The first 10 genes were selected from QTLs associated with partial resistance to Phytophthora sojae infection in the soybean cultivar “Conrad”. Promoter regions of these genes were cloned, sequenced for validation and finally fused to the green fluorescent protein gene (gfp) to study their expression profiles and responses to P. sojae infection. Promoter constructions were evaluated using the 3 different validation tools described in the proposal. Although the promoters from all of the first set of candidate genes were active and GFP expression was observed in all cases, pathogen induction was not observed. The promoters were “functional” in soybean tissues but did not seem to be pathogen inducible in the tissues that we evaluated. Whole transgenic plants have been generated and we will evaluate induction in these plants over the next few months. Efforts are being redirected to identify more candidate genes, which will allow us to isolate more candidate promoters. Additional genes were identified on chromosome 18 of Williams82 which is associated with partial resistance to Phytophthora sojae. For gene identification, a new research assistant was hired and will begin to work on this project in June, 2012. Identification of defense response genes using mapping approaches seems to yield results that may not be too reliable. All of the promoters for genes in a specific region need to be evaluated rather than more precisely identified gene promoters. RNAseq data may allow more accuracy in selection of relevant genes and their associated promoters.

Analysis of RNAseq data is progressing but has not been completed due to computer access and programming issues. The RNAseq data following Sclerotinia treatment has been analyzed, while the RNAseq data from treatment with Pseudomonas syringae and Fusarium virguliforme is ongoing. Using microarray expression data, 11 soybean genes have been identified that are fairly specifically induced only by pathogens. We are attempting to transfer some of the 11 soybean genes that were pathogen specific into Arabidopsis to test if these genes can enhance defense to pathogens in Arabidopsis, and thereby provide supporting evidence that these are indeed defense-related genes. We have successfully cloned 2 of the genes, and will soon introduce them into Arabidopsis, where we will conduct disease assays. If we obtain positive results, efforts will follow with soybean. To complement this effort, we will also clone and characterize the promoters for each of these 11 soybean genes.