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:
The goal of this collaboration is to verify that genes that show an expression pattern suggestive of a defense-related gene are actually affecting defense responses. Genes were identified as soybean candidate defense-related genes to Sclerotinia sclerotiorum and oxalate oxidase, its virulence factor. We conducted functional analysis of the candidate genes by confirming microarray data with qRT-PCR, and transforming soybean with candidate genes to allow disease studies. Similar experiments are also being conducted in Arabidopsis. We have confirmed nine candidate-defense related genes by qRT-PCR, constructed six homozygous Arabidopsis overexpression lines by semi-quantitative RT-PCR, and transformed soybean with DNA constructs designed to silence the expression (RNAi) of four candidate defense-associated genes. For the soybean transgenics, three events of the RNAi GPCR have T2 seed and homozygous lines have been identified. The other transformation experiments are at an earlier stages of development, prior to plant regeneration. Our experiments with Arabidopsis suggests that it may be a poor reporter plant for screening of disease resistance to Sclerotinia. Therefore, we are continually working to improve infection assays in Arabidopsis, and we also investigating Nicotiana benthamiana as an alternative host with more consistent responses.