Location: Plant Gene Expression Center
2020 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 2020.
The first objective involves identifying 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 involves characterizing and mapping 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 to determine the extent of resistance compared to a susceptible cultivar. It demonstrated high levels of resistance to bacteria in both seedling and adult plants. For one wild species, a previously generated mapping population was obtained and 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 involves introducing prioritized resistance genes into tomato and characterizing 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 and will be used for transformation once the facility reopens.
Accomplishments
1. Natural diversity in immune receptors affects recognition of bacteria in plants. Immune receptors are very effective in controlling disease by plant pathogens. Plant populations show genetic diversity due to natural selection from their environment and the pathogens that infect them, among other factors. ARS researchers in Albany, California, in collaboration with researchers at the University of California Berkeley, Cornell University, and the Romanian Institute of Biochemistry, identified natural diversity in two immune receptors that affects their ability to recognize a protein from the pathogenic bacterium Pseudomonas syringae. This work will help inform the design of immune receptors for broader recognition of plant pathogens. This information will be used by scientists to improve food security, which will ultimately benefit stakeholders, growers and consumers.
2. Gene editing of plant-infecting bacteria. Many bacteria, such as Pseudomonas (P.) syringae, cause disease in crop plants by injecting a suite of effector proteins. However, it is difficult to show how each effector protein contributes to bacterial virulence because effectors can have overlapping functions. ARS researchers in Albany, California, in collaboration with researchers at the University of California, Berkeley, and the University of California, San Francisco, established a gene editing technique in P. syringae to remove entire clusters of effector genes and showed that bacterial growth was affected. This work will help identify the function of effector proteins and reduce disease. This information will be used by scientists to improve food security, which will ultimately benefit stakeholders, growers and consumers.
3. Plant protein is targeted by multiple bacterial proteins for disease. The pathogenic bacterium Pseudomonas syringae injects effector proteins into plants, where they manipulate host proteins and promote disease. Identification of these host proteins could allow the development of techniques to block pathogens from modifying host proteins and therefore reduce disease. ARS researchers in Albany, California, in collaboration with researchers at the University of California, Berkeley, found a plant protein that is targeted by multiple effector proteins to promote host susceptibility to infection. This work will help identify plant proteins that are important for broad-spectrum disease resistance. This information will be used by scientists to improve food security, which will ultimately benefit stakeholders, growers and consumers.
Review Publications
Baudin, M., Schrieber, K., Martin, E.C., Petrescu, A.J., Lewis, J.D. 2019. Structure-function analysis of ZAR1 immune receptor reveals key molecular interactions for activity. Plant Journal. 101(2):352-370. https://doi.org/10.1111/tpj.14547.
Chen, Y., Bendix, C., Lewis, J.D. 2020. Comparative genomics screen identifies microbe-associated molecular patterns from Candidatus Liberibacter spp. that elicit immune responses in plants. Molecular Plant-Microbe Interactions. 33(3:539-552. https://doi.org/10.1094/MPMI-11-19-0309-R.
Hassan, J.A., Chau-Ly, I.J., Lewis, J.D. 2020. High-throughput identification of resistance to Pseudomonas syringae pv. tomato in tomato using seedling flood assay. Journal of Visualized Experiments. 157.Article e60805. https://doi.org/10.3791/60805.
