A high-throughput, in-vitro assay for Bacillus thuringiensis insecticidal proteins

Area of Science:

• Biochemistry
• Molecular Biology
• Insect Toxicology

Background:

• Bacillus thuringiensis (Bt) Cry proteins are insecticidal toxins.
• Understanding Cry protein-receptor interactions is crucial for developing effective pest control strategies.
• DNA shuffling is a powerful tool for generating diverse protein variants.

Purpose of the Study:

• To develop and validate a high-throughput in vitro assay for screening Bt Cry protein variants.
• To identify the specific insect receptors responsible for the toxicity of the Cry1Ab variant IP1-88.
• To investigate the role of different receptor proteins in mediating Cry toxin activity.

Main Methods:

• Development of an automation-amenable insect cell-based assay.
• Expression of insect Cry protein receptors (On-Cad, On-APN1, On-APN3, Sf-Cad) in insect (Sf21) and mammalian (Expi293F) cell lines.
• Ligand blotting, immunofluorescence microscopy, and cell viability assays to determine toxin-receptor binding and cellular toxicity.

Main Results:

• The Bt Cry1Ab variant IP1-88 bound to Ostrinia nubilalis cadherin-like protein (On-Cad) and aminopeptidase-N 1 (On-APN1).
• IP1-88 showed toxicity only in cells expressing On-Cad, in both insect and mammalian cell lines.
• On-Cad alone was sufficient to confer sensitivity to IP1-88, suggesting it is a primary receptor.

Conclusions:

• A novel high-throughput assay for screening Bt Cry proteins was successfully developed and validated.
• The European corn borer cadherin-like protein (On-Cad) was identified as a functional receptor for the Cry1Ab variant IP1-88.
• The findings suggest that On-Cad plays a critical role in the mode of action of this Bt toxin, independent of secondary receptors.