Location: Sunflower Improvement Research
Project Number: 3060-21220-034-014-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2021
End Date: Dec 31, 2024
Objective:
1. To validate the temporal expression of soybean phenylpropanoid pathway genes following Sclerotinia infection; 2. To silence the phenylpropanoid pathway using Virus Induced Gene Silencing (VIGS) to check for resistance/susceptibility to Sclerotinia stem rot (SSR) disease; 3. To overexpress target genes in Nicotiana benthamiana or Arabidopsis thaliana to check for resistance/susceptibility to SSR; 4. To bioengineer soybean plants for resistance to SSR; and 5. To conduct an estimation of phenylpropanoid intermediates in bioengineered plants.
Approach:
For Objective 1, the temporal expression of soybean genes following infection with S. Sclerotiorum will be estimated using quantitative PCR. For Objective 2, to characterize the function of target soybean genes, we will use virus-induced gene silencing (VIGS) using Bean pod mottle virus (BPMV) with the ‘Traff’ variety of soybean. Soybean leaves will be bombarded with gold particles labelled with constructs using a gene gun, and their expression will be checked with qRT-PCR. For Objective 3, we will use the overexpression of target phenylpropanoid pathway genes in the model plant Arabidopsis thaliana or Nicotiana benthamiana to characterize gene function. A Sclerotinia disease assay will also be performed to check for resistance/susceptibility in each transgenic line. For Objective 4, we will engineer soybean lines with enhanced resistance to Sclerotinia stem rot (SSR) in two different ways: (1) for the subset of genes that confer enhanced SSR resistance upon VIGS silencing, we will use targeted mutagenesis with CRISPR/Cas9 to knockout and/or knockdown the expression of certain genes. The edited plants will be compared to control plants that either (a) were derived from the same family, but segregated to wild-type or (b) represent background genotype without having experienced the CRISPR/Cas9 transformation; or (2) for genes that confer reduced SSR resistance upon VIGS silencing, we will use overexpression with an appropriate promoter to drive increased expression of the target gene product. Transgene-positive plants will be compared with control segregating plants that no longer carry the transgene to assess the effect of the overexpression on resistance to SSR. For Objective 5, we will harvest stems of four week old, bioengineered plants of both infected and uninfected control lines and send the frozen sample to West Coast Metabolomics (UC, Davis) facility for metabolite estimation using gas chromatography mass spectrometry.
