Location: Plant Gene Expression Center
Project Number: 2030-21000-050-000-D
Project Type: In-House Appropriated
Start Date: Mar 20, 2018
End Date: Feb 24, 2023
Objective:
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.
