Location: Horticultural Crops Research
Project Number: 2072-22000-044-09-S
Project Type: Non-Assistance Cooperative Agreement
Start Date: Jul 1, 2020
End Date: Jun 30, 2022
1) Identify biological targets from key pests (spotted wing drosophila, slugs, thrips, etc.). 2) Design and synthesize bioactive peptides for target pests. 3) Evaluate bioactive peptides impact on target pests.
Identification and expression of GPCRs: Since 2003, we have identified various insect GPCRs, characterized, and functionally expressed them in insect Sf9 or Sf21 cells. We will identify target GPCRs for SWD, thrips, and slugs. Then, the GPCR proteins will be functionally expressed in Sf9 insect cells. Screening process and design bioactive peptides: Phage display describes a biopanning technique where a peptide or protein is fused with the coat protein of a bacteriophage, resulting in exposure of the fused protein on the surface of the virion. The peptides of the phage displays are randomly generated at a specific number of amino acids creating a vast library of peptides. This allows the rapid identification of peptide ligands with a high affinity for a target cell-surface receptor, by an in vitro selection process. Purified phage DNA from the culture will be sequenced to identify nucleotide sequences encoding proteins. Based on the identified phage peptide sequences various small peptides will be designed and synthesized to evaluate the insecticidal activity on the target pests. Once small peptides are screened, a small amount of peptides (~5 mg) will be synthesized. Evaluation of bioactive peptides: Each peptide will be evaluated on whether they have an agonistic (bind to GPCR as natural ligand) or antagonist (block natural ligand binding to GPCR) effect on their own GPCR in Sf9 cells. In the binding test, bioactive peptides will be added before real ligands in 96-well cells, then evaluated for the potential to interfere with the binding interaction between the ligand and GPCR. Various concentrations of small peptides or water (control) will be injected into target insects, and insects will be monitored for survival.