Source: UNIVERSITY OF CALIFORNIA, DAVIS submitted to
PLANT INTERACTIONS WITH HUMAN PATHOGENS AND THE IMPLICATIONS TO FOOD SAFETY
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
TERMINATED
Funding Source
HATCH
Reporting Frequency
Annual
Accession No.
1009164
Grant No.
(N/A)
Project No.
CA-D-PLS-2327-H
Proposal No.
(N/A)
Multistate No.
(N/A)
Program Code
(N/A)
Project Start Date
Jan 15, 2016
Project End Date
Sep 30, 2020
Grant Year
(N/A)
Project Director
Melotto, MA.
Recipient Organization
UNIVERSITY OF CALIFORNIA, DAVIS
410 MRAK HALL
DAVIS,CA 95616-8671
Performing Department
Plant Sciences
Non Technical Summary
Consumption of fresh- or minimally processed produce including lettuce has increased during recent decades as a part of an integrated strategy for a healthy human diet. Concurrently, the incidence of outbreaks related to human bacterial pathogens on fresh produce has increased and it is now a growing concern in industrialized countries, of both the health sector and agro food sector. Contamination of fresh produce might occur at any stage of the food-chain, yet unlike food of animal origin, there is currently no efficient decontamination treatment. Difficulties in bacterial eradication of leafy greens are thought to be related, at least partially, to resistance of internally localized bacteria to surface decontamination. Hence, prevention of internal contamination is an important goal as part of a multi-hurdle approach to mitigate fresh produce contamination. Salmonella enterica is a major foodborne pathogen that is associated with contamination of fresh produce. Evidence has accumulated indicating that S. enterica serovars may exploit the plant as a transient host during its cycling in the field between animal and human hosts. Salmonella enterica serovar Typhimurium (STm) can adhere to lettuce leaf surface and utilizes stomata as a portal of entry into internal leaf tissues. Chemotaxis and motility were demonstrated to take part in this process. Shortly after bacterial adherence to the lettuce leaf surface, the plant responds by closing stomata to prevent bacterial entry; however, our recent data have shown that STm can overcome this response and force stomatal opening within a few hours. The bacterial mechanisms and host immunity factors that are involved in these interactions are still unknown. In the present study we will focus on understanding how S. enterica adapts to the lettuce phyllosphere The specific objectives are: (1) Identify STm-derived effectors that induce stomatal opening; (2) Identify genes required for STm internalization and endophytic persistence; and (3) Identify guard cell genes that are regulated by STm. The results should provide new insights into mechanisms involved in the adaptation of a STm to lettuce and should pave the way toward the development of new breeding programs as well as agro-technologies aimed at mitigating epiphytic and endophytic colonization of S. enterica in lettuce leaves and other crops. The study should also yield basic knowledge regarding innate immunity mechanisms utilized by a crop plant to limit colonization by human pathogens.
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
2011430108050%
2124010116050%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1430 - Greens and leafy vegetables; 4010 - Bacteria;

Field Of Science
1160 - Pathology; 1080 - Genetics;
Goals / Objectives
lettuce as a model crop. The specific objectives are:1. Identify STm-derived effectors that induce stomatal opening. 2. Identify genes required for STm internalization and endophytic persistence.3. Identify lettuce guard cell genes regulated upon STm infection.
Project Methods
Our preliminary data suggest that STm effectors may be involved in stomatal reopening. Thus, we will use a targeted genetic screen approach to identify which effector(s) are responsible for this phenotype. We will select mutants from our single-gene deletion collection that are known to be transported by SPI-1 and/or SPI-2, and screen individual mutants for those unable to reopen stomata using the medium-throughput thermo-imaging system developed by us. We will screen a library of 200 multi-gene deletion (MGD) mutants in STm 14028s covering 3476 genes of STm, to identify DNA regions containing genes that are critical for persistence in the lettuce phyllosphere. Each mutant will be tested individually using the detached leaf model for endophytic persistence. Our preliminary data indicate that the stomatal response and endophytic persistence are similar in both detached and intact lettuce leaves, so the former will be used to allow a rapid medium-throughput screen. Once a phenotype is identified in a multi-gene deletion mutant (either low persistence or low internalization), we will test mutants in individual genes present in the multi-gene region of interest, using the collections of targeted single gene deletion we have developed. Putative STm lettuce phyllosphere adaptation genes will be genetically complemented to confirm their function in vivo. Once a phenotype is established, we will try to identify the affected colonization stage, by testing leaf adherence, aggregation near stomata, internalization, and resistance to ROS. Note that this screen cannot use pools of mutants because pools would have a wild type effect on stromata opening, so only this hierarchical screening method can find the class of genes we are seeking.We have developed a method to routinely isolate guard cells protoplasts (GCPs) suitable for high throughput RNA sequencing. We will employ an RNA-seq approach to identify a subset of lettuce guard cell genes that appear to be regulated upon STm infection that could explain, at least in part, the ability of STm to manipulate stomatal movement. This objective is only now possible, as the lettuce genome has only recently been released (lgr.genomecenter.ucdavis.edu).

Progress 01/15/16 to 09/30/20

Outputs
Target Audience:For the entire period of this project (2016-2020), PD Melotto, as well as graduate students and post-docs involved in this research, have reached several audiences in different setting as follows: 1) Yearly participation, as invited speaker and panelist, in the "Education Conference on One Health for Food Safety and Security" hosted by Nanjing Agicultural University, China and the Western Institute for Food Safety and Security (WIFSS), University of California, Davis. The audience is composed by undergraduate and graduate students representing the colleges of Resources and Environmental Sciences, Horticulture, Plant Protection, Veterinary Medicine, Life Science, Agro-grassland Sciences, Food Science and Technology, and Animal Science and Technology (~60 attendees per year for 5 years). 2) Delivery of over 20 oral or 18 poster presentations during national and international scientific conferences, California commodity board meetings, USDA stakeholder and PD meetings, and UC Davis research conferences. The audience included scientists from the industry and academia, professors, post-doctoral fellows, students, as well as professionals and board members of the horticultural industry. All of these presentations were public and/or recorded for a wide distribution among scientific society members. Although it is difficult to estimate the total number of attendees, they ranged between 40 to 200 attendees per event. 3) Organization of 6 workshops targeted to educate students and provide opportunities for scientists to present and discuss the current state of knowledge in this field. The audience included students, post-doctoral fellows, and professionals from the industry, government, and academia. Total number of attendees was ~170. Changes/Problems:Due to the COVID-19 pandemic, our laboratory has been operating at a minimum capacity since March 2020 only to maintain plant lines and sensitive equipment. Therefore, we were not able to run experiments originally planned under objective 3. Nonetheless, our group has been highly productive in this project which resulted with the publication of 10 peer-reviewed articles (5 of which in 2020), 11 conference papers, 1 book chapter, and 1 thesis. As work conditions go back to normal, we will be able to continue this project, which has been approved already. What opportunities for training and professional development has the project provided?Training activities: PD Melotto was the major professor to 4 graduate students and mentor to 2 post-doctoral fellows, who worked in this project from 2016 to 2020. She provided guidance for them to develop skills required for a successful career in science. Specifically, the PD held bi-weekly meetings with each student and post-doc to discuss results, troubleshoot research problems and their alternatives, and plan new experiments using sound experimental design for robust statistical analyses. This project also allowed us to train 3 undergraduates per year in principles of scientific enquiry and responsibility for timely delivery of progress reports to the group. Professional development: all graduate students and post-docs involved in this project participated in at least one scientific conference to present their results, in addition to author publications. Furthermore, our group participates in a monthly conference with nine other research groups on campus that share our interest in host-microbe interactions. This venue provide opportunities to increase knowledge, networking, and develop professional presentation skills with instant feedback. How have the results been disseminated to communities of interest?Beyond the dissemination of our results through peer-reviewed publications and oral presentations in scientific and educational conferences, this project allowed us to be engaged in outreach activities to increase community awareness of fresh produce safety. We supervised one high school student per year during 9 weeks through the American Chemistry Society (ACS) program "Summer Experiences for the Economically Disadvantaged" (SEED). Students were introduced to the goals of this project as well as the methods required to accomplish the goals. Upon completion of the internship, the students presented the project to their peers amplifying the impact of our activities. What do you plan to do during the next reporting period to accomplish the goals? Nothing Reported

Impacts
What was accomplished under these goals? IMPACT: Despite extensive efforts to deliver safe and nutritious food, disease outbreaks associated with lettuce (especially romaine) continue to occur, which are linked to contamination on the farm. This project addressed the possibility that the human pathogen Salmonella enterica (STm) colonizes lettuce leaves and survives through the crop productions chain until it reaches the consumers. Knowledge generated through this research highlights the genetic mechanisms by which STm persists in lettuce providing new opportunities for mitigation strategies, such as breeding for enhanced crop safety. A comprehensive review on this topic is available to interested audiences (Melotto et al., 2020; https://www.frontiersin.org/articles/10.3389/fpls.2020.00428/full). Accomplishments under each objective are described below. Objective 1. Identify STm-derived effectors that induce stomatal opening. Opening of the stomatal pore on the leaf surface provides a port of entry for bacterial pathogens into leaf tissues, where they can be protected from sanitation procedures. To understand how STm modulates stomatal movement, we have conducted a genetic screen of STm multi-gene deletion (MGD) mutants for the ability to open lettuce stomatal pores. We identified 10 regions in the STm genome that are required for this phenotype and further characterized the mutant strains for other phenotypes (objective 2). Targeted mutant analyses are still required to assign the phenotype to the proteins encoded by these regions. These additional experiments are technically challenging and time consuming; thus, it could not be completed during this grant period. Nonetheless, all results gathered under this objective were published (Montano et al. 2020). Objective 2. Identify genes required for STm internalization and endophytic persistence. The 10 MGD strains selected under objective 1 were also tested for their ability to survive in the leaf apoplast, overcome plant defenses, and obtain nutrients leached to the apoplast using a series of experimental approaches in molecular biology and microbiology. With these analyses, we have identified STm genomic regions that controls (directly or indirectly) the ability of the bacterium to internalize and persist in lettuce leaves (Montano et al., 2020). Additionally, we expanded the originally proposed analysis to identify plant genes and proteins required for stomatal closure in response to STm. We found that the plant proteins EXO70H4 and SCE1 are involved in bacterial-specific responses, while BBE8, STP1, and LSU2 may be required for stomatal response to a broad range of bacteria such as Salmonella and Pseudomonas (Oblessuc et al., 2019). Furthermore, we discovered that salicylic acid signaling and biosynthesis via the plant proteins NPR1 and ICS1, respectively, are involved in stomatal and apoplastic defense, as well as callose deposition, against STm (Oblessuc et al., 2020). Objective 3. Identify lettuce guard cell genes regulated upon STm infection. Isolation of guard cells from lettuce leaves and gene expression analysis (RT-qPCR) were planned to be completed in 2020. However, our laboratory has been closed since the beginning of the year due to the COVID-19 pandemic and these experiments could not be performed. However, other results from our lab related to this objective highlight the existence of plant genetic components important for STm infection of lettuce and other leafy greens. For instance, we discovered that STm is not adapted to overcome the basic stomatal immune response of all leafy vegetables, stomatal response to STm may be dependent on the environmental conditions, and internalized bacteria can survive inside lettuce leaves until senescence (Roy and Melotto 2019). Furthermore, the ability of STm to colonize lettuce leaves depends on the plant genotype and increased STm persistence in leaves is associated with weak plant defenses (Oblessuc et al., 2020, Jacob and Melotto 2020). Altogether, these results underscore an exciting opportunity to capitalize on plant genetics to control STm colonization of edible leaves.

Publications

  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Jacob C, Melotto M. 2020. Human pathogen colonization of lettuce dependent upon plant genotype and defense response activation. Front Plant Sci 10:1769
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Oblessuc PR, Matiolli CC, Melotto M. 2020. Novel molecular components involved in callose-mediated defense against human pathogens. BMC Plant Biology 20:16.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Montano J, Rossidivito G, Torreano J, Porwollik S, Sela S (Saldinger), McClelland M, Melotto M. 2020. Salmonella enterica serovar Typhimurium 14028s genomic regions required for colonization of lettuce leaves. Front Microbiol 11:6.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Melotto M, Brandl MT, Jacob C, Jay-Russell M, Micallef SA, Warburton M, Van Deynze A. 2020. Breeding crops for enhanced food safety. Front Plant Sci 11:428.
  • Type: Journal Articles Status: Published Year Published: 2020 Citation: Oblessuc PR, Melotto M. 2020. A simple assay to assess Salmonella enterica persistence in lettuce leaves after low inoculation dose. Front Microbiol 11:1516.
  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Oblessuc PR, Bisneta MV, Melotto M. 2019. Common and unique Arabidopsis proteins involved in stomatal susceptibility to Salmonella enterica and Pseudomonas syringae. FEMS Microbiol Letters 366:fnz197.
  • Type: Other Status: Published Year Published: 2019 Citation: Melotto M. 2019. Bringing Food Safety and Plant Breeding Together Can Create Solutions. SeedWorld. https://seedworld.com/maeli-melotto-thinks-bringing-food-safety-and-plant-breeding-together-can-create-solutions/?bulk_email_rid=575&bpmtrackid=12&bpmreplica=0
  • 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: 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: Van Deynze A, Melotto M, Jay-Russell M. 2019. NIFA: Breeding for food safety. Annual Meeting of the National Association of Plant Breeders. August 25-29, Pine Mountain, GA, USA.
  • 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: 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: Journal Articles Status: Published Year Published: 2018 Citation: Matiolli CC, Melotto M. 2018. A comprehensive Arabidopsis yeast two-hybrid library for protein-protein interaction studies: a resource to the plant biology research community. Mol. Plant-Microbe Interact. 31:899-902.
  • 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: 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: Theses/Dissertations Status: Published Year Published: 2017 Citation: Montano J. 2017. Salmonella pathogenicity island 2 is essential for survival in lettuce. (MSc. Thesis)
  • 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: 2017 Citation: He SY, Melotto M, 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: 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: 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: Melotto M, Zhang F, Oblessuc PR, He SY. 2017. Stomatal defense a decade later. Plant Physiol. 174: 531-571.
  • 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: 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. American Phytopathological Society Annual Meeting. Tampa, FL, USA.
  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 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.


Progress 10/01/18 to 09/30/19

Outputs
Target Audience:During this reporting period, PI Melotto delivered oral presentations to the following audiences: 1) A webinar to the USDA Plant Breeding for Food Safety Meeting. September 3, 2019, Washington, D.C. The audience included USDA food safety working group (~19 attendees). 2) Plant Health 2019: Annual National Meeting of the American Phytopathological Society. August 3-7, Cleveland, Ohio, USA. The audience included scientist, students, and professionals from the industry. Attendance: ~150 3) One Health for Food Safety and Security Conference. January 29, 2019. UC Davis, CA. USA. The audience included scientist, students, extension specialists, and academic professors from UC Davis and Nanjing Agricultural University, China. Attendance: ~100. 4) Department of Plant Sciences, Mentor-Mentee Seminar Series. December 5, 2018. UC Davis, CA. USA. The audience included students and professors. Attendance ~35. 5) UC-ANR Vegetable Crops Program Meeting. November 26-27, 2018. University of California, ANR. Davis. The audience included scientist, students, professionals from industry, extension specialists, and farmer advisors. Attendance: ~100 PI Melotto also organized scientific meetings as follows: 1) NIFA Conference: Breeding Crops for Enhanced Food Safety. June 4-5, 2019. UC Davis, CA. USA. The audience included scientist, students, professionals from industry, extension specialists, farmer advisors, and USDA national program leaders. Attendance: 65 on-site and 15 remote. Co-organizers: Michele Jay-Russell and Allen Van Deynze. 2) Satellite Meeting: The Interaction between Plants and Human Pathogens. July 14-18, 2019. International Society for Molecular Plant-Microbe Interactions (IS-MPMI) Glasgow, Scotland. The audience included students and professors. Attendance ~30. Co-organizer: Nicola Holden. Changes/Problems:We were not able to identify single gene deletion mutants of Salmonella enterica with altered phenotypes for stomatal opening and bacterial persistence in lettuce leaves. Therefore, objectives 1 and 2 cannot be completed entirely during this grant period. Nonetheless, we have made progress in identifying Salmonella genomic regions required for overcoming stomatal defense and persistence in leaves. Furthermore, the function of these regions were experimentally validated and the results were widely disseminated to diverse audiences. What opportunities for training and professional development has the project provided?Completed training: - A post-doctoral fellow, Dr. Paula Oblessuc, was hired by a company in Portugal as a director of the crop protection division. - A research associate, Dr. Cleverson Matiolli, is now a post-doctoral fellow in Lisbon, Portugal. Ongoing training: - Three PhD students (Brianna Fochs, Cristian Jacob, and David Bridges) are being trained in this project. How have the results been disseminated to communities of interest?The results of these project has been disseminated through peer-reviewed papers (1), podcast (1), conference presentations with published abstracts (3), invited seminars to the interested public (5), and scientific meetings (2). What do you plan to do during the next reporting period to accomplish the goals?We will continue the experiments to complete the objective 3 of this proposal.

Impacts
What was accomplished under these goals? This study was designed to gain understanding on how Salmonella enterica (STm) adapts to the plant phyllosphere, employing lettuce as a model crop. The specific objectives and accomplishments for this period are: Objective 1. Identify STm-derived effectors that induce stomatal opening. In previous years, we have completed a genetic screen of STm multi-gene deletion (MGD) mutants for the ability to open lettuce stomatal pores and selected 10 MGD mutants for further phenotypic analyses. Among the selected MGD mutant strains, two strains (Mut3 and Mut9) lacking the entire Salmonella pathogenicity island (SPI) 1 and 2, respectively, in addition to a few adjacent genes and operons were included. The identification of Mut3 and Mut9 led us to hypothesize that effector proteins delivered by the type-three secretion apparatuses encoded within these islands are required to overcome stomatal defenses. We evaluated single gene deletion (SGD) mutants in these regions; however, they showed the same phenotype as the wild type bacterium. We concluded that functional redundancy among the effectors might exist and higher order mutants would be required to exhibit altered phenotypes. Alternatively, the lack of stomatal opening by the Mut3 and Mut9 might be due to indirect effects of the mutation. Therefore, this objective cannot be completed entirely. Objective 2. Identify genes required for STm internalization and endophytic persistence. This objective is complete. Additionally, we were able to expand the originally proposed analysis to identify plant genes and proteins required for stomatal closure in response to Salmonella. Using readily available resources for plant analysis (i.e., Arabidopsis mutants), we found that the plant proteins EXO70H4 and SCE1 are involved in bacterial-specific responses, while BBE8, STP1, and LSU2 may be required for stomatal response to a broad range of bacteria (Salmonella and Pseudomonas). The identification of new molecular components of the guard cell movement induced by bacteria will enable a better understanding of the initial stages of plant colonization and facilitate targeted prevention of leaf contamination with harmful pathogens (Oblessuc et al., 2019). A manuscript reporting the results obtained under objectives 1 and 2 was accepted for publication. Current citation is: Montano J, Rossidivito G, Torreano J, Porwollik S, Sela S, McClelland M, Melotto M. 2020. Salmonella enterica serovar Typhimurium 14028s genomic regions required for colonization of lettuce leaves. Front Microbiol (accepted) Objective 3. Identify lettuce guard cell genes regulated upon STm infection. Isolation of guard cells from lettuce leaves to run gene expression analysis (RT-qPCR) is under way. This is a technically challenging procedure and will require adjustments to minimize wound responses that could interfere with the analysis.

Publications

  • Type: Journal Articles Status: Published Year Published: 2019 Citation: Oblessuc PR, Bisneta MV, Melotto M. 2019. Common and unique Arabidopsis proteins involved in stomatal susceptibility to Salmonella enterica and Pseudomonas syringae. FEMS Microbiol Letters 366:fnz197.
  • Type: Other Status: Published Year Published: 2019 Citation: Melotto M. 2019. Bringing Food Safety and Plant Breeding Together Can Create Solutions. SeedWorld. https://seedworld.com/maeli-melotto-thinks-bringing-food-safety-and-plant-breeding-together-can-create-solutions/?bulk_email_rid=575&bpmtrackid=12&bpmreplica=0
  • 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: 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: Van Deynze A, Melotto M, Jay-Russell M. 2019. NIFA: Breeding for food safety. Annual Meeting of the National Association of Plant Breeders. August 25-29, Pine Mountain, GA, USA.


Progress 10/01/17 to 09/30/18

Outputs
Target Audience:During this reporting period, PI Melotto delivered oral presentations to the following audiences: 1) USDA-NIFA Project Directors Meeting held in Salt Lake City, Utah. The audience included scientists and research professors. Attendance: ~100. 2) Plant-Microbe Interaction Forum hosted by Pioneer DuPont in Hayward, CA. The audience included scientist, students, and professionals from industry from the Bay Area. Attendance: ~50 in addition to remote audience (webinar). 3) Seed Central's "DISCOVER Research at UC Davis", held at the University of California, Davis. The audience included professionals from the seed industry in California. This presentation was recorded for broadcasting to members of Seed Central, the seed industry surrounding UC Davis (http://www.seedcentral.org/). 4) Pioneer Plant Science Symposia Series held at the University of California, Davis. The audience included scientist,students, professionals from industry, extension specialists, and farmer advisors. Attendance: ~400. PI Melotto also organized and hosted the "Plant Science Day" as part of the "NAU-UC Davis Education Conference on One Health for Food Safety and Security" held at the University of California Davis. China. The audience included scientist, students, extension specialists, and academic professors from UC Davis and Nanjing Agricultural University, China. Attendance: ~100. Changes/Problems:Based on our genetic screen results, it became evident that the phenotypes we are studying (bacterial internalization and persistence in lettuce leaves) are controlled by a very complex genetic mechanism. It is possible that single gene mutations do not interfere with the bacterium's ability to colonize lettuce and we will need to use alternative strategies to assign gene function, such as genetic complementation of the MGD mutants with entire operons. What opportunities for training and professional development has the project provided?Completed training: - Two undergraduate research assistants, Samuel Koehler and Stephanie Giori, completed their Bachelors degree at UC Davis. They worked in this project for almost one year to acquire technical skills in basic microbiology. - In the summer of 2018, PI Melotto mentored and provided research internship to one high school student as part of the "Summer Experience for the Economically Disadvantage" (SEED) program. He gave an oral presentation on his research activities to a broad interested audience composed by students, faculty members, industry professionals, and members of the local community. - Andrew Valejo, the high school student who participated in this project in the summer of 2017, is now a freshmen in Stanford University. His experience in the PI's laboratory enhanced his competitiveness among the applicants to this prestigious university. - Mariana Vas Bisneta, a visiting PhD student, was trained in microscopy and microbiology while engaged in activities proposed in this project. She is an author of our conference paper mentioned above. Ongoing training: - A post-doctoral fellow, Dr. Paula Oblessuc, is being trained in this project with the objective to become a university professor. - Three PhD students (Brianna Fochs, Cristian Jacob, and Gabrielle Rossidivito) are being trained in this project. - A research associate, Dr. Cleverson Matiolli, is being trained in molecular biology. During this grant period, he was able to develop a yeast-two-hybrid system to study protein-protein interaction that will be valuable for the next steps in this project. This resource was published in a high reputable journal (Matiolli and Melotto, 2018). How have the results been disseminated to communities of interest?The results of these project has been disseminated through peer-reviewed papers (2), conference presentations with published abstracts (2), and invited seminars to the interested public. What do you plan to do during the next reporting period to accomplish the goals?Although we have defined several genomic regions that contribute to STm colonization of the phyllosphere, we have not landed on specific genes responsible for this phenotypes. In the next grant period, we will phenotype single gene deletion mutants to assign functionality to specific genes.

Impacts
What was accomplished under these goals? This study was designed to gain understanding on how Salmonella enterica (STm) adapts to the plant phyllosphere, employing lettuce as a model crop. The specific objectives and accomplishments for this period are: Objective 1. Identify STm-derived effectors that induce stomatal opening. In previous years, we have completed a genetic screen of STm multi-gene deletion (MGD) mutants for the ability to open lettuce stomatal pores and selected 10 MGD mutants for further phenotypic analyses. Among the selected MGD mutant strains, two strains (Mut3 and Mut9) lacking the entire Salmonella pathogenicity island (SPI) 1 and 2, respectively, in addition to a few adjacent genes and operons were included. The identification of Mut3 and Mut9 led us to hypothesize that effector proteins delivered by the type-three secretion apparatuses encoded within these islands are required to overcome stomatal defenses. We have started evaluating single gene deletion (SGD) mutants in these regions and so far all mutants tested showed the same phenotype as the wild type bacterium. We will continue testing new SGD mutants to identify the gene responsible for the stomatal opening phenotype. In this report period, we also sequenced the genome of the selected 10 MGD mutants using a MiSeq platform and verified the missing functional units in these strains. In silico annotation revealed that the major metabolic functions of the deleted regions are associated with sensing the environment, bacterium movement, transport through the membrane, and biosynthesis of surface appendices. Previously, these functions have been found to be associated with epiphytic fitness of bacterial phytopathogens. Furthermore, Kroupitski et al. 2009 have observed that STm SL1344 aggregates near open stomata and uses chemotaxis and motility for internalization through lettuce stomata. Additionally, we found that darkness prevents STm SL1344's ability to re-open the stomatal pore and internalization into the leaves possibly due to the lack of chemoattractant leaching through closed stomata. These findings suggest that close proximity to the stomate may be required for Salmonella to induce opening of the pore. Therefore, STm invasion of the apoplast may be a consequence of a combined behavior of the bacterium on the phylloplane that can be modulated by plant-derived cues and, with this study, we have defined key genomic regions involved in this complex process. Objective 2. Identify genes required for STm internalization and endophytic persistence. The 10 selected MGD strains were also tested for their ability to survive in the leaf apoplast, overcome plant defenses, and obtain nutrients leached to the apoplast using a series of experimental approaches in molecular biology and microbiology. Although we were not able to pinpoint specific genes or operons that are responsible for these phenotypes, we successfully identified STm genomic regions that controls (directly or indirectly) bacterium internalization of and persistence in lettuce leaves. Interestingly, seven of these mutants showed increased population titers inside the leaf until 21 days post inoculation, two mutants showed similar titers as the wild type bacterium, whereas one mutant with a large deletion that includes the SPI-2 (Mut9) 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 described under Objective 1) 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 Mut3, was significantly impaired in the leaf apoplast wash fluid (AWF) suggesting that the STm persistence in the apoplast may be linked to nutrient acquisition capabilities or overallacterial fitness in this niche, which are dependent on the gene(s) deleted in the Mut9 strain. These results are reported in a manuscript under reviewed by "Applied and Environmental Microbiology" Journal. We will continue our search for specific genes within these regions by characterizing single gene deletion mutants already available in our laboratory. Objective 3. Identify lettuce guard cell genes regulated upon STm infection. We have not started this objective yet.

Publications

  • 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: 2018 Citation: Matiolli CC, Melotto M. 2018. A comprehensive Arabidopsis yeast two-hybrid library for protein-protein interaction studies: a resource to the plant biology research community. Mol. Plant-Microbe Interact. 31:899-902.
  • Type: Journal Articles 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: Journal Articles 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.


Progress 10/01/16 to 09/30/17

Outputs
Target Audience:During this report period, PI Melotto gave an oral presentation of this project to the following audiences: 1) UC-ANR Vegetable Crops Program Meeting held at the University of California, Davis. The audience included scientist,students, professionals from industry, farmer advisors. Attendance: ~30. 2) One Health for Food Safety Symposium organized by Nanjing Agricultural University, China. The audience included scientist, students, extension specialists, and academic professors from UC Davis and China. Attendance: ~100. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Completed training: - A graduate student, Jeanine Montano, involved in this project has completed a Masters degree in Plant Pathology, UC Davis and secured a job in the industry. - An undergraduate research assistant, Joseph Torreano, completed his Bachelors degree at UC Davis. He worked in this project for almost one year to acquire technical skills in microbiology. His training in this project helped him to get employed as a lab technician at Palo Alto Medical Foundation, CA. - In the summer of 2017, PI Melotto mentored and provided research internship to two high school students. One of them was a member of the "Summer Experience for the Economically Disadvantage" (SEED) program and the other was developing a competitive college application to major in Plant Science-related fields. Both gave an oral presentation on their research activities to a broad interested audience composed by students, faculty members, industry professionals, and members of the local community. Ongoing training: - A post-doctoral fellow, Dr. Paula Oblessuc, is being trained in this project with the objective to become a university professor. - Two PhD students (Cristian Jacob and Gabrielle Rossidivito) and a visiting scholar (Mariana Bisneta) are being trained in this project. How have the results been disseminated to communities of interest?PI Melotto and her graduate students have given invited seminars at scientific conferences discussing the various aspects of this research. Additionally, our team have published 2 peer-reviewed articles and one book chapter in highly reputable sources. We have also hosted and organized a one-day workshop for two student delegations from Nanjing University, China to foster collaborations and engage the next generation of scientists in the various aspects of food safety. High school students gave two seminars open to the interested public. What do you plan to do during the next reporting period to accomplish the goals?For the next period, we plan on completing objective 1 and continuing objective 2.

Impacts
What was accomplished under these goals? This study was designed to gain understanding on how Salmonella enterica (STm) adapts to the plant phyllosphere, employing lettuce as a model crop. The specific objectives and accomplishments for this period are: Objective 1. Identify STm-derived effectors that induce stomatal opening. We completed a genetic screen of STM multi-gene deletion (MGD) mutants for the ability to modulate stomatal aperture. A total of 337 MGD mutants were analyzed and we selected 10 MGD mutants for further analyses, including motility and persistence within the lettuce apoplast. In particular, a mutant lacking the entire Salmonella Pathogenicity Island 2 (SPI2) was not able to persist within the lettuce apoplast as well as the wild type. 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. Interestingly, upon investigation of single-gene deletion (SGD) mutants within SPI2, we observed that the ones lacking type three secretion system (T3SS) structural genes have increased apoplastic titers. These results indicates that non-structural genes on SPI2 are the major contributors to of STM apoplastic persistence. Objective 2. Identify genes required for STm internalization and endophytic persistence. Lettuce is highly sensitive to environmental conditions for growth. Due to its high phenotypic plasticity, it is crucial to define specific conditions to perform the experiment so that robust results can be obtained. During this period, we have established the optimal growth of lettuce and plant inoculation conditions under controlled environment chamber following BLS2 standards. Protocols are in place to access Stm internalization and endophytic persistence in lettuce leaves in the next grant period. Objective 3. Identify lettuce guard cell genes regulated upon STm infection. We have not started this objective yet.

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)
  • 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: 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: 2017 Citation: He SY, Melotto M, 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.


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

Outputs
Target Audience:During this report period, PI Melotto presented this project to the following audiences: 1) Symposium entitled "Decoding the exchange between human pathogens and plants: attachment, metabolism and recognition" organized by the International Association for Food Protection (IAFP) in St. Louis, MO. The audience included scientist, students, professionals from industry. Attendance: ~60. 2) Seminar series organized by the Department of Biological Sciences, California State University, Chico, CA. The audience included scientist, students, and academic professors. Attendance: ~50. 3) Plant Microbe Interaction Symposium organized by the Joint Genome Institute, US DOE, Walnut Creek, CA. The audience included scientist, students, and academic professors. Attendance: ~30. 4) One Health for Food Safety Symposium organized by Nanjing Agricultural University, China. The audience included scientist, students, and academic professors from UC Davies and China. Attendance: ~100. All this presentations were by invitation, which speak for the broad interest in new technologies and advances in scientific discoveries to mitigate leafy vegetables-related foodborne illnesses in the US and abroad. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Two PhD students and two undergraduate research assistants are being trained in this project. How have the results been disseminated to communities of interest?PI Melotto and the graduate students have given 3 invited seminars at scientific conferences discussing the various aspects of this research. We have also hosted and organized a one-day workshop for two student delegations from Nanjing University, China to foster collaborations and engage the next generation of scientists in the various aspects of food safety. What do you plan to do during the next reporting period to accomplish the goals?For the next period, we plan on continuing with objective 1 and start objective 2.

Impacts
What was accomplished under these goals? This study was designed to gain understanding on how Salmonella enterica (STm) adapts to the plant phyllosphere, employing lettuce as a model crop. 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. The specific objectives and accomplishments for this period are: Objective 1. Identify STm-derived effectors that induce stomatal opening. We have completed a genetic screening of a 200 multi-gene deletion (MGD) mutants in STm 14028s covering 3476 genes of STm to identify DNA regions containing genes that are critical for inducing stomatal opening. Ten mutant strains were selected for further characterization. In addition to not being able to open stomata, these selected strains posses differential ability to internalize and colonize the lettuce leaf or obtain nutrients from the leaf internal tissues. Objective 2. Identify genes required for STm internalization and endophytic persistence. We have not started this objective yet. Objective 3. Identify lettuce guard cell genes regulated upon STm infection. We have not started this objective yet.

Publications

  • Type: Conference Papers and Presentations Status: Published Year Published: 2016 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: 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. American Phytopathological Society Annual Meeting. Tampa, FL, USA.
  • 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.