Location: Plant Gene Expression Center
2023 Annual Report
Objectives
Objective 1: Determine the mechanisms of newly discovered transcriptional regulatory networks that control the expression of host pathogen resistance and defense genes in Solanaceae crops; develop new strategies for counteracting pathogen regulation of host target processes.
Sub-objective 1.A: Construct CRISPR/Cas9 gene-edited lines with null mutations in components of the DCL4/DCL2/RDR6 network, to determine the mechanism and role of the network in pathogen defense.
Sub-objective 1.B: Validate new plant lines using genetic and molecular genetic approaches.
Sub-objective 1.C: Determine the mechanisms of pathogen regulation of plant defense systems.
Sub-objective 1.D: Develop new strategies for counteracting pathogen regulation of host target processes.
Objective 2: Screen wild tomato germplasm and breeding materials for new sources of resistance to bacterial pathogens; characterize resistance traits and genetically map genes for immunity using appropriate tools and approaches that enable breeders to incorporate resistance into their breeding program.
Sub-objective 2.A: Screen wild tomato germplasm and breeding materials for new sources of resistance to bacterial pathogens.
Sub-objective 2.B: Characterize resistance traits in Solanaceous species.
Sub-objective 2.C: Genetically map genes for immunity using appropriate tools and approaches that enable breeders to incorporate resistance into their breeding program.
Objective 3: Utilize root and shoot phenotyping analyses to characterize a sorghum microbial isolate collection’s impact on plant phenotype and fitness under drought; utilize host-mediated microbiome engineering under drought to guide the development of assembled communities capable of improving crop performance under drought; and utilize genome-resolved metagenomics to identify microbial genes that are responsible for beneficial bacterial enrichment under drought stress and other environmental challenges.
Sub-objective 3.A: Utilize root and shoot phenotyping analyses to characterize a sorghum microbial isolate collection’s impact on plant phenotype and fitness under drought.
Sub-objective 3.B: Utilize host-mediated microbiome engineering under drought to guide the development of assembled communities capable of improving crop performance under drought.
Sub-objective 3.C: Utilize genome-resolved metagenomics to identify microbial genes that are responsible for beneficial bacterial enrichment under drought stress and other environmental challenges.
Approach
Crops are facing increasing pressure from both biotic and abiotic stresses in the environment, and improved information is needed regarding the molecular processes that govern these interactions. A combination of molecular, genetic, genomic, and bioinformatic approaches will be used to address this multifaceted problem. Particular emphasis will be placed on identifying signaling components, regulatory genes and transcriptional networks involved in controlling plant responses to biotic and abiotic signals, and the impact of the microbiome on plant genotypes and farming practices will be determined. On an ongoing basis, cutting-edge strategies and technologies such as next generation sequencing and computational modeling, will be assimilated and developed, networks involved in controlling plant responses to developmental, biotic and abiotic signals. The interaction of the microbiome on plant genotypes and farming practices will be determined. On an ongoing basis, productivity-enhancing strategies and technologies, such as next generating sequencing and computational modeling, will be assimilated and employed to enhance predictive agriculture. More specifically, we intend to apply three distinct but complimentary strategies to explore plant response to immunity and drought stress. First, we will investigate newly discovered transcriptional regulatory networks that control the expression of host pathogen resistance and defense genes in the Solanaceae family, which includes tomato, an important crop with wild relatives that host a wealth of disease resistance genes that can be used in breeding programs. The resulting data will be used to develop new strategies for counteracting pathogen regulation of host target processes. Second, we will screen tomato germplasm for new sources of resistance to pathogens. These newly identified resistance traits will be characterized to enable incorporation of improved protection into breeding programs. Third, we will use new phenotyping capabilities to explore the role of root-associated bacteria in promoting fitness under drought stress in the grain and forage crop Sorghum bicolor, an important forage and bioenergy crop that is often planted on marginal and drought-prone lands. A combination of host-mediated microbiome engineering and genome-resolved metagenomics will be used to select the best microbes and microbial genes for supporting sorghum’s response to drought stress and other environmental challenges. Collectively, the knowledge generated from these experiments can be used by breeders and growers, as well as other ARS, academic and industry partners, for manipulation of crop fitness.
Progress Report
This report documents progress for project 2030-12210-003-000D, Enhancing Crop Resilience to Biotic and Abiotic Stress Through Understanding the Immune and Microbiome Signaling Mechanisms, which started February 2023 and continues research from projects 2030-12210-002-000D, 2030-21000-052-000D, 2030-21000-050-000D, and 2030-22000-034-000D. The goal of this research is to generate a better understanding of the ways in which crops interact with their biotic environments.
As part of Objective 1, we began the process of constructing Clustered Regularly Interspaced Short Palindromic Repeats (CRISPR)/Cas9 gene-edited lines with null mutations in components of the targeted gene network, to determine the mechanism and role of the network in pathogen defense. We have also begun designing Agrobacterium vectors for generating transgenic overexpression lines and for transient expression assays. Collectively, we have performed a portion of work associated with our first three milestones, including the design of CRISPR/CAS9 gene editing constructs, genotyping of previously constructed lines, and isolation of RNA for analysis.
In support of Objective 2, we have screened a substantial portion of the 75 lines from our diversity panel that we had selected to screen.
In support of Objective 3, we have begun preparing and characterizing isolates for inoculation experiments and completed the binning and assembly of metagenomic datasets.
As this project was only officially approved in February of fiscal year (FY) 2023, we have not completed all milestones for the first year of the project, but substantial progress has been made on each.
Accomplishments
