THE ALIMENTARY TRACK OF THE GLASSY-WINGED SHARPSHOOTER AS A TARGET FOR CONTROL OF PIERCE’S DISEASE AND DEVELOPMENT OF MIMETIC INSECTICIDAL PEPTIDES FOR GLASSY-WINGED SHARPSHOOTER CONTROL Project Leader:

Toxin technology using Bacillus sphaericus has been used to control the important vectors of human diseases, the Aedes aegypti and Culex quinquefasciatus mosquito species. Agricultural pests such as caterpillars and beetles have been targeted by transgenic insecticidal crops expressing Bacillus thuringiensis (Bt) toxin. However, a Bt technology would be ineffective in controlling xylem-and phloem-sucking insect pests, such as the glassy-winged sharpshooter, Homalodisca coagulata (GWSS), because their feeding habit circumvents the tissues in which the toxins are expressed in transgenic plants and they are likely immune to the Bt toxins. Our research is developing mimetic peptides to target the exposed active domains of transport proteins on the surface of the GWSS midgut microvillar membrane and GWSS salivary enzymes. We are using a genomics approach to develop proteins cloned from the variable binding domains of immunoglobulin molecules specific to GWSS gut and salivary proteins. To isolate targets we have used degenerate PCR to amplify genes characterized in other insect species, and are screening a cDNA microarray to identify novel gut and saliva protein encoding genes. Our first mimetic peptides are targeting the GWSS V-ATPase c subunit. INTRODUCTION Examples of medical uses of mimetic technology include the inactivation of disease-related enzymes (Burke et al. 2001), blockage of metabolic receptors important to disease (Berezov et al. 2000), and the use of antibodies developed against disease constituents (Moe et al. 1999). Human cancers (Monzayi-Karbassi and Keiber-Emmons, 2001), diabetes (Deghenghi, 1998), and heart disease (Lincoff et al. 2000) all have been treated successfully through these applications of mimetics. Antibody proteins have been synthesized successfully in plants (Larrick et al. 2001; Stoger et al. 2002), and promoters directing expression to the cell wall and vascular structures of plants (Shi et al. 1994; Springer, 2000), are available, so we can assure that our antibody peptides are synthesized in a tissue-specific manner in transformed plant lines. During the last year we isolated two full length cDNA clones of the V-ATPase c subunits of GWSS. The V-ATPase c protein is an excellent target for mimetic inactivation in insects because they provide the electromoive force which drives H/K transport, which in turn maintains gut pH. V-ATPases have been successfully targeted by highly specific bafilomycin/concanamycin antibiotics in the inhibition of human tumor cells (Boyd et al. 2001) and osteoporosis (Farina et al., 2001), and these antibiotics have been shown to specifically bind to the V-ATPase c subunit, demonstrating that binding and blocking the active site of this subunit can effectively inactivate the transporter. OBJECTIVES 1. Determine the structure and cell types in the midgut epithelium and salivary glands of the glassy-winged sharpshooter (GWSS), Homalodisca coagulata; 2. Prepare a normalized cDNA microarray of GWSS using pooled cDNAs isolated from each developmental stage. 3. Screen the microarray using cDNA probes derived from midgut and salivary gland tissue-specific probes to determine the tissue-specific expression of key midgut microvillar and saliva proteins; 4. Clone and sequence genes encoding one or more key midgut microvillar and saliva proteins and determine their suitability as targets for a molecular biological approach to GWSS and Pierce’s disease control. 5. Predict functional domains of key GWSS midgut epitheliumand salivary gland-specific proteins based on sequences of genes using bioinformatics; 6. Express functional domain peptides for antibody production; 7. Clone single-chain fragment variable antibody genes into recombinant phage libraries and screen the libraries; 8. Conduct feeding studies to identify efficacious mimetic peptides effective in killing or deterring GWSS. RESULTS While we are still in the process of screening our cDNA microarray to identify clones encoding novel midgut and salivary gland protein genes, we have isolated two full length cDNAs of the V-ATPase c subunit from our GWSS cDNA library using clone capture (Shepard and Rae, 1997). These clones differ in several positions in the coding region, as well as in the 3’and 5’-untranslated regions (Figure 1.). The protein sequences align significantly with the V-ATPase c subunit proteins of several other insect species (Figure 2). Antibodies to the gut lumen exposed N-terminus and external loop have been produced in rabbits and affinity purified by New England Peptide. These antibodies are being used in feeding studies to determine the efficacy of our approach. Antibody RNAs were extracted from the spleens of the immunized rabbits and the