In vivo competition assays between Vip3 proteins confirm the occurrence of shared binding sites in Spodoptera littoralis

Due to their different specificity, the use of Vip3 proteins from Bacillus thuringiensis in combination with the conventionally used Cry proteins in crop protection is being essential to counteract the appearance of insect resistance. Therefore, understanding the mode of action of Vip3 proteins is crucial for their better application, with special interest on the binding to membrane receptors as the main step for specificity. Derived from in vitro heterologous competition binding assays using (125)I-Vip3A and other Vip3 proteins as competitors, it has been shown that Vip3 proteins share receptors in Spodoptera frugiperda and Spodoptera exigua brush border membrane vesicles (BBMV). In this study, using (125)I-Vip3Aa, we have first extended the in vitro competition binding site model of Vip3 proteins to Spodoptera littoralis. With the aim to understand the relevance (in terms of toxicity) of the binding to the midgut sites observed in vitro on the insecticidal activity of these proteins, we have performed in vivo competition assays with S. littoralis larvae, using disabled mutant (non-toxic) Vip3 proteins as competitors for blocking the toxicity of Vip3Aa and Vip3Af. The results of the in vivo competition assays confirm the occurrence of shared binding sites among Vip3 proteins and help understand the functional role of the shared binding sites as revealed in vitro.