Location: Sunflower Improvement Research
Project Number: 3060-21220-034-022-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2022
End Date: Dec 31, 2024
Objective:
1. Characterize key virulence genes used by Sclerotinia sclerotiorum during distinct phases of the soybean infection; 2. Evaluate key virulence genes for their value as host-induced gene silencing (HIGS) targets and extend RNAi strategies to plant components involved in resistance; and 3. Develop transgenic soybean plants with silencing constructs targeting the host and pathogen genes most important in Sclerotinia stem rot development.
Approach:
For Objective 1, a CRISPR-based gene knockout system will be used to confirm the role of various fungal virulence genes in the development of Sclerotinia stem rot (SSR) infection. Two genes will be targeted: a secreted laccase (Sslac2; Sscle03g023030) and an alcohol oxidase (SsAOX; Sscle03g024060). For Objective 2, to characterize the utility of the genes identified in Objective 1 as targets for HIGS, we will first use virus induced gene silencing (VIGS) through the comovirus Bean pod mottle virus (BPMV). This system uses BPMV as vehicle to recruit the plant silencing machinery and thus produce small RNAs (sRNAs) targeting the above fungal genes. The downregulation of these genes will be confirmed using reverse transcriptase quantitative PCR (RT-qPCR). To maximize SSR resistance, targeted genes which demonstrate the greatest reduction in disease will be combined on a single vector to silence multiple virulence genes concurrently. For Objective 3, RNAi binary vectors will be constructed for each target gene and introduced into soybean using agrobacterium transformation standard protocols. Transgenic plants will be challenged with S. sclerotiorum via petiole inoculations under controlled conditions, and lesion size, fungal biomass, and mRNA levels of targets will be evaluated. Upon successful confirmation of resistance in the greenhouse and requisite seed increases, we will move forward with field trials to test for SSR resistance.
