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 – Lectins were tested for effect on Hessian fly larvae. Expression assays using 454 sequencing allowed identification of several genes to be targeted for further study and eventual silencing. Transcription factors that may coordinate the expression of suites of defense genes are among the most interesting. The Hfr-3 promoter was cloned for potential use in driving transgenes. Cloning of a heat shock gene promoter is under way. Promoter motifs common to 5 Hessian fly-induced wheat genes were identified and annotated. Genes in wheat and Hessian fly polyamine and amino acid biosynthesis were annotated. Objective 2 – Using the Hessian fly genome sequence we have annotated genes and pathways that are essential to survival of the insect and its interactions with wheat. Genes annotated include those in the RNA interference and microRNA pathways, metabolic pathways, synthesis of non-essential amino acids, detoxification/antioxidant defense, and digestion. An assay that allows toxic proteins to be evaluated for transgenic resistance in wheat to Hessian fly was developed and published. Using this assay we have discovered a toxic recombinant protein that has potential for resistance in wheat. Currently, wheat is being transformed with the transgene encoding the protein. Future studies will evaluate other toxic proteins to include Bacillus thuringiensis endotoxins (BT toxins). 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. A macrogeographic study of Hessian fly populations in the southeastern United States has been completed and published. In addition, a microgeographic study with populations in Alabama is currently underway to gain better understanding of gene flow within small geographic distances.
1. Effects of antinutrient proteins on Hessian fly larvae. The most effective control of Hessian fly is through deployment of genetically resistant wheat; unfortunately, the deployment of resistance places a selection pressure on Hessian fly populations that leads to the appearance of biotypes that can overcome resistance. One strategy to enhance the durability of native resistance genes is to combine them with genetically engineered resistance (i.e. GMO resistance). The Hessian fly is an obligate parasite of wheat and related grasses and no protocol for making toxic proteins accessible to feeding larvae was available. ARS researchers at West Lafayette, Indiana have developed a feeding assay for Hessian fly larvae and are using the assay to identify toxic proteins for genetically engineered resistance in wheat. These studies will help breeders and scientists devise innovative approaches to ensure the durability of resistance in wheat. Crop producers, commodity groups, and consumers will benefit from improved pest control that increases yield and quality without increasing costs.