Location: Plant Gene Expression Center
2019 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 FY19.
The first objective was to identify new sources of resistance to bacterial pathogens in tomato. A high-throughput plate-based assay was used to screen 50 additional 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. These lines are being grown to test for resistance in the next generation. Lines that exhibit heritable resistance will be 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 resistance trait and will allow us to prioritize specific lines for further analysis. Resistant lines will be tested using bacterial growth assays to determine the extent of resistance compared to a susceptible cultivar. For one wild species, a previously generated mapping population was obtained, and is being tested to determine which chromosomal regions are associated with resistance. 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. Tomato cultivars were tested for resistance to bacteria carrying HopZ1a, and several were identified that are susceptible to bacteria carrying HopZ1a. ZED1, a pseudokinase that works with the resistance protein ZAR1 for recognition of HopZ1a, was introduced into appropriate vectors for plant transformation.
Accomplishments
1. Interactions regulate activity of immune receptors. Plants can be protected from plant pathogens using immune receptors. However, immune receptors must be carefully regulated so that they do not induce immunity in the absence of pathogens. ARS researchers in Albany, California, in collaboration with researchers at the University of California Berkeley and the Romanian Institute of Biochemistry, have identified key molecular interactions that regulate the activity of immune receptors. This work will help inform the design of immune receptors for broader recognition of plant pathogens. The information will be used by scientists to improve food security, which will benefit stakeholders, growers and consumers.
2. Small molecules induce defense responses in plants. Citrus greening, or Huanglongbing (HLB), has caused devastating losses to the U.S. citrus industry. No resistant varieties of citrus are available and control measures are highly limited. ARS researchers in Albany, California, in collaboration with researchers at the University of California Berkeley, have identified small molecules associated with protein-coding genes that trigger immune responses in multiple plant species, including citrus species. By boosting immunity in citrus, these molecules could help prevent or reduce HLB infection. This work will help scientists develop resistant citrus plants, which will benefit stakeholders, growers and consumers.
Review Publications
Hassan, J.A., De La Torre-Roche, R., White, J., Lewis, J.D. 2018. Soil mixture composition alters Arabidopsis susceptibility to Pseudomonas syringae infection. Plant Direct. 2:1-13. https://doi.org/10.1002/pld3.44.
Schreiber, K.J., Lewis, J.D. 2019. Protein acetylation in pathogen virulence and host defense: In vitro detection of protein acetylation by radiolabeled acetyl coenzyme A. Methods in Molecular Biology. 1991:23-32. https://doi.org/10.1007/978-1-4939-9458-8_3.
