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
0%
Research Effort Categories
Basic
90%
Applied
10%
Developmental
0%
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.