Location: Plant Gene Expression Center
2023 Annual Report
Objectives
The long-term goal of this research is to identify and characterize new sources of plant resistance, in order to protect plants from disease. The specific objectives of this project plan are:
Objective 1: Using a high-throughput plate-based assay on wild tomato species and accessions, identify new sources of resistance to bacterial pathogens in tomato.
• Subobjective 1A: Screen wild tomato accessions for resistance.
• Subobjective 1B: Test for heritability of resistance and incidence of resistance.
Objective 2: Characterize and map unique resistance genes in tomato; transfer trait and marker information to breeders.
• Subobjective 2A: Characterize resistance responses in candidate accessions.
• Subobjective 2B: Begin mapping resistance in candidate accessions.
Objective 3: Introduce prioritized resistance genes into tomato, and characterize resistance responses.
• Subobjective 3A: Introduce candidate genes into cultivated tomato.
• Subobjective 3B: Characterize defense responses induced by candidate genes.
Approach
Objective 1, Subobjective 1A:
Hypothesis: Wild tomato accessions will exhibit differential recognition of P. syringae pv. tomato (Pst T1), a race 1 strain.
Experimental Design: We will use a plate-based flooding assay to screen wild tomato accessions for resistance to Pst T1.
Contingencies: We have already optimized the system and there are extensive genetic resources that can be tested.
Objective 1, Subobjective 1B:
Hypothesis: Environmental and genetic factors will influence the resistance phenotype.
Experimental Design: We will test the progeny of candidate resistant lines for the heritability of resistance and the incidence of resistance. We will prioritize lines with heritable resistance that is observed in the majority of the population.
Contingencies: We do not anticipate any issues as we have already established the assay.
Objective 2, Subobjective 2A:
Hypothesis: Resistance may be due to classical monogenic Resistance (R) genes or quantitative disease resistance (QDR).
Experimental Design: We will characterize resistance responses, including hypersensitive response (HR), ion leakage and bacterial growth, in candidate resistant lines at both the seedling and adult stages.
Contingencies: It may be difficult to select an appropriate negative control for the ion leakage assays, however we think it is worthwhile to test this as a quantitative measure of the HR.
Objective 2, Subobjective 2B:
Hypothesis: Outcrossing candidate accessions to a sequenced cultivar will introduce sufficient diversity to map the causative loci.
Experimental Design: We will outcross the candidate wild accession(s) to Heinz 1706, screen the F2 population for resistance, and map single nucleotide polymorphisms associated with resistance.
Contingencies: Ren-Seq is a next-generation mapping approach that is designed to specifically amplify nucleotide binding site leucine rich repeat (NBS-LRR)-like genes, and is another option, should we run into difficulties.
Objective 3, Subobjective 3A:
Hypothesis: Tomato cultivars are missing functional ZAR1 and/or ZED1 genes.
Experimental Design: Transform tomato cultivar with constructs encoding ZAR1 and/or ZED1.
Contingencies: It may be necessary to introduce both genes at the same time in a single vector into tomato.
Objective 3, Subobjective 3B:
Hypothesis: Tomato carrying ZAR1 and ZED1 will confer enhanced recognition of pathogens.
Experimental Design: We will test transgenic ZAR1 and/or ZED1 lines with P. syringae carrying HopZ1a.
Contingencies: If the cultivar carries the Pto/Prf locus, we can also use a PstDC3000 strain that lacks AvrPto and AvrPtoB, and introduce HopZ1a into this strain.
Progress Report
This is the final report for project 2030-21000-050-000D, Mining Collections of Wild Germplasm and Novel Defense Regulators for Enhanced Plant Defenses, which has been replaced by new project 2030-12210-003-000D, Enhancing Crop Resilience to Biotic and Abiotic Stress Through Understanding the Microbiome and Immune Signaling Mechanisms. For additional information, see the new project report.
Objective 1 was to identify new sources of resistance to bacterial pathogens in tomato. A high-throughput plate-based assay was used to continue screening wild tomato species for resistance to Pseudomonas syringae. Wild tomato species are important reservoirs of genetic diversity and their genetic composition reflects adaptation to various environments, habitats and pathogens. Pathogen pressure on hosts leads to natural diversity in genes regulating the innate immune response. Through our screen, several wild species were identified with resistance. The progeny of these lines exhibited heritable resistance. Lines with a high frequency of resistance were the focus of Objective 2.
Objective 2 was to characterize and map unique resistance genes in tomato. Characterization of resistance will determine the genetics of the resistance trait, and will allow us to prioritize specific lines for further analysis. Several resistant lines were tested using bacterial growth assays and resistance assays to determine the extent of resistance compared to a susceptible cultivar. They demonstrated high levels of resistance to the bacteria in both seedling and adult plants. An inbred backcross population generated from a resistant wild tomato line and a susceptible cultivar was tested for resistance. Five quantitative trait loci on four chromosomes were identified by screening lines in the seedling flood assay. Next-generation sequencing was carried out on two pools of individuals from a cross between a resistant wild accession and a susceptible cultivar, that showed high resistance or high susceptibility to infection. Computational analysis identified a small number of candidate genes associated with resistance. Identification of genomic regions associated with resistance will provide tools for plant breeders to introduce resistance into cultivars.
Objective 3 was to introduce prioritized resistance genes into tomato, and to characterize resistance responses. ZAR1 is an ancient resistance gene that is found in a broad array of plant species, and is important for the recognition of multiple bacterial proteins, including HopZ1a. ZED1, a pseudokinase that works with the resistance protein ZAR1 for recognition of HopZ1a, was transformed into a tomato cultivar. Ten new transgenic lines were generated for ZED1. While the plants produced fruit, they unexpectedly did not produce seed and thus further experiments could not be carried out.
Accomplishments
1. Immune receptor interacts with multiple kinases. Immune receptors are highly effective in protecting plants from disease. The immune receptor HopZ Activated Resistance 1 (ZAR1) is found in most flowering plants and can recognize a wide variety of effectors from different pathogens through plant kinases. ARS researchers in Albany, California, in collaboration with researchers at the University of California, Berkeley, identified multiple kinases that can interact with ZAR1, suggesting that these kinases may recognize pathogen proteins. Using computational approaches, the interaction of one kinase with ZAR1 was rationally designed and strengthened. This work identifies new genes that can contribute to plant immunity and protect crops from infection, benefitting stakeholders, growers and consumers.
2. Genetic markers are associated with resistance to Pseudomonas syringae. Plant diseases cause serious crop losses under permissive environmental conditions. Genetic resistance is highly effective in protecting plants from infection. ARS researchers in Albany, California, in collaboration with researchers at the University of California, Berkeley, identified a wild tomato accession with good resistance to the bacterial pathogen Pseudomonas syringae. Using different genetic mapping approaches, five genetic regions were identified as being associated with resistance. This work provides important molecular markers for genetic resistance, which will reduce plant disease and benefit plant breeders, stakeholders, growers and consumers.
