1a. Objectives (from AD-416)
To functionally characterize soybean response to pathogens, with focus on Sclerotinia sclerotiorum. Candidate defense-related genes will be identified from soybean response to the pathogen or products of the pathogen. Functional characterization will include repression and/or enhancement of expression of the candidate genes in soybean and/or Arabidopsis.
1b. Approach (from AD-416)
The ARS scientist will identify candidate defense genes, whereas the collaborator's lab will assist with construct design for over and under expression of genes in host plants and will generate transgenic soybean material. The ARS scientist will assist in propagation of the soybean transgenics and will conduct disease assays on this material to determine effect on defense.
3. Progress Report
Based on earlier microarray clustering analysis from fungal inoculations and oxalic acid infiltration studies of soybean, we selected 9 putative defense response genes with strong DNA homology between soybean and Arabidopsis. Arabidopsis knock-out lines were obtained for these genes from ABRC–Salk Institute and the lines were planted and homozygous progeny was identified. We also identified 10 additional putative defense related genes to clone and transform into Arabidopsis to test over-expression. Simultaneously we were running additional biological and time course experiments on soybean; the last set of microarray clustering did not identify the same 9 knockout genes as being of interest, but the genes selected for over-expression were consistent. Select genes of interest (a G-protein coupled receptor, and a 14-3-3) were bombarded into soybean tissue as an RNA interference construct to produced transformed plants. We generated 34 GPCR RNAi lines, of which only 5 plants produced seed. For the 14-3-3, we are in the process of advancing 56 clones to the embryo stage. Activities of this project were monitored through personal meetings, phone calls and e-mails.