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Project description

Development of mimetic insecticidal peptides for glassy-winged sharpshooter control. (03XA005)
Program Exotic Pests and Diseases Research Program
B.A. Federici, Entomology, UC Riverside
Host/habitat Unspecified
Pest Glassy-Winged Sharpshooter Homalodisca coagulata; Pierce's Disease Xylella fastidiosa
Disciplines Entomology, Plant Pathology
Agricultural Systems
Start year (duration)  2003 (One Year)
Objectives Predict functional domains of key GWSS midgut epithelium- and salivary gland-specific proteins based on sequences of genes using bioinformatics.

Synthesize functional domain peptides for antibody production.

Clone single-chain fragment variable (ScFv) antibody genes into recombinant phage libraries and screen the libraries.

Conduct feeding studies to identify efficacious mimetic peptides effective in killing or deterring GWSS feeding.

Sequences from cloned glassy-winged sharpshooter, Homalodisca coagulata, genes will be analyzed using bioinformatics tools to identify the surface exposed-epitopes and active sites of the encoded proteins. Peptides based on the sequences that encode these regions will be synthesized and used to produce antibody libraries to these peptides. The antibody functional domains will be cloned into a phage display system, which will be screened for specificity and binding activity of the antibody peptides to the GWSS peptide antigens. Those antibody peptides having the best binding activity will be synthesized and used in feeding studies to identify those that kill or deter the feeding of GWSS.
Final report In situ hybridizations have been performed using paraffin embedded thick sections of dissected GWSS guts. These demonstrate the expected expression of the V-ATPase subunits and membrane transporter throughout the alimentary tract and salivary glands. Polymerase chain reaction (PCR) products of each of the 10,848 clone insert have been purified and are in the process of being quantified for normalization before microarray spotting. We have begun microarray screening with a limited array of 1,536 clones and targets prepared from sharpshooters exposed to sublethal and LD50 levels of the insecticide esfenvalerate. These control experiments were initially performed at the Custom Microarray Facility at the University of Arizona during a microarray training course and are being repeated using the new Hyb4 hybridization station purchased from Genomic Solutions by our laboratory. RNA has been extracted from GWSS dissected guts and salivary glands for target preparations. A single GWSS gut yields sufficient RNA for the preparation of a single target. We have pooled several dissected gut and salivary gland samples for extraction so as to have sufficient RNA for dye swap experiments. A minimum of three pooled samples has been prepared for each target type. Antibodies are being prepared by Orbigen, Inc., to the GWSS V-ATPase c subunit and a novel amino acid transporter recovered from our cDNA library using the clone capture technique. The clone capture technique allows isolation of the full-length cDNA of any gene in the library using complementary oligo probes that have been biotinylated. After hybridization, the captured clones are removed by association with streptavidin-coated magnetic beads. Once the antibodies to these two proteins have been prepared, we will clone the variable regions into the Eznet Display cDNA Library Screening Kit from Maxim Biotech, Inc. We should be able to conduct feeding screenings for efficacious antibody peptides by the end of the summer.

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