Using computational methods, we have analyzed gene activity (RNA-seq) in the iron efficient soybean line Clark, 1 and 6 hours after iron stress. We have identified 974 genes that respond to iron stress in either leaves or roots. In a separate experiment, we performed RNA-seq on Clark leaves and roots 30, 60 and 120 minutes after iron stress. As this experiment was done after a new version of the genome was out, we were able to identify more genes responding to iron stress; 7957 in the leaves and 7320 in the roots across the time points. In the leaves, we are interested in transcription factors and signaling proteins that signal to the root that more iron is needed for continued growth and development. In the root, we are interested in the genes that receive the shoot signal and initiate the uptake of additional iron from the soil and genes that transport iron from the root to shoot.

We have also taken advantage of data published by our group over the last ten years identifying hundreds of genes involved in IDC responses in IDC tolerant and susceptible lines. While the studies focused on late responses to IDC, they can still be used to identify genes that could improve or worsen IDC symptoms. By cross-referencing these experiments with the Soybean Virus Induced Gene Silencing (VIGS) website (http://soybase.org/SoyVIGS) we identified 130 VIGS constructs that were already available. So far, we have screened VIGS constructs for 52 different genes and 31 have yielded a visible phenotype ranging from alterations in growth, to reduced chlorophyll content and IDC-like symptoms. We have also tested 14 of the VIGS constructs on plants grown in well watered and drought conditions to see if any of the genes are associated with drought tolerance. Five of the constructs altered the severity of drought symptoms relative to controls. In addition, our laboratory is now making our own VIGS silencing constructs designed specifically from the early stress data.

We have made significant progress over the project. A manuscript detailing the 1 and 6 hour gene expression data has been published. Both PIs have been invited to speak about the research at National and International Meetings. We are collaborating with other groups at Iowa State to determine if genes identified in this proposal protect against other plant stresses including pathogens. In addition, we are contributing to the training of undergraduate and graduate students by involving them in the analysis of expression data and VIGS screens.