A mutation in Plutella xylostella ABCC2 causes resistance to Bacillus thuringiensis Cry1Ac by interfering with its receptor function

Plutella xylostella is the most destructive pest in cruciferous crops and Bacillus thuringiensis (Bt) has been widely used as a control measure. However, sequential application of Bt insecticides resulted in development of Cry1Ac-resistant P. xylostella. In order to use Bt effectively and continuously, it is urgent to address the mechanism of how P. xylostella has acquired resistance against Cry1Ac. A recent report suggested a mutation in the ATP-binding cassette (ABC) transporter gene (ABCC2) was related to Cry1Ac-resistance. However, there was no direct evidence of interaction between ABCC2 and resistance. In this study, we examined the receptor function of PxABCC2 against Cry1Ac toxin to understand the interaction resulting in resistance. We overexpressed PxABCC2 ectopically on Sf9 cells using a baculovirus (AcMNPVs) expression system, and assessed Cry1Ac susceptibility. 10nM of Cry1Ac had an effect on Sf9 cells that expressed PxABCC2 and swelled 53.84% of the effected cells. We also established S9 cells expressed PxABCC2 with a mutation and treated with Cry1Ac, resulted in no swelling cells, and no susceptibilities were observed. These results suggest PxABCC2 functions as a receptor and the mutation of PxABCC2 caused a loss of receptor function, which is responsible for Cry1Ac resistance. Furthermore, we visualized Cry1Ac and PxABCC2 using double immunostaining methods to observe the interaction between them. When Cry1Ac was treated with Sf9, cells expressed PxABCC2, Cry1Ac signals were scattered to surround PxABCC2 signals. Cry1Ac bound to PxABCC2 on the cell membrane. Conversely, no Cry1Ac signals were observed around mutated PxABCC2 signals, as no susceptibility against Cry1Ac was observed. These results indicate that PxABCC2 would lose its Cry1Ac binding ability when a mutation occurs, and this results in the loss of receptor function. This study demonstrates a mutation in PxABCC2 which involves Cry1Ac resistance and the loss of bindings between cry toxins and receptors might be mode of action to acquire resistance.