporting period to accomplish the goals?There are no changes to the approved project initiation document.

Impacts
What was accomplished under these goals? IMPACT: Pathogens that cause foodborne illness pose a challenge to food safety and security, as vegetable crops that are vectors for these human diseases may appear healthy, resulting in their integration to marketplaces throughout the global food supply chain. Salmonella enterica serovar Typhimurium strain SL1344 has been shown to disrupt plant innate immune signaling (including stomatal closure) and survive for long periods of time inside the leaf. It is crucial to understand the underlying mechanism of Salmonella persistence in these leafy greens to employ science-based mitigations strategies relevant to the fresh produce industry. During this reporting period, we have made significant progress towards achieving each objective as described below. Objective 1 is nearly completed and we expect to have an additional peer-reviewed publication to report our findings. Our results indicate that the Salmonella Pathogenicity Island 2 (SPI-2), but not SPI-1, is required for Salmonella persistence in lettuce apoplast. It is important to note that SPI-2 is a genomic segment of roughly 40kb, with 42 open reading frames. Besides encoding the TTSS-2 secretion apparatus, effectors, and a two-component regulatory system that regulates the expression of SPI-2, SPI2 also carries genes coding for a tetrathionate reductase complex, a cysteine desulfurase enzyme complex, membrane transport proteins, murein transpeptidases, and genes with uncharacterized functions. Thus, it is possible that they also may have a function in the bacterium persistence in the leaf. We have not been able to complete Objective 2 due to difficulties in the RNA-seq pipeline. Both the PI and co-PI's labs are working together to streamline the protocol to obtain simultaneous transcriptomic profiles of the plant and the bacterium over the course of infection. The main difficulty has been to obtain enough bacterial-associated sequence reads from infected plant tissue due to the overwhelming amount of plant transcripts, including highly abundant chloroplast-derived RNAs. As such, we are still testing several inoculation doses and RNA purification procedures to optimize the recovery of bacterial RNA from infected plants. We anticipate that the non-cost extension of the project recently approved for this project will allow us to complete this objective.

Publications

  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Melotto M, Zhang F, Oblessuc PR, He SY. 2017. Stomatal defense a decade later. Plant Physiol. 174: 531-571.
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Panchal S, Melotto M. 2017. Stomate-based defense and environmental cues. Plant Signaling & Behavior 12(9): e1362517.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2017 Citation: Montano J, Jacob C, Porwollik S, McClelland M, Melotto M. 2017. Human pathogens interaction with lettuce. Plant Microbe Interaction Symposium, Joint Genome Institute (JGI), US DOE, Walnut Creek, CA. April 21, 2017.
  • Type: Theses/Dissertations Status: Published Year Published: 2017 Citation: Montano J. 2017. Salmonella Pathogenicity Island 2 is essential for survival in lettuce. (MSc. Thesis).


Progress 04/01/16 to 03/31/17

Outputs
Target Audience:Besides giving invited seminars at various Universities and Scientific Society Meetings, this project was presented to undergraduate and graduates students engaged in the One Health Initiative at the UC Davis campus. This year, we organized a series of talks during the NAU-UC Davis Graduate Education Conference on One Health https://www.wifss.ucdavis.edu/wp-content/uploads/documents/PROGRAM-2017StudentConf.pdf). Additionally, PI Melotto delivered a talk at the UC-ANR Vegetable Crops Meeting attended by farm advisors and cooperative extension specialists. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?One undergraduate student, two PhD students, one post-doctoral fellow, and one technician are being trained in this project. How have the results been disseminated to communities of interest?We have given invited seminars at various universities and scientific conferences discussing the various aspects of this research. This year in particular, this project was highlighted in the following conferences: · UC-ANR Vegetable Crops Program Meeting. UC Davis. Dec 5-6, 2016. · International Association for Food Protection (IAFP) Annual Meeting, August 2016, St. Louis, MO. · Department of Biological Sciences, California State University, April 22, Chico, CA. · Plant Microbe Interaction Symposium, Joint Genome Institute, US DOE, Walnut Creek, CA, April 15, 2016. What do you plan to do during the next reporting period to accomplish the goals?There are no changes to the approved project initiation document.

Impacts
What was accomplished under these goals? IMPACT: Pathogens that cause foodborne illness pose a challenge to food safety and security, as vegetable crops that are vectors for these human diseases may appear healthy, resulting in their integration to marketplaces throughout the global food supply chain. Salmonella enterica serovar Typhimurium strain SL1344 has been shown to disrupt plant innate immune signaling (including stomatal closure) and survive for long periods of time inside the leaf. It is crucial to understand the underlying mechanism of Salmonella persistence in these leafy greens to employ science-based mitigations strategies relevant to the fresh produce industry. During this report period, we have made significant progress towards achieving each objective as described below. Objective 1: is focused on identifying the genetic determinants conferring the ability of Salmonella enterica serovar Typhimurium 14028S (hereafter 14028S) to modulate stomatal aperture and persist within the apoplast of Lactuca sativa cv. Salinas. The original goal was based on S. enterica serovar Typhimurium SL1344 (hereafter SL1344), but due to better genetic resources with 14028S the focus was changed from the former strain to the latter. Initial correlation of stomatal aperture modulation between 14028S and SL1344 indicated that these two strains induce the same stomatal reopening at 4-hours post inoculation and analysis could proceed with 14028S. To achieve the aforementioned goal we began by conducting a genetic screen of 14028S multi-gene deletion (MGD) marker exchange mutants for the ability to modulate stomatal aperture at 4-hours post inoculation. A total of 337 MGD mutants were analyzed, resulting in 10 MGD mutants of interest. These 10 mutants were phenotypically characterized for several factors including persistence within the apoplast, motility (e.g., swim, swarm, and chemotaxis), epiphytic survival, and ability to grow on apoplastic wash fluid. All assays were performed on the lettuce cultivar Salinas. This phenotypic characterization identified two mutants of interest who differ from the wild type in the ability to persist within the apoplast (one significantly less than and one more than) and ability to grow on apoplastic wash fluid. The mutant that is not able to persist within the apoplast as well as the wild type lacks the entire Salmonella Pathogenicity Island 2 (SPI2), indicating that SPI2 is vital for establishment of a durable population within the apoplast. We have also evaluated the mutant lacking Salmonella Pathogenicity Island 1 (SPI1) for the same phenotypes, and have determined that it is non-essential. Upon investigation of single-gene deletion (SGD) mutants within SPI2, it has been determined that the SPI2 Type Three Secretion System (T3SS) structural genes are not required for durable apoplastic persistence. Furthermore, mutants lacking these structural genes have increased apoplastic titers indicating that expression of the SPI2 T3SS is detrimental to the survival of 14028S. The SPI2 is a large pathogenicity island containing approximately 50 genes that are involved in processes including expression of the SPI2 T3SS, iron regulation, peptidoglycan rearrangement, tetrathionate metabolism, and secreted effectors involved in animal-cell infection. The determination that loss of the entire SPI2 results in ineffective colonization of the apoplast, while loss of only SPI2 T3SS structural genes leads to increased colonization indicates that non-structural genes on SPI2 are the major contributors to effective establishment of apoplastic populations by 14028S. Current work on this project revolves around complementing these SGD mutants to show restoration of the wt phenotype, investigating the expression of the SPI2 T3SS in vivo and further analysis of non-structural SGD SPI2 mutants. Objective 2: The optimal growth conditions for lettuce cultivar Salinas and its inoculation method with human pathogens was established in the laboratory. Five- to six-week old plants were inoculated by syringe infiltration to obtain uniform leaf inoculation suitable for RNA-expression studies. We observed that both the Salmonella and E. coli apoplastic populations survives at least 28 days in the lettuce apoplast. Additionally the PI and co-PI's laboratory are working together to finish the plant-bacterium simultaneous RNA-seq analysis. A UC Davis Ph.D. student visited MSU to coordinate the experiment. Using the optimized inoculation procedure, we are preparing inoculated plant tissue samples to be sent to MSU facilities for sequencing and downstream bioinformatic analyses.

Publications

  • Type: Journal Articles Status: Accepted Year Published: 2017 Citation: Melotto M, Zhang F, Oblessuc PR, He SY. 2017. Stomatal defense a decade later. Plant Physiol. DOI:10.1104/pp.16.01853 (in press).
  • Type: Journal Articles Status: Published Year Published: 2017 Citation: Zhang L, Zhang F, Melotto M, Yao J, He SY. 2017. Jasmonate signaling and manipulation by pathogens and insects. J. Exp. Bot. erw478. doi: 10.1093/jxb/erw478.
  • Type: Book Chapters Status: Published Year Published: 2017 Citation: Montano J, Melotto M. 2017. Stomatal bioassay to characterize bacterial-stimulated PTI at the pre-invasion phase of infection. In: Shan L, He P (eds). Methods in Molecular Biology, vol. 1578, Plant Pattern Recognition Receptors, Springer Protocols, Chapter 19: pp. 233-241.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Montano J, Porwollik S, McClelland M, Melotto M. 2016. Determining the genetic basis for stomatal aperture modulation by Salmonella enterica serovar Typhimurium strain 14028. Phytopathology 106 (12):149.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Montano J, Porwollik S, McClelland M, Melotto M. 2016. Mechanism of Salmonella enterica serovar Typhimurium 14028S invasion and persistence in lettuce. UC Davis Research Retreat on Host Microbe Interaction, Tahoe City, CA. October 27-28, 2016.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 Citation: Roy D, Montano J, Jacob C, Melotto M. 2016. Differential immune response of lettuce to Escherichia coli O157:H7 and Salmonella enterica serovar Typhimurium. Plant Microbe Interaction Symposium, Joint Genome Institute (JGI), US DOE, Walnut Creek, CA. April 15, 2016.


Progress 04/01/15 to 03/31/16

Outputs
Target Audience:Besides giving invited seminars at various Universities and Scientific Society Meetings, this project has caught the attention of scientific delegations. In particular, the A*STAR Singapore delegation (www.a-star.edu.sg) directly requested a presentation on our food safety research during their two-week tour in the USA. These activities speak for the broad interest in new technologies and advances in scientific discoveries to mitigate leafy vegetables-related foodborne illnesses. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD students at UC Davis and one technician at MSU are being trained in this project. How have the results been disseminated to communities of interest?We have given seven invited seminars at various universities and scientific conferences discussing the various aspects of this research. PD Melotto has started collaboration with the Western Institute for Food Safety and Security (WIFSS) at UC Davis to facilitate implementation of a joint One Health for Food Center to be located at Nanjing Agricultural University (NAU). She has presented this research at the 2015 Annual Symposium of One Health and Food Safety (http://www.wifss.ucdavis.edu/?p=10299) and had opportunities to interact with several scientists to identify areas where UC Davis and NAU faculty can collaborate. We have hosted and organized activities for undergraduate and graduate student delegations visiting UC Davis to foster collaborations and engage the next generation of scientists in the emerging field of plant interactions with human pathogens. What do you plan to do during the next reporting period to accomplish the goals?There are no changes to the approved project initiation document.

Impacts
What was accomplished under these goals? IMPACT: Pathogens that cause foodborne illness pose a challenge to food safety and security, as vegetable crops that are vectors for these human diseases may appear healthy, resulting in their integration to marketplaces throughout the global food supply chain. Salmonella enterica serovar Typhimurium strain SL1344 has been shown to disrupt plant innate immune signaling (including stomatal closure) and survive for long periods of time inside the leaf. It is crucial to understand the underlying mechanism of Salmonella persistence in these leafy greens to employ science-based mitigations strategies relevant to the fresh produce industry. In this first year of this project, we have obtained university's authorization to work with mutant variants of pathogenic Salmonella strains for the genetic screening (objective 1), as well as recruited and trained new personnel to work in a BSL2 environment. Progress towards achieving each objective is described below. Objective 1: we have set up a medium throughput genetic screen to elucidate the mechanism(s) by which Salmonella modulates lettuce stomatal movement using a mutant library with high-coverage and multi-gene deletions created with lambda-red mediated gene-replacement. Individual Salmonella mutant strains were analyzed for their inability to stimulate stomatal opening (i.e. unable to overcome stomatal immunity). We observed that two mutant strains lacking operons involved in secretion could not open stomata. Further characterization of identified mutants will aim to identify the individual genes in the deleted genome region that are pertinent for stomatal opening and their biological function(s). Objective 2: during this year, the sequenced genome of the lettuce cultivar Salinas became available to us. Due to its large developmental plasticity in response to environmental variables, we had to adjust the experimental design to use laboratory-grown lettuce plants to run transcriptome analysis. Additionally, we are optimizing a novel dual RNAseq procedure to profile both lettuce and bacterial transcriptomes in inoculated leaves to elucidate temporal regulation of these transcriptomes simultaneously.

Publications

  • Type: Book Chapters Status: Under Review Year Published: 2016 Citation: Montano J, Melotto M. 2016. Stomatal bioassay to characterize bacterial-stimulated PTI at the pre-invasion phase of infection. In: Shan L, He P (eds). Methods in Molecular Biology, vol. Plant Pattern Recognition Receptors, Springer Protocols.