1a. Objectives (from AD-416)
The long-term objectives of this project are to facilitate development of durable and effective pest controls through host resistance either selected or genetically engineered and to minimize the risks to deployment of new genes for resistance. Over the next 5 years we will focus on the following specific objectives: (1) Better understand the molecular bases of resistance and susceptibility in wheat; (2) Reveal insight into the molecular basis of virulence in Hessian fly; (3) Elucidate Hessian fly population structure and risks to new genes for resistance. Despite their economic importance, little is known about the molecular interactions between Hessian fly and wheat that result in resistance or susceptibility, the molecular mechanism of resistance in wheat, or the effects of these interactions on the genetic structure of fly populations.
1b. Approach (from AD-416)
Objective 1: Gene expression in compatible and incompatible wheat-Hessian fly interactions will be assessed by microarray technology and 454FLX sequencing. Gene function will be assessed with BLAST. Enzyme and substrate binding activities will be verified by protein expression and biochemical analyses. Promoter regions will be identified by various bioinformatic softwares. Viral-induced gene silencing (VIGS) will assess the involvement of wheat genes during compatible and incompatible interactions. Objective 2: Microarray technology and 454FLX sequencing will reveal gene expression in the larval Hessian fly during compatible and incompatible interactions with wheat. The morphology of midgut and salivary gland tissues will be examined by transmission electron microscopy. Comparative transcriptomics will identify Hessian fly genes involved in parasitism of wheat. The role of Hessian fly genes in host susceptibility or resistance will be assessed through RNAi knockdown. Objective 3: Microsatellite markers will be used to assess heterogeneity and gene flow in Hessian fly populations. Changes in allelic variation will assist in assessing the risks to deployed resistance. Differentiation at different geographic scales will be assessed by Fst and Rst values. Estimation of effective population size (Ne) will be used to measure the strength of genetic drift in populations.
3. Progress Report
Objective 1 – Changes in cellulose content were determined in resistant and susceptible wheat attacked by Hessian fly larvae. Brachypodium plants were tested for resistance and susceptibility to Hessian fly larvae. Isolated Bt toxin Cry11A and confirmed its activity on mosquito larvae. The list of potential promoter donor genes was evaluated and one gene was selected for cloning based on expression at larval feeding sites and no seed expression. The first stages of promoter cloning have begun. Objective 2 – Expression analysis of genes encoding secreted salivary gland proteins (SSGPs) has been conducted in collaboration with USDA-ARS scientists at Manhattan, KS using a Hessian fly Affymetrix array. Results from the array analysis have been validated by quantitative real-time PCR (qPCR) for selected SSGPs. Initial analysis involved three laboratory lines of Hessian fly (vH9, vH13, and white eye) and two populations (Israel and Alabama). We are continuing microinjection of double stranded RNA (dsRNA) into embryos for RNA interference (RNAi) in 1st-instar larvae. A tobacco protoplast system is being used to discover the target sites of SSGPs in plant cells and putative function of SSGPs. Gene expression in different compatible/incompatible interactions is being documented by 454 sequencing. A new research direction involving a bioassay for Hessian fly larvae has been developed that was not available when the objectives and milestones for the Project Plan were developed. Objective 3 – The microsatellites that have been chosen for use in population studies have been characterized and mapped to the polytene chromosomes. These microsatellites have been used to analyze population structure globally by assessing gene flow between populations. In addition, a microgeographic study with populations (third year collections) in Alabama is currently underway to gain better understanding of gene flow within small geographic distances. Currently, we have a few samples from Alabama from this year, but are awaiting more samples in order to finish our analysis.
1. New approaches for resistance in wheat to Hessian fly. Genetic resistance is the most effective method of protecting wheat from Hessian fly damage; however, new genotypes of the insect appear that can overcome formerly resistant wheat. In an evaluation of 21 of the 33 identified Hessian fly resistance (R) genes in wheat we discovered that only 5 of the R genes would provide effective protection of wheat in the southeastern United States. These results suggest that new strategies for deployment of R genes and genetically engineered resistance are needed. Using a bioassay for Hessian fly larvae we are testing lectins and Bacillus thuringiensis Cry toxins for toxicity against Hessian fly larvae. Results will impact the discovery of genes encoding toxic proteins for transgenic resistance in wheat ensuring the durability of resistant wheat and benefiting the agricultural community (crop producers and commodity groups) with improved pest control that increases yield and quality without increasing cost.
Shukle, R.H., Subramanyam, S., Williams, C.E. 2010. Ultrastructural Changes in the Midgut of Hessian Fly Larvae Feeding on Resistant Wheat. Journal of Insect Physiology. 56:754-760.