Location: Plant Gene Expression Center
2022 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
Progress was made on all three objectives in fiscal year (FY) 2022.
The first objective 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 are the focus of Objective 2.
The second objective was to characterize and map unique resistance genes in tomato. Characterization of resistance will determine the genetics of the trait and allow us to prioritize specific lines for further analysis. One resistant line was tested using bacterial growth assays and resistance assays to determine the extent of resistance compared to a susceptible cultivar. It demonstrated high levels of resistance to the bacteria in both seedling and adult plants. Next-generation sequencing was carried out on two pools of individuals showing high resistance or high susceptibility. Computational analysis is ongoing. Identification of genomic regions associated with resistance will provide tools for plant breeders to introduce resistance into cultivars.
The third objective 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. New transgenic lines have been generated for ZED1. The plants are beginning to produce fruit and will be screened for homozygosity in FY 2023.
Accomplishments
1. Bacterial genes promote disease in Brassica crops. Pathogens cause substantial crop losses in susceptible hosts under the right environmental conditions. Many Brassica plants, such as cauliflower and broccoli, have little genetic resistance to the bacterial pathogen Xanthomonas campestris. ARS researchers in Albany, California, in collaboration with researchers at the University of California Berkeley, the French research agency, National Institute for Agricultural Research (INRA), and Lawrence Berkeley National Labs, carried out a barcoded sequencing-based assay to identify bacterial genes that were important for causing disease in cauliflower. Many of these genes had not been previously identified. This work identified bacterial genes to target for reducing disease in crops. This work contributes to a more resilient food production system that benefits stakeholders, growers, and consumers.
Review Publications
Lewis, J.D., Knoblauch, M., Turgeon, R. 2022. The phloem as an arena for plant pathogens. Annual Review of Phytopathology. 60:77–96. https://doi.org/10.1146/annurev-phyto-020620-100946.
Luneau, J.S., Baudin, M., Monnens, T., Carrere, S., Bouchez, O., Jardinaud, M., Gris, C., Francois, J., Ray, J., Torralba, B., Arlat, M., Lewis, J.D., Lauber, E., Deutschbauer, A.M., Noel, L.D., Boulanger, A. 2022. Genome-wide identification of fitness determinants in the Xanthomonas campestris bacterial pathogen during early stages of plant infection. New Phytologist. https://doi.org/10.1111/nph.18313.
