Source: UNIVERSITY OF CALIFORNIA, BERKELEY submitted to NRP
DISSECTING EFFECTOR-INDEPENDENT IMMUNE RESPONSES TRIGGERED BY AND SUPPRESSED BY PSEUDOMONAS SYRINGAE IN WILD AND DOMESTICATED SOLANACEOUS SPECIES
Sponsoring Institution
National Institute of Food and Agriculture
Project Status
ACTIVE
Funding Source
AFRI COMPETITIVE GRANT
Reporting Frequency
Annual
Accession No.
1032833
Grant No.
2024-67013-43069
Cumulative Award Amt.
$958,480.00
Proposal No.
2024-06679
Multistate No.
(N/A)
Project Start Date
Aug 1, 2024
Project End Date
Jul 31, 2027
Grant Year
2024
Program Code
[A1171]- Plant Biotic Interactions
Recipient Organization
UNIVERSITY OF CALIFORNIA, BERKELEY
(N/A)
BERKELEY,CA 94720
Performing Department
(N/A)
Non Technical Summary
Plant pathogens cause serious worldwide effects on crop production and yield. Pseudomonas syringae is a bacterial pathogen that causes disease in many plants, including tomato. P. syringae injects effector proteins into the plant using a needle-like syringe. Effector proteins typically suppress plant immune pathways to promote disease, however they can be recognized in resistant plant hosts. Using a strain of P. syringae that lacks effectors, we discovered it causes a strong immune response in wild tomato, that is dependent on a functional needle-like syringe. This was a surprising result as the immune response is usually triggered by effector proteins and this strain lacks all effectors. In addition, we found that recognition is stronger and more prevalent in wild tomato compared to domesticated tomato, and that effectors can suppress this recognition. Understanding recognition could be very useful for engineering new sources of resistance in cultivars because the needle-like syringe system is found in many plant-infecting bacteria, potentially allowing for recognition of multiple bacterial species. Identifying causative genes for recognition of the needle-like syringe may provide additional tools for crop protection and food security.To better understand the recognition of this strain, we will test existing or create new P. syringae mutants and determine if the immune phenotype is retained or lost. We will temporarily express candidate bacterial genes in Solanaceous (tomato, tobacco, etc) species and see if they cause the immune response. We will test specific effector genes for suppression of the immune response, and characterize plant-bacterial interactions and host responses. We will test a wide range of tomato lines for their ability to recognize the effectorless strain of P. syringae. In addition, we will test genes of interest in tomato or tobacco species for their role in the immune response. We will continue to train diverse postdocs, graduate and undergraduate scientists, and collaborate with a HBCU. We will connect scientific training and agricultural outreach through interactions between scientists and growers, as well as the general public at PubScience events. This project will help identify new potential targets for crop protection, in order to reduce crop losses from disease and strengthen food security. This work will also help train, expand and diversify the scientific workforce, bridge connections between scientists and growers, and enhance science communication with the general public.
Animal Health Component
10%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
Classification

Knowledge Area (KA)Subject of Investigation (SOI)Field of Science (FOS)Percent
2121460110040%
2011460110030%
2011460106030%
Knowledge Area
212 - Pathogens and Nematodes Affecting Plants; 201 - Plant Genome, Genetics, and Genetic Mechanisms;

Subject Of Investigation
1460 - Tomato;

Field Of Science
1060 - Biology (whole systems); 1100 - Bacteriology;
Goals / Objectives
Pathogens cause substantial annual crop losses of ~$220B worldwide. Pseudomonas syringae is a bacterial pathogen that injects type III secreted effector (T3SE) proteins into the host. T3SEs have dual roles; they promote bacterial virulence by suppressing host immunity in susceptible hosts and they elicit host immunity in resistant hosts. We found that an effectorless strain (P. syringae D36E) lacking the 36 T3SEs found in P. syringae pv. tomato DC3000, elicits a strong hypersensitive response (HR) in the wild tomato relative Solanum pimpinellifolium and a weaker HR in tomato cultivars, and that the HR is dependent on a functional type III secretion system (T3SS). This HR phenotype is particularly interesting because the HR is typically associated with effector recognition, but P. syringae D36E does not carry any T3SEs. Our preliminary data suggest that a) plants evolved to recognize structural components of the T3SS, b) T3SEs in P. syringae suppress this recognition, c) differences in host recognition may be affected by the domestication of tomato.The major goals of this project are to dissect the recognition of structural components of the T3SS in wild tomato and its suppression by T3SEs, to train highly qualified scientists and to disseminate information to farmers and the general public. Understanding T3SS recognition could be very useful for engineering new sources of resistance in cultivars because the T3SS is highly conserved across many plant-infecting bacteria, potentially allowing for recognition of multiple bacterial species. Identifying causative genes for recognition of the T3SS may provide additional tools for crop protection.Our specific objectives and subobjectives are:1. Identify molecules from effectorless P. syringae D36E strain that trigger HR in wild tomato1.1 Test additional S. pimpinellifollium accessions with different P. syringae mutants1.2 Test for role of shared T3SS components that are secreted and translocated1.3 Test candidate genes through Agrobacterium-mediated transient expression or in protoplasts2. Identify effectors that suppress the P. syringae D36E-triggered HR2.1 Quantify contribution of effectors to HR suppression2.2 Identify effector(s) that suppresses the HR3. Identify tomato genes responsible for recognition of P. syringae D36E and characterize diversity of recognition in wild tomato3.1: Small-scale screen of tomato cultivars for recognition of P. syringae D36E3.2: Screen natural diversity panel for recognition of P. syringae D36E3.3: Validate candidate genes using Virus-Induced Gene Silencing (VIGS) or overexpression3.4: Determine conservation of P. syringae D36E recognition across wild tomato4. Broader impacts through diversifying the next generation of scientists, connecting scientists and farmers, and public outreach on science.4.1: Equitable and inclusive training of diverse undergraduate and graduate students4.2: Diversifying and expanding the STEM pipeline by partnering with Fisk University4.3: Outreach to facilitate connections with growers and the general public
Project Methods
To identify molecules from P. syringae that trigger HR in wild tomato, we will use standard unmarked allelic exchange to generate knockout mutants. P. syringae mutants will be evaluated for sensitivity to kanamycin, and by amplifying and sequencing the regions flanking the deletion. We will test P. syringae mutants for their ability to cause the HR phenotypically and using ion leakage assays. The HR-elicitor will be identified by finding mutants that fail to cause the HR, do not result in rapid ion leakage and can still deliver a control effector protein, AvrPto. As an alternative strategy, we will express candidate genes by Agrobacterium-mediated transient expression in plants or in protoplasts. We will evaluate plants for the HR and using ion leakage assays. We have optimized methods for transient expression in wild tomato species and shown that AvrPto triggers an HR in resistant backgrounds. We will confirm expression using Western blot analysis. These methods are standard in the lab. These efforts will allow us to identify strains that differ in their type III secretion components and to determine which bacterial genes are necessary for recognition. The HR experiments will be excellent experiential learning opportunities for undergraduate students. Graduate students and/or the postdoc will also gain expertise in these methods.To determine the contribution of effector proteins to HR suppression, we will test different polymutants and evaluate the HR using ion leakage assays. We will also carry out quantitative RT-PCR for HR markers, which has not been done in wild tomato. We expect that effectors suppressing the HR will have a similar strong effect in suppressing ion leakage and HR marker gene expression. To identify the effector that suppresses the P. syringae D36E-triggered HR, we will generate unmarked allelic exchange mutants as previously mentioned. We will also express candidate effectors in a P. syringae expression vector to determine if they can complement HR suppression. If multiple effectors are needed, we can infect plants with multiple strains of P. syringae D36E, with each carrying one T3SE, or by expressing several T3SE genes on one plasmid. These efforts will allow us to validate HR marker genes in S. pimpinellifolium, to characterize the level of HR suppression, identify effectors that are necessary for HR suppression and determine their prevalence in different strains of P. syringae. The HR experiments will be excellent experiential learning opportunities for undergraduate students. Graduate students and/or the postdoc will also gain expertise in these methods.To identify host proteins that are responsible for recognition, we will test tomato cultivars and a natural diversity panel for the HR. We will carry out GWAS to identify single nucleotide polymorphisms that are associated with the HR. We will evaluate candidate genes using Virus-Induced Gene Silencing or overexpression in tomato and/or Nicotiana species. We will also evaluate whether recognition of P. syringae D36E is conserved across wild tomato. These efforts will identify tomato cultivars and/or wild species that recognize P. syringae D36E, identify SNPs that are associated with recognition, identify candidate genes associated with recognition, and determine if the strength of response correlates with tomato domestication. The HR experiments will be excellent experiential learning opportunities for undergraduate students. Graduate students and/or the postdoc will also gain expertise in these methods.We will continue to recruit diverse undergraduate students and teach them to do high-impact biological research. We will host an undergraduate student from Fisk University each summer and engage them in significant research experiences. The Fisk student will complete assessments with other summer students to evaluate their experience anonymously. We will also collaborate with Dr. Damo at Fisk University for computational modeling of bacterial proteins and will give a virtual presentation in their seminar series each year. We will carry out an outreach program that will connect farmers and student scientists through farm visits. We will carry out pre- and post-surveys to evaluate the effectiveness of the outreach. We will post growers' questions and answers from the literature to public websites. We will also help facilitate talks to the general public on science through PubScience. These efforts will diversity the next generation of scientists, develop stronger connections to HBCUs, connect scientists and growers, disseminate information through public websites, expand public awareness of scientific topics, and train undergraduate and graduate students and the postdoc.Taken together, understanding T3SS recognition could be very useful for engineering new sources of resistance in cultivars because the T3SS is highly conserved across many plant-infecting bacteria, potentially allowing for recognition of multiple bacterial species. Our data also suggest that recognition of the T3SS is more common in wild tomato relatives and was lost or weakened as domestication occurred. This suggests that plant pathogens evolved into better pathogens as tomato was domesticated, which could be due to basal defense-related genes being bred out of the wild germplasm or less genetic diversity in cultivars. These changes during domestication likely contribute to some of the devastating diseases we see today. Identifying causative genes for recognition of the T3SS may provide additional tools for crop protection.

Progress 08/01/24 to 07/31/25

Outputs
Target Audience: Scientific community. The scientific community was reached through seminars, development of new methods, and the lab website. Postdoc. A postdoc was hired for this project. She is being trained in techniques used in the field of plant-microbe interactions, and is developing oral and written communication skills. Graduate students. One graduate student is working on this project. This individual is being trained in scientific techniques, oral and written communication. Undergraduate students at UC Berkeley. Individuals are recruited to the lab through two main undergraduate research programs. They receive training in scientific techniques, oral and written communication. 8 undergraduate students were trained through this project. Growers. Farmers and UC Berkeley scientists interacted through a farm visit, where participants exchanged information and learned more about each other's work. We posted growers' questions and answers from the literature on the lab website. General public. Student scientists presented scientific topics of interest at PubScience, which reaches the general public at local breweries. This outreach event has been very well received and covers a wide range of scientific topics, with opportunities for open discussion. Changes/Problems: Nothing Reported What opportunities for training and professional development has the project provided?Through this project, one postdoc, one graduate student, and 8 undergraduate students have been trained in plant-microbe interactions, plant biology and microbiology. Three lab members participated in the Bay Area Plant Hub meeting to expand their professional networks. One graduate student served as an ASPB (American Society of Plant Biologists) Ambassador at the meeting. One undergraduate student presented her research at an undergraduate poster session. Three lab members gave seminars in the PGEC seminar series. One graduate student presented their work in the Genetics Training Grant symposium. Three lab members interacted with farmers through our farm visit. How have the results been disseminated to communities of interest?The PI gave a talk on this research in the UC Davis plant pathology seminar series. Three lab members gave talks in the PGEC seminar series and/or the Genetics Training Grant symposium. One undergraduate student presented her research in an undergraduate poster session. PubScience ran five events on diverse scientific topics, including plant diseases. Lab members discussed their research with farmers during a farm visit. One lab member served as an ASPB Ambassador. The Lewis Lab website was regularly updated with news and resources, as well as questions & answers from the farm visit. What do you plan to do during the next reporting period to accomplish the goals?For objective 1, we will quantify the HR induced by EtHAn and P. syringae D36E using conductivity assays. We will continue generating single mutants of the remaining harpin genes as well as higher order mutants lacking all harpins. We will test for protein expression of the 10 candidate bacterial genes that were transiently expressed in SP. We will expand our candidate list of bacterial genes for testing in SP using Agrobacterium-mediated transient expression assays. For objective 2, we will carry out qRT-PCR on the different polymutant strains to quantify the level of HR suppression in these different strains. We will also quantify the level of HR suppression using conductivity. We will begin knocking out effectors that contribute to HR suppression. For objective 3, we will finish screening additional lines in the natural diversity panel of tomato lines. We will continue analyzing candidates from the GWAS analysis using different sequence and protein databases, and compare the candidates between full sequenced accessions with or without an HR. We will conduct synteny analysis of the regions of interest between SLL and SP. We will begin cloning the strongest candidates. For objective 4, we will continue training undergraduate students in scientific research. We will continue reaching the public through PubScience and farmers/growers through our farm visits. We will recruit a Fisk student for summer research, and embed them in a different undergraduate summer research program if needed.

Impacts
What was accomplished under these goals? Our first objective was to identify molecules from effectorless P. syringae D36E strain that trigger HR in wild tomato. We tested several Solanum pimpinellifolium (SP) accessions for their ability to recognize EtHAn and P. syringae D36E and found that both strains caused a similar hypersensitive response (HR) phenotype. This result supports our conclusion that the recognized molecule is shared between the two strains. To determine whether a pathogen-associated molecular pattern (PAMP) is recognized, we carried out HR assays with heat-killed bacteria grown in rich media or hrp-inducing media. The type III secretion system (T3SS) is induced in hrp-inducing media but is not induced in rich media. We found that heat-killed bacteria grown in either media did not induce the HR, suggesting that a PAMP is not recognized. It is possible that the P. syringae D36E secretes an active protein that produces damage-associated molecular patterns, or that P. syringae D36E translocates a molecule into the plant that is recognized. To determine whether translocation is necessary for the HR, we have been generating knockout mutants in harpin genes, which are needed for translocation. Single mutants of hopAK1, hrpZ1 and hopP1 still showed an HR, as well as double mutants of hopP1 and hopAK1, or hopP1 and hrpZ1. However, a double hopAK1 hrpZ1 mutant did not cause an HR and was impaired in translocation, as the hopAK1 hrpZ1 double mutant carrying the recognized effector protein AvrPto, did not cause an HR in SP. These results support that translocation of an unknown molecule is necessary for the P. syringae D36E HR. Lastly, we have been testing 10 candidate proteins for their ability to induce the HR when transiently expressed in SP by Agrobacterium tumefaciens. Although the positive control, AvrPto, induced a strong HR in all experiments, none of ten tested candidates caused an HR. These candidates included five harpin or harpin-like proteins and five T3SS proteins that are translocated to the plant. We still need to validate that these proteins are expressed by Western blotting. Our second objective was to identify effectors that suppress the P. syringae D36E-triggered HR in SP. We have carried out preliminary qRT-PCR experiments to examine induction of two HR marker genes, hin1 and hsr203J, compared to a ubiquitin housekeeping gene, and found that both are induced during the HR with P. syringae D36E or EtHAn but not P. syringae D36EΔhrcC. This is consistent with our expectation that a functional type III secretion system is necessary for immune induction. Our third objective was to identify tomato genes responsible for recognition of P. syringae D36E and characterize diversity of recognition in wild tomato. We tested ~150 accessions of S. lycopersicum var. lycopersicum (SLL), S. lycopersicum var. cerasiforme (SLC) and SP for their ability to recognize P. syringae D36E. For 115 of these, we tested 20-30 cotyledons per line and observed reproducible phenotypes in multiple experiments. For the remaining lines, the number of tested cotyledons is too low or we have observed inconsistent phenotypes in different experiments. We will continue to test additional accessions to increase the number of cotyledons, test a new seed source for lines with inconsistent phenotypes, and test more divergent wild tomato species. We found that the majority of SLL lines lacked recognition of P. syringae D36E, and most SLC lines had no or weak recognition. Most SP accessions were able to recognize P. syringae D36E. We carried out GWAS using binary or continuous phenotypes, and identified the strongest signal on four different chromosomes. We have identified genes of interest on one chromosome with the strongest signal and have begun comparing the sequences in accessions with or without an HR. Our fourth objective was to train the next generation of scientists, connect scientists and farmers and communicate with the public about science. We trained 8 UC Berkeley undergraduate students through various research programs. Students were involved in genetic screens to identify accessions resistant to P. syringae. We worked with Dr. Steven Damo and Dr. Jose Vasquez-Medina to coordinate a summer research experience with a student at Fisk University. Unfortunately, the NSF REU program funding was not awarded until May 30, 2025 and the prospective Fisk student had already made other plans for the summer. Since the NSF REU program has now been funded, we will be able to recruit the Fisk students for subsequent years and embed them in this program. We carried out a farm visit to Live Earth Farm as part of the "Fresh from the Farm: A conversation with growers" program. The visit involved 6 scientists from undergraduate students, graduate students, technicians, postdocs and staff researchers. In pre-assessment surveys, respondents ranked their knowledge of farming as low (average score 2/5, where 1 is little knowledge and 5 is highly knowledgeable). They expressed interest in learning about the challenges faced by farmers. In post-assessment surveys, respondents ranked their knowledge of farming much higher (average 4.2/5). They enjoyed learning about techniques employed by the farm, including the use of compost tea, grafting and dry farming. We posted questions from the growers and our responses to the lab website, where they are publicly accessible. In addition, we were involved in the PubScience program, where student scientists talk about their research to the public. Five PubScience events occurred in this reporting period.

Publications