1a. Objectives (from AD-416):
1) Discover and characterize host genes and pathways that respond to Hessian fly in wheat; 1a) Compare expression profiles among different experiments to identify genes specific to interaction type (compatible vs. incompatible), response type (standard vs. hypersensitive/oxidative burst-based resistance), tissue location (global vs. feeding site-specific), host type (wheat vs. Brachypodium) and temporal expression (time-course comparisons); 1b) For a target subset of loci (to be determined after preliminary analyses in Sub-objective 1a), characterize gene sequences and/or their encoded products in appropriate functional assays; 1c) Identify and characterize promoters and their motifs for driving transgenes with the potential applications of testing insecticidal efficacy of antinutrient/toxin-encoding genes, or utility in driving as yet undetermined transgenes for use in breeding lines/commercial cultivars; 2) Identify and analyze the efficacy of R gene intervention in Hessian fly populations; 2a) Predict the durability of resistance by assessing the frequency of virulent genotype changes with the deployment of an R gene; 2b) Document the durability of resistance in a gene pyramid; 2c) Document the fitness cost of virulence in Hessian fly; 3) Discover potential transgenes for resistance in wheat to Hessian fly; 3a) Evaluate effects of antinutrient and toxic proteins on growth of Hessian fly larvae.
1b. Approach (from AD-416):
Objective 1: Host genes and pathways will be discovered and characterized by comparing data from various expression assays including microarrays, Illumina Whole Transcriptome Sequencing (RNA-Seq) and quantitative real-time PCR (qPCR). Functional assays will utilize viral-induced gene silencing coupled with infestations to detect changes in resistance and qPCR to verify alterations in gene expression. Gene promoters will be cloned by genome walking and analyzed by promoter motif software. Biolistic transformation will generate transgenic plants for functional analyses of promoter elements. Objective 2: The efficacy of resistance (R) gene intervention in Hessian fly populations will be assessed by documenting the change in frequency of virulence alleles in populations across the southeastern United States as a function of R gene deployment. Frequency of virulence in populations will be determined by diagnostic PCR based markers for virulence/avirulence alleles to specific R genes. The durability of resistance in a R gene pyramid will be documented by screening populations having low levels of virulence to the R genes in growth chamber tests. The number of generations required before significant increases in the level of virulence occur in the test populations will be used to evaluate the efficacy of the gene pyramid. Documentation of fitness cost of virulence in Hessian fly to R genes in wheat will be assessed by determining effects on survival to adult and fecundity during selection for virulence to specific R genes over multiple generations. Objective 3: An in planta translocation feeding assay that allows toxic proteins to be ingested by Hessian fly larvae will be used to discover potential transgenes for resistance in wheat to this pest. The most efficacious toxic proteins documented in the feeding assay will be selected for biolistic transformation of wheat in collaboration with a cooperator at Kansas State University and tested for efficacy of resistance in transgenic plants to Hessian fly as well as the other major insect pest of wheat (i.e. green bug, Russian wheat aphid and wheat stem sawfly). With Hessian fly, the ultimate goal is to have transgenes for resistance under the control of Hessian fly responsive promoters so that toxic proteins are expressed only at the feeding site of larvae and not ubiquitously in the plant. If issues arise with evaluation of toxic proteins with the Hessian fly feeding assay we will evaluate candidate toxic proteins in bioassays with Drosophila melanogaster. If Hessian fly responsive promoters do not become available, we will use other wheat promoters, such as a phloem specific promoter, to avoid ubiquitous expression in the plant. A phloem specific promoter should also be effective against aphid pest of wheat.
3. Progress Report:
Objective 1. Analysis of RNA-Seq data from different experiments completed or near completion. A new approach to VIGS is being implemented. Feeding assay is fully optimized. Transgenic wheat carrying different promoters obtained from collaborator, seed increase initiated. Cloning of additional promoters initiated. Objective 2. Evaluation of vH13 frequency in laboratory populations initiated. Evaluation of virulence alleles to H6 and H9 initiated. F2 lines carrying H25H26 identified. Selection of fly lines virulent to H25 and H26 initiated. Hessian fly collections from Georgia and Idaho will be made this fall. Objective 3. Additional lectins have been evaluated for effects on Hessian fly larvae and one of these has been documented as being promising for transgenic resistance. Cry toxins have been evaluated and it appears lectins and other toxic proteins will be more efficacious for transgenic resistance in wheat to Hessian fly attack.