Binding site alteration is responsible for field-isolated resistance to Bacillus thuringiensis Cry2A insecticidal proteins in two Helicoverpa species.

Silvia Caccia,William James,Juan Ferré,Nadine Bautsoens,Sharon Downes,Jeroen Van Rie,Rod J Mahon,Carmen Sara Hernández-Rodríguez

doi:10.1371/journal.pone.0009975

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.

Highlights

The agronomical impact of bioinsecticides based on Bacillus thuringiensis (Bt) has increased significantly since the late 1950s when commercial sprays based on this bacterium were first developed
We previously demonstrated for both species that these Cry2Ab resistant insects are susceptible to Cry1Ac [26,27], and for H. armigera that Cry2Ab resistant insects are cross-resistant to Cry2Aa [26]
Susceptibility assays Surface treatment bioassays performed on neonates of Cry2Ab resistant H. armigera and H. punctigera showed that these strains were completely resistant to the maximum concentration of Cry2Ab employed (Table 1)

Summary

Introduction

The agronomical impact of bioinsecticides based on Bacillus thuringiensis (Bt) has increased significantly since the late 1950s when commercial sprays based on this bacterium were first developed. The major current interest of Bt insecticidal proteins (Cry proteins) resides on the large scale use of insect-resistant engineered plants expressing these proteins Such genetically modified Bt-crops today represent the most widely adopted transgenic crops after those with herbicide tolerance [2]. Cry 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. Colonies established from survivors of F2 screens against Cry2Ab are highly resistant to this toxin, but susceptible to Cry1Ac

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Conclusion