Promoter regions were initially cloned from the genes identified from the QTL regions associated with partial resistance to P. sojae infection in cv. Conrad. The promoter regions were isolated from cv Jack, fused to the green fluorescent protein (gfp) reporter gene and reintroduced into soybean. Soybean hairy roots expressing these promoter-gfp constructs were also generated but the promoters did not show either pathogen-inducible or wound-inducible gene expression in hairy roots. Transgenic plants containing the following promoter:gfp constructs were generated: Transmembrane amino acid transporter (GmTAAT), Heat shock protein (GmHSP), Asparaginase (GmASP) , ABC amino acid transporter (GmABC) and Ethylene Response Factor 3 (GmERF3).

These promoters were selected as they gave high levels of expression using transient expression and in hairy roots. Seeds were recovered from three GmTAAT events, six GmHSP events, two GmGmASP events, three GmABC events, and six GmERF3 events. GFP expression was observed in tissues from all promoter constructs except GmTAAT. Unfortunately, for the remaining promoter constructs that did give some expression in transgenic soybean, GFP expression was not induced in any of the progeny seedlings by Fusarium graminearum or P. sojae inoculation. The GmERF3 promoter displayed wound induction in above ground soybean seedling materials (leaves and cotyledons) but not in roots. GmERF3 wound induction was atypical as the peak induction time was 24-48 h instead of 1-4 h. An element within the promoter was isolated and identified as a new class of elements called “late wound inducible” (LWI) elements. Aside from the one GmERF3 promoter, where a novel element was identified, the other promoters from this first group do not appear to be inducible and may not be useful for the project.

Additional soybean genes associated with pathogen response have been identified by the Dorrance lab (30 additional genes identified through microarray) and the Clough lab (51 additional genes identified through microarray and qRT-PCR). From the 51 genes identified by the Clough lab, 11 soybean genes, that seem to be pathogen specific, were selected for further analysis. The 11 genes are mostly unknowns but an O-methyltransferase, ferric reductase, cinnamoyl-CoA transferase and another ABC transporter were identified in this group of 11 genes. The cDNA of 5 of these genes were cloned, and the cDNA for the cinnamoyl-CoA transferase gene was introduced into Arabidopsis. Studies on the response of the Arabidopsis T1 progeny plants to Pseudomonas syringae are underway.

Analysis of RNAseq data continues. We remain hopeful that RNAseq will provide more robust data for gene expression and allow us to identify the most useful genes and promoters that are inducible by specific pathogen attack.