RESUMEN
Microbially derived, protein-based biopesticides offer a more sustainable pest management alternative to synthetic pesticides. Vegetative insecticidal proteins (Vip3), multidomain proteins secreted by Bacillus thuringiensis, represent a second-generation insecticidal toxin that has been preliminarily used in transgenic crops. However, the molecular mechanism underlying Vip3's toxicity is poorly understood. Here, we determine the distinct functions and contributions of the domains of the Vip3Aa protein to its toxicity against Spodoptera frugiperda larvae. We demonstrate that Vip3Aa domains II and III (DII-DIII) bind the midgut epithelium, while DI is essential for Vip3Aa's stability and toxicity inside the protease-enriched host insect midgut. DI-DIII can be activated by midgut proteases and exhibits cytotoxicity similar to full-length Vip3Aa. In addition, we determine that DV can bind the peritrophic matrix via its glycan-binding activity, which contributes to Vip3Aa insecticidal activity. In summary, this study provides multiple insights into Vip3Aa's mode-of-action which should significantly facilitate the clarification of its insecticidal mechanism and its further rational development.