In many countries the refuge strategy has been used to avoid development of Bt-resistant populations. This strategy proposes to use refuge zones, where non-Bt crops are cultivated adjacent to Bt plants. This procedure aims to maintain a population of susceptible insects to mate with resistant insects, resulting in progeny that are susceptible to the toxin, and thus delaying the appearance of resistance in the field⁴. This practice is in part responsible for forestalling Bt resistance in insects, even after eleven years of extensive use of Bt crops^4,5.

However, insects with mutations linked to the resistance trait have been found in Bt fields. Since resistance is recessive, these insects are not resistant, but mating between heterozygous insects could result in the generation of homozygous resistant offspring. Therefore, the appearance of resistant insects seems to be imminent⁴. Laboratory studies on Bt resistance in selected insect lines have indicated that the most common mechanism involves mutations in the cadherin receptor of Bt toxins. In three different cotton insect pests, resistant populations resistant to Cry1Ac were isolated, and in all these cases the resistance was linked to mutations in the cadherin gene⁴.

Mode of action of Cry toxins and mechanism of insect resistance
An understanding of the mode of action of Cry toxins is crucial to devising strategies to prevent insect resistance. Our research group studied Cry toxins at the molecular level, particularly how Cry1Ab and Cry1Ac kill their targets. Cry toxins form pores in the apical membrane of larvae midgut cells, destroying the cells and hence killing the larvae.

Cry toxins are synthesized as protoxins. When susceptible larvae ingest the protoxin, it is solubilized and activated by gut proteases, generating a toxic fragment. The activated toxin then binds to two different receptors in a sequential manner. Both receptors are localized in the microvilli membrane of cells that form the midgut epithelium. The first contact of the toxin is with the cadherin receptor. This interaction induces a conformational change in the toxin, cleaving a small fragment from the amino-terminal region—the helix α-1. This cleavage exposes previously buried hydrophobic regions and triggers the formation of a tetrameric oligomer structure^4,6. The oligomer then has an increased affinity to the second receptor, aminopeptidaese N (APN). APN facilitates the insertion of the oligomer into the membrane, forming a lytic pore that leads to cell disruption and ultimately insect death⁶. The first symptoms of intoxication are the immediate paralysis of intestinal movement and feeding cessation. Then the midgut cells are disrupted and the insect dies from destruction of the midgut tissue^4,6.

Resistance to Cry toxins could occur by blocking any one of the steps in this complex mechanism of action. However, as mentioned above, the most common mechanism of resistance reported until now involves mutations in the cadherin gene.

Genetically engineered Cry toxins with activity against resistant insects
In our model, the interaction of a Cry protein with the cadherin receptor facilitates cleavage of the helix α-1 region and the formation of an oligomeric structure composed of four subunits. Based in our data, we hypothesized that Cry1A toxins that lack a helix α-1 region would form the oligomeric structures without first interacting with the cadherin receptor. Therefore the oligomer could contact the second APN receptor and kill the larvae, even if the cadherin protein is mutated or absent in the larvae gut. We proposed that genetically engineered Cry1A toxins lacking the helix α-1 region could be lethal to Cry1A-resistant insects whose resistance is linked to either mutations in or to dsRNA-induced silencing of the cadherin gene.

Our data showed that genetically engineered Cry1Ab and Cry1Ac toxins (Cry1AbMod and Cry1AcMod) were able to form oligomeric structures in vitro in the absence of the cadherin receptor⁷. Most importantly, we demonstrated that two species of lepidopteran insects (Manduca sexta and Pectinophora gossypiella) that are resistant to Cry1Ab and Cry1Ac toxins due to lower production of cadherin protein by RNAi or to mutations in the cadherin gene, respectively, became susceptible to Cry1AbMod and Cry1AcMod toxins⁷.

Figure 2 depicts a model of the mode of action of the Cry1A toxin and a comparison with the Cry1AMod toxin. The main difference is the formation of the oligomeric structure that in one case requires the interaction with the cadherin receptor and in the other is independent of this receptor.