Location: Plant Gene Expression Center
2021 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) 2021.
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. These lines were grown up to test for resistance in the next generation. Several 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 resistance trait and will allow us to prioritize specific lines for further analysis. One resistant line was tested using bacterial growth assays and additional methods 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. For one wild species, a previously generated mapping population was 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. ZED1, a pseudokinase that works with the resistance protein ZAR1 for recognition of HopZ1a, was transformed into a tomato cultivar. Although the lines contained the gene of interest, protein expression could not be detected. ZED1 was introduced into a different vector for plant transformation. The transformations have resumed and the lines will be screened for homozygosity in FY 2022.
Accomplishments
1. Genetic markers associated with resistance to Pseudomonas syringae. Plant disease causes substantial crop losses under appropriate environmental conditions. Genetic sources of resistance are powerful tools to prevent disease. ARS researchers in Albany, California, in collaboration with researchers at the University of California, Berkeley, identified a wild tomato accession with resistance to the pathogenic bacterium Pseudomonas syringae. This work identifies genetic tools to protect plants from infection and improve food security. This work will benefit plant breeders, stakeholders, growers and consumers.
2. Gene makes plants more susceptible to infection by Pseudomonas syringae. Bacteria, such as Pseudomonas syringae, cause disease in plants by injecting a suite of effector proteins into their hosts. Effector proteins target pathways or processes in the plant to promote infection. ARS researchers in Albany, California, in collaboration with researchers at the University of California, Berkeley, identified a protein that is targeted by effector proteins to promote bacterial virulence. This work identifies a gene that is important for broad spectrum resistance against pathogens. This work can be used to improve food security, which will benefit stakeholders, growers and consumers.
Review Publications
Baudin, M., Martin, E., Sass, C., Hassan, J.A., Bendix, C.L., Sauceda, R., Diplock, N., Specht, C.D., Petrescu, A., Lewis, J.D. 2020. A natural diversity screen in Arabidopsis thaliana reveals determinants for HopZ1a recognition in the ZAR1-ZED1 immune complex. Plant Cell and Environment. 44(2):629-644. https://doi.org/10.1111/pce.13927.
Csorgo, B., Leon, L., Chau-Ly, I., Vasquez-Rifo, A., Berry, J., Mahendra, C., Crawford, E., Lewis, J.D., Bondy-Denomy, J. 2020. A compact Cascade-Cas3 system for targeted genome engineering. Nature Methods. 17:1183-1190. https://doi.org/10.1038/s41592-020-00980-w.
Schreiber, K.J., Hassan, J.A., Lewis, J.D. 2020. Arabidopsis Abscisic Acid Repressor 1 (ABR1) is a susceptibility hub that interacts with multiple Pseudomonas syringae effectors. Plant Journal. 105(5):1274-1292. https://doi.org/10.1111/tpj.15110.
Schreiber, K.J., Lewis, J.D. 2020. Identification of a putative DNA-binding protein in Arabidopsis that acts as a susceptibility hub and interacts with multiple Pseudomonas syringae effectors. Molecular Plant-Microbe Interactions. 34(4). https://doi.org/10.1094/MPMI-10-20-0291-R.
Schreiber, K.J., Chau-Ly, I., Lewis, J.D. 2021. What the wild things do: Mechanisms of plant host manipulation by bacterial type III-secreted effector proteins. Microorganisms. 9(5). Article 1029. https://doi.org/10.3390/microorganisms9051029.
