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 – The first phase of bioinformatic analysis of a set of wheat microarrays is complete. The expression of 243 wheat class III peroxidase genes and other potential reactive oxygen species-producing genes were extracted from microarray data files and quantification of a subset was verified by quantitative real-time PCR (qRT-PCR). Peroxidase enzymatic activity was correlated with transcript expression and hydrogen peroxide (H2O2) production in resistant and susceptible wheat infested with Hessian fly. All of the above were compared to similar non-host resistance experiments using rice and Hessian fly. The effect of Hessian fly on wheat leaf cuticle, the first line of defense against attack, was examined using gas chromatography. Wax composition was quantified, including fatty acids, aldehydes, primary alcohols and alkanes. Cutin monomers of various lengths were also quantified. Microarray and qRT-PCR data indicated a correlation between wax- and cutin-associated biosynthetic gene transcript levels and their products. Objective 2 – Expression analysis of genes encoding secreted salivary gland proteins has been conducted in collaboration with USDA-ARS scientists at Manhattan, KS using a Hessian fly Affymetrix array. Results were validated by quantitative real-time PCR. We are using a viral induced gene silencing vector to express small interfering RNA in wheat plants for RNA interference in Hessian fly larvae. Gene expression in different compatible/incompatible interactions is being documented by 454 sequencing. A new research direction involving a feeding assay for Hessian fly larvae has been developed. We have used this feeding assay to assess the efficacy of nine different lectins and three different Cry delta endotoxin proteins on the growth and development of Hessian fly larvae. One lectin, snowdrop lectin (SDL), was the most efficacious of the antinutrient proteins evaluated in delaying larval growth and development. In collaboration with scientists at Durham University, UK we have obtained clones of SDL plus a chimeric construct to express a fusion protein of SDL and an additional toxin for transformation of wheat. The efficacy of SDL and the fusion protein in conferring Hessian fly resistance will be documented in transgenic wheat. 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. Assessing Hessian fly population structure in North America and the Old World. The Hessian fly (HF) is the most important insect pest of wheat in the southeastern wheat production region of the United States. Genetically resistant wheat is the most effective means for preventing yield losses due to HF. While the use of resistant wheat is an effective means for controlling HF, it places a selective pressure on populations and has led to the appearance of biotypes that can overcome the resistance. In this study, we used molecular markers to document the population structure of HF and to understand the relationships between Old and New World populations. Published results indicated HF introduced had into North America had the genetic diversity to adapt to various climates and to rapidly overcome deployed resistance in wheat. This knowledge developed by ARS scientists at West Lafayette, IN will benefit scientists and breeders facing the challenge of devising more durable deployment strategies for resistance in wheat to the HF. Wheat producers and commodity groups will also benefit from this knowledge with improved pest control without increased cost.
2. Better understanding the molecular bases of resistance and susceptibility in wheat. Susceptible wheat plants deliver nutrients to Hessian fly larvae via increased permeability and lysis of plant cells at the larval feeding site. Yet the mechanisms involved in these physical changes were unknown, as were the mechanisms that help resistant plants maintain cell integrity. ARS scientists in West Lafayette, IN demonstrated that quantities of wheat leaf waxes as well as the expression of genes involved in their synthesis decrease over the first 8 days of larval attack in susceptible plants. These decreases were not seen in plants with induced resistance, which are able to maintain wax structure and increase the deposition of certain classes of wax that may function as physical barriers and feeding deterrents. Genetic and molecular manipulation of components of plant leaf cell waxes may provide a stronger physical barrier that, coupled with biochemical plant defenses, can increase the longevity of deployed insect-resistance genes in wheat cultivars. This knowledge will benefit scientists and breeders facing the challenge of devising more effective strategies for resistance in wheat to the Hessian fly that improved pest control without increased cost.
Arrueta, L., Shukle, R.H., Wise, I.L., Mitapalli, O. 2011. Gene characterization of two digestive serine proteases in orange blossom wheat midge (Sitodiplosis mosellana). The Canadian Entomologist. 142:(6)532-545.