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 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 (SSGPs) has been conducted at West Lafayette, IN by quantitative real-time PCR and in collaboration with USDA-ARS scientists at Manhattan, KS using a Hessian fly Affymetrix tiling array. Expressed sequence tag (EST) sequencing and comparison of transcripts encoding SSGPs in populations of Hessian fly from Israel has documented diversity in SSGPs not found in populations from the United States. A gene (MdesL1) encoding a secreted lipase-like protein expressed in the salivary glands of the larval Hessian fly has been cloned and characterized. The protein MdesL1 could be involved in extra-oral digestion and changes in host-cell permeability. Ribonucleic acid interference (RNAi) is being evaluated for efficacy in knockdown of transcripts for genes encoding virulence factors in the interaction of the larval Hessian fly with wheat. Objective 3 – Ninety-four microsatellite markers have been mapped to polytene chromosomes and over 30 have been selected for population studies. These microsatellites have been used to analyze population structure in the southeastern United States, assess gene flow between populations, and document the relationship between population structure and landscape ecology. In the southeastern United States, it appears that the primary driving force for gene flow between Hessian fly populations is the amount of wheat grown for hay. We have also determined that there is a small but significant amount of gene flow across the southeastern United States and it has created two distinct populations across this geographic area. In addition, a microgeographic study with 13 populations (second year collections) in Alabama has also begun in order to understand fine scale gene flow in populations.
1. 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 epidermal cells at the larval feeding site. Yet the mechanisms involved in these physical changes are unknown, as are mechanisms that help resistant plants maintain epidermal cell integrity. We demonstrated that quantities of wheat cuticular waxes and cutins 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 are not seen in plants with induced resistance, which are able to maintain cuticle structure and increase the deposition of certain critical classes of wax that may function as physical barriers and feeding deterrents. Genetic and molecular manipulation of components of plant cuticle may provide a stronger physical barrier that, coupled with biochemical plant defenses, can increase the durability of deployed insect-resistance genes in wheat cultivars.
2. Understanding Hessian fly secreted salivary gland effectors. The salivary glands and midgut of the larval Hessian fly are the primary interfaces with wheat. Little is known about the roles of these two organs in the interactions between larval Hessian fly and wheat. However, secreted salivary gland proteins (SSGPs) in the larval Hessian fly are hypothesized to be the effectors reprogramming host-plant tissues in compatible interactions with susceptible wheat and to be the avirulence gene products eliciting resistance in incompatible interactions with resistant wheat. We have cloned and characterized a gene, designated MdesL1, encoding a secreted lipase-like protein expressed in the salivary glands of the larval Hessian fly. Data from genomic, transcriptomic, and proteomic analyses suggest the protein encoded by MdesL1 is likely secreted into host-plant cells during larval feeding and could be involved in extra-oral digestion and changes in host-cell permeability or in generating a second messenger in a host-cell signaling cascade. Results from this study have been published in the Journal of Insect Physiology. Additionally, initial comparison of the transcripts encoding SSGPs in populations of Hessian fly from Israel has revealed greater diversity than is present in populations from the United States. Some of these divergent SSGPs in Israeli populations fall basal in phylogenetic analyses to clades containing families of SSGPs, suggesting these divergent SSGPs may represent ancestral types. Through comparative analyses, bioinformatics, and functional analyses we have gained insight into the possible evolution of these SSGP effectors and their roles in the interactions between larval Hessian fly and wheat. These studies are discovering new aspects of the wheat-Hessian fly interactions that will enable novel approaches to genetically engineered resistance to this pest.Shukle, R.H., Mittapalli, O., Morton, P.K., Chen, M. 2009. Characterization and expression analysis of a gene encoding a secreted lipase-like protein expressed in the salivary glands of larval Hessian fly, Mayetiola destructor (Say). Journal of Insect Physiology. 55(2):104-111.