Project Number: 5020-22000-018-01
Start Date: Jul 01, 2012
End Date: Jun 30, 2015
We already have a large time-course microarray and a laser-capture micro-dissection microarray that have been analyzed but not completely mined for location-specific differences and compatible versus incompatible interaction differences. We will perform RNA-Seq on Hamlet wheat plants that generate a hypersensitive response, giving a different type of resistance from previous experiments for comparison of incompatible and compatible interactions. We will generate RNA-Seq data for Brachypodium response to Hf, which gives an intermediate interaction: plants are stunted some but still make seed, larvae develop farther than on wheat but do not emerge as adults. Through bioinformatics analyses, we will compare expression profiles among different data sets to identify genes specific to interaction type (compatible vs. incompatible) response type (standard versus hypersensitive/oxidative burst-based resistance), tissue location (global versus feeding site-specific) and host type (wheat versus Brachypodium) to yield a more complete view of resistance and new genes that were not detected in early experiments. In addition, these results will give us raw material for selecting genes for promoter studies. Feeding assays utilizing our new technique (Hessian fly in situ translocation assay) to deliver compounds to the larvae via the plant, will use endogenous wheat antinutrient proteins and proteins from unrelated organisms to determine whether these proteins contribute to resistance by acting as acute toxins, disrupting/binding larval midgut, inducing behavioral changes in larvae or limiting their growth. We have already cloned Hessian fly-responsive wheat promoters for Hfr-1, Wci-1, Dir, Wci-2 and Hfr-3. We are considering 20 additional genes and will target those with low expression in the seed under many different stress conditions. A collaborator is producing T1 plants with Wci-1, Hfr-3 and Wci-2 promoters driving GUS for future analysis of positional expression in response to Hessian fly attack. We will clone some Hessian fly-responsive promoters from genes discovered in other microarrays and RNA Seq experiments. With collaborators willing to transform wheat, we will hook endogenous wheat antinutrient genes to promising promoters to test the efficacy of the constructs as a new type of resistance. We will analyze promoter sequences to identify motifs common to Hessian fly-responsive genes and elements characterized in other plant genes.