Recipient Organization
WASHINGTON STATE UNIVERSITY
240 FRENCH ADMINISTRATION BLDG
PULLMAN,WA 99164-0001
Performing Department
(N/A)
Non Technical Summary
Thrips-transmitted tomato spotted wilt virus (TSWV) is one of the top 10 most economically important plant viruses in the world. The deployment of tomato cultivars with Sw-5 gene that confers broad-spectrum resistance to TSWV has been very effective for reducing the impact of TSWV. However, this management tactic is being compromised due the emergence of resistance breaking (RB) strains of TSWV in the US and globally. An integrative analyses of RB strain protein interactors in tomato genotypes with Sw-5 will be used to gain insights into the RB strains-host interactome. We propose to (1) mechanistically understand the biology of these interactions and the host factors that either positively or negatively regulate viral replication and pathogenesis; (2) identify host proteins that elicit the plant immune response or that confer resistance against viral infection; and (3) identify the plant proteins that could play a role in the viral infection and accumulation, as these proteins could be potential targets for engineering TSWV resistance. Our proposed work integrates molecular, genetic, proteomic and metabolomic approaches to systematically investigate the potential mechanisms and pathways responsible for TSWV infection, interactions with hosts and the underlying interactions between the host and RB strains. The outcomes of this research will illustrate the mechanism by which plants respond and defend themselves during viral infection and pathogenesis. Additionally, this research will help delineate the molecular events that occur during different stages of virus infection and provide insights into how TSWV RB strains potentially overcome the host resistance.
Animal Health Component
0%
Research Effort Categories
Basic
100%
Applied
0%
Developmental
0%
Goals / Objectives
Objective 1: Identification of host (tomato) interacting partners of TSWV proteinsObjective 2: Investigate the biological importance of the identified molecular host-virus interactions.Objective 3: Investigate tomato genes that are responsive to the RB strains of TSWV at the metabolome level
Project Methods
Objective 1: Identification of host (tomato) interacting partners of TSWV proteins.Task 1a. Identification of host interacting protein partners of TSWV-encoded proteins by yeast two-hybrid (Y2H) library screen. We will use Sw-5 tomato cDNA library to identify host interacting factors against NSm, N, and NSs of a TSWV RB strain. The tomato cultivar Defender (containing Sw-5) will be used to prepare a cDNA library for this purpose.Task 1b. Validation of the putative protein-protein interactions by targeted Y2H, Bimolecular fluorescence complementation (BiFC), and pull-down assays. Putative host interacting partners identified by the Y2H library screen from Task 1a will be validated by targeted Y2H assay. For targeted Y2H, ORFs encoding the selected host interacting partner(s) will be amplified and cloned. BiFC assay will be performed to facilitate the analysis of protein-protein interactions in vivo, and ORFs encoding the selected host interacting partner(s) will be amplified and cloned into pSITE vectors. For in vitro protein-protein interactions, pull-down assays will be performed using GST-, His- and/or MBP-tagged proteins will be expressed in and purified from E. coli.Objective 2: Investigate the biological importance of the identified molecular host-virus interactions.Task 2a. Analyze the roles of molecular interactions in viral pathogenesis in gene knockout lines. Silencing of the target gene(s) from Objective 1 will be carried out using modified Tobacco rattle virus (TRV) vectors. For this purpose, the target genes will be amplified from the tomato genome for making gene-specific silencing constructs. TSWV-induced symptom development and virus accumulation will be determined in agroinfiltrated tomato plants. Selected genes will be stably knocked out in tomato by CRISPR/Cas9 for functional analysis.Task 2b. Study the effects of target genes in viral pathogenesis in the over-expressing lines. Transgenic tomato plants (generated via stable transformation) overexpressing the target genes from Objective 1 will be generated by Agrobacterium-mediated transformation. The role of target protein(s) in viral pathogenesis will be further analyzed using the overexpression lines.Objective 3: Investigate tomato genes that are responsive to the RB strains of TSWV at the metabolome level. Task 3. Investigate the downstream pathways of molecular plant-virus interactions at metabolomics level. Transcriptional dynamics that occur during plant-virus interactions must be translated into proteins and other metabolites to affect viral pathogenesis. Several host factors influence viral pathogenesis at different stages of viral infection. As defense response signaling is mediated by different metabolites, we will determine the metabolomic outcomes of the host-TSWV interaction. Metabolomic analysis will be carried out using knockout lines and over-expressing lines (Tasks 2a&b) that were infected by the RB strain.