Binding Site Alteration Is Responsible for Field-Isolated Resistance to Bacillus thuringiensis Cry2A Insecticidal Proteins in Two Helicoverpa Species

Abstract

BackgroundEvolution of resistance by target pests is the main threat to the long-term efficacy of crops expressing Bacillus thuringiensis (Bt) insecticidal proteins. Cry2 proteins play a pivotal role in current Bt spray formulations and transgenic crops and they complement Cry1A proteins because of their different mode of action. Their presence is critical in the control of those lepidopteran species, such as Helicoverpa spp., which are not highly susceptible to Cry1A proteins. In Australia, a transgenic variety of cotton expressing Cry1Ac and Cry2Ab (Bollgard II) comprises at least 80% of the total cotton area. Prior to the widespread adoption of Bollgard II, the frequency of alleles conferring resistance to Cry2Ab in field populations of Helicoverpa armigera and Helicoverpa punctigera was significantly higher than anticipated. Colonies established from survivors of F2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac.Methodology/Principal FindingsBioassays performed with surface-treated artificial diet on neonates of H. armigera and H. punctigera showed that Cry2Ab resistant insects were cross-resistant to Cry2Ae while susceptible to Cry1Ab. Binding analyses with 125I-labeled Cry2Ab were performed with brush border membrane vesicles from midguts of Cry2Ab susceptible and resistant insects. The results of the binding analyses correlated with bioassay data and demonstrated that resistant insects exhibited greatly reduced binding of Cry2Ab toxin to midgut receptors, whereas no change in 125I-labeled-Cry1Ac binding was detected. As previously demonstrated for H. armigera, Cry2Ab binding sites in H. punctigera were shown to be shared by Cry2Ae, which explains why an alteration of the shared binding site would lead to cross-resistance between the two Cry2A toxins.Conclusion/SignificanceThis is the first time that a mechanism of resistance to the Cry2 class of insecticidal proteins has been reported. Because we found the same mechanism of resistance in multiple strains representing several field populations, we conclude that target site alteration is the most likely means that field populations evolve resistance to Cry2 proteins in Helicoverpa spp. Our work also confirms the presence in the insect midgut of specific binding sites for this class of proteins. Characterizing the Cry2 receptors and their mutations that enable resistance could lead to the development of molecular tools to monitor resistance in the field.

Resistance to Bacillus thuringiensis Toxin Cry2Ab in Trichoplusia ni Is Conferred by a Novel Genetic Mechanism

“…Although the intoxication pathways and molecular interactions of Cry toxins with insect midgut proteins are not fully understood, it has been well documented that the specific binding of different toxins to the midgut brush border membrane proteins varies, depending on the toxins and host insects (4,33,50). Cry1A and Cry2A toxins are known to bind to different binding sites in the insect midgut, and consequently, Cry1A resistance caused by binding site alteration (also known as "reduced toxin binding") usually does not exhibit cross-resistance to Cry2A toxins (22,23,51). In T. ni, it has been determined that binding site alteration-mediated Cry1Ac resistance does not confer resistance to Cry2Ab (18,32).…”

“…To delay the development of resistance to Bt toxins in insect populations, the toxin Cry2Ab has been used to pyramid with Cry1Ac in the new generation of Bt crops (1,6,21), as Cry2Ab is known not to share the same binding sites with Cry1Ab or Cry1Ac in insects (22)(23)(24). The gene pyramiding strategy to combine Cry1Ac and Cry2Ab has proven to be effective in delaying the development of insect resistance to Bt toxins (25,26).…”

“…At present, the understanding of mechanisms of resistance to Bt toxins is primarily on the resistance to Cry1Ab and Cry1Ac. Understanding of the molecular mode of action of Cry2Ab and the mechanism of resistance to Cry2Ab remains very limited (22,28,32,37). As Cry2Ab is a major Cry toxin in the current Bt crops widely deployed in the field, it is desirable to understand the characteristics of Cry2Ab resistance in insects and the underlying mechanisms that confer resistance to Bt crops.…”

Potential shortfall of pyramided transgenic cotton for insect resistance management

“…Redundant killing could also be undermined if selection for resistance to one of the toxins causes cross-resistance to the other toxin (5,14,19,21). Cry1Ac and Cry2Ab have been considered a good combination for pyramided Bt crops because they have low amino acid homology and bind to different target sites in the larval midgut (22,23). However, in field-derived strains of H. zea and Helicoverpa armigera, responses to Cry1Ac and Cry2Ab were genetically correlated, indicating potential crossresistance (5,(24)(25)(26)(27).…”

Biological activity of Bt proteins expressed in different structures of transgenic corn against Spodoptera frugiperda

“…Furthermore, the high probability of cross-resistance between Cry1F and Cry1A.105, which share the same binding site, also contributes to the resistance evolution (HERNÁNDEZ-RODRÍGUEZ et al, 2013). In this context, the long-term efficacy of YieldGard VT PRO™ and PowerCore™ corn technologies is partly dependent of the Cry2Ab2 protein, which does not exhibit crossresistance with Cry1 proteins (FERRÉ & VAN RIE, 2002;CACCIA et al, 2010). However, Cry2Ab2 protein showed low toxicity against S. frugiperda (SIVASUPRAMANIAM et al, 2008).…”