Extensive use of chemical insecticides adversely affects both environment and human health. One of the most popular biological pest control alternatives are bioinsecticides based on Bacillus thuringiensis .

The Vip3Aa insecticidal protein from Bacillus thuringiensis (Bt) is produced by specific transgenic corn and cotton varieties for efficient control of target lepidopteran pests. The main threat to this technology is the evolution of resistance in targeted insect pests and understanding the mechanistic basis of resistance is crucial to deploy the most appropriate strategies for resistance management. In this work, we tested whether alteration of membrane receptors in the insect midgut might explain the >2000-fold Vip3Aa resistance phenotype in a laboratory-selected colony of Heliothis virescens (Vip-Sel). Binding of ¹²⁵I-labeled Vip3Aa to brush border membrane vesicles (BBMV) from 3rd instar larvae from Vip-Sel was not significantly different from binding in the reference susceptible colony. Interestingly, BBMV from Vip-Sel larvae showed dramatically reduced levels of membrane-bound alkaline phosphatase (mALP) activity, which was further confirmed by a strong downregulation of the membrane-bound alkaline phosphatase 1 (HvmALP1) gene. However, the involvement of HvmALP1 as a receptor for the Vip3Aa protein was not supported by results from ligand blotting and viability assays with insect cells expressing HvmALP1.

ABC proteins are primary-active transporters that require the binding and hydrolysis of ATP to transport substrates across the membrane. Since the first report of an ABCC2 transporter as receptor of Cry1A toxins, the number of ABC transporters known to be involved in the mode of action of Cry toxins has increased. In Spodoptera exigua, a mutation in the SeABCC2 gene is described as genetically linked to resistance to the Bt-product Xentari^TM. This mutation affects an intracellular domain involved in ATP binding, but not the extracellular loops. We analyzed whether this mutation affects the role of the SeABCC2 as a functional receptor to Cry1A toxins. The results show that Sf21 cells expressing the truncated form of the transporter were susceptible to Cry1A toxins. Moreover, specific Cry1Ac binding was observed in those cells expressing the truncated SeABCC2. Additionally, no differences in the irreversible Cry1Ac binding component (associated with the toxin insertion into the membrane) were observed when tested in Sf21 cells expressing either the full-length or the truncated form of the SeABCC2 transporter. Therefore, our results point out that the partial lack of the nucleotide binding domain II in the truncated transporter does not affect its functionality as a Cry1A receptor.

The Asian corn borer, Ostrinia furnacalis (Guenée, 1854), is a highly damaging pest in Asia and the Pacific islands, and larvae feed mainly from corn crops. To determine the suitability of Bt-corn technology for the future control of this pest, understanding the potential to develop resistance to Cry1Ab and the basis of cross-resistance to other Cry1 proteins is of great interest. Here, we have explored the binding of Cry1A proteins to brush border membrane vesicles from two O. furnacalis colonies, one susceptible (ACB-BtS) and one laboratory-selected with Cry1Ab (ACB-AbR). The insects developed resistance to Cry1Ab and showed cross-resistance to Cry1Aa, Cry1Ac, and Cry1F. Binding assays with radiolabeled Cry1Ab and brush border membrane vesicles from susceptible insects showed that Cry1A proteins shared binding sites, though the results were not conclusive for Cry1F. The results were confirmed using radiolabeled Cry1Aa. The resistant insects showed a reduction of the specific binding of both Cry1Ab and Cry1Aa, suggesting that part of the binding sites were lost or altered. Competition binding assays showed full competition between Cry1Ab and Cry1Aa proteins in the susceptible colony but only partial competition in resistant insects, confirming the alteration of some, but not all, binding sites for these two proteins. The binding site model for Cry1A proteins in O. furnacalis is in agreement with the occurrence of multiple membrane receptors for these proteins.

The ABC transporters are membrane proteins that can act as putative receptors for Cry proteins from Bacillus thuringiensis (Bt) in the midgut of different insects. For the beet armyworm, Spodoptera exigua, ABCC2 and ABCC3 have been found to interact with Cry1A proteins, the main insecticidal proteins used in Bt-crops, as well as Bt-based pesticides. The ABCC2 has shown to have specific binding towards Cry1Ac and is involved in the toxic process of Cry1A proteins, but the role of this transporter and how it relates with the Cry1A proteins is still unknown. Here, we have characterized the interactions between the SeABCC2 and the main proteins that bind to the receptor. By labelling the Cry1Aa protein, we have found that virtually all of the binding is in an oligomeric state, a conformation that allowed higher levels of specific binding that could not be achieved by the monomeric protein on its own. Furthermore, we have observed that Cry1A proteins can hetero-oligomerize in the presence of the transporter, which is reflected in an increase in binding and toxicity to SeABCC2-expressing cells. This synergism can be one of the reasons why B. thuringiensis co-expresses different Cry1 proteins that can apparently have similar binding preferences. The results from in vitro competition and ex vivo competition showed that Cry1Aa, Cry1Ab and Cry1Ac share functional binding sites. By using Cry1Ab-Cry1Ac chimeras, the presence of domain I from Cry1A proteins was revealed to be critical for oligomer formation.