Dual resistance to Bacillus thuringiensis Cry1Ac and Cry2Aa toxins in Heliothis virescens suggests multiple mechanisms of resistance

Abstract

One strategy for delaying evolution of resistance to Bacillus thuringiensis crystal (Cry) endotoxins is the production of multiple Cry toxins in each transgenic plant (gene stacking). This strategy relies upon the assumption that simultaneous evolution of resistance to toxins that have different modes of action will be difficult for insect pests. In B. thuringiensis-transgenic (Bt) cotton, production of both Cry1Ac and Cry2Ab has been proposed to delay resistance of Heliothis virescens (tobacco budworm). After previous laboratory selection with Cry1Ac, H. virescens strains CXC and KCBhyb developed high levels of cross-resistance not only to toxins similar to Cry1Ac but also to Cry2Aa. We studied the role of toxin binding alteration in resistance and cross-resistance with the CXC and KCBhyb strains. In toxin binding experiments, Cry1A and Cry2Aa toxins bound to brush border membrane vesicles from CXC, but binding of Cry1Aa was reduced for the KCBhyb strain compared to susceptible insects. Since Cry1Aa and Cry2Aa do not share binding proteins in H. virescens, our results suggest occurrence of at least two mechanisms of resistance in KCBhyb insects, one of them related to reduction of Cry1Aa toxin binding. Cry1Ac bound irreversibly to brush border membrane vesicles (BBMV) from YDK, CXC, and KCBhyb larvae, suggesting that Cry1Ac insertion was unaffected. These results highlight the genetic potential of H. virescens to become resistant to distinct Cry toxins simultaneously and may question the effectiveness of gene stacking in delaying evolution of resistance.

FIG. 1.

Specific binding saturation of ¹²⁵I-Cry1Aa (A), ¹²⁵I-Cry1Ab (B), ¹²⁵I-Cry1Ac (C), and ¹²⁵I-Cry2Aa (D) to BBMV proteins from YDK (•), CXC (▿) or KCBhyb (▪) larvae. BBMV proteins (10 μg) were incubated with increasing amounts of labeled toxins for 1 h. Nonspecific binding was calculated in the presence of 1,000 nM of the respective unlabeled homologous toxin and was subtracted from total binding to obtain specific binding. Binding reactions were stopped by centrifugation. Bound labeled toxin (nanomolar concentration) was calculated using the RADLIG software. Each data point is a mean based on at least two experiments done in quadruplicate. Error bars depict standard deviation of the mean values.

FIG. 2.

Homologous binding competition of ¹²⁵I-Cry1Aa (A), ¹²⁵I-Cry1Ab (B), and ¹²⁵I-Cry1Ac (C) to BBMV proteins from YDK (•), CXC (▿), or KCBhyb (▪) larvae. BBMV proteins (10 μg) were incubated with labeled toxins (0.1 nM) in the presence of increasing concentrations of homologous unlabeled competitor for 1 h. Binding reactions were stopped by centrifugation. Binding was expressed as a percentage of the amount of toxin bound in the absence of competitor. Each data point is a mean based on at least two independent trials done in duplicate. Error bars depict standard deviation of the mean values.

FIG. 3.

Irreversible binding of ¹²⁵I-Cry1Ac toxin to BBMV from YDK (•), CXC (▿), or KCBhyb (▪) larvae. Binding reactions were started by mixing BBMV proteins (10 μg) with 0.1 nM ¹²⁵I-Cry1Ac. One hour after initiation of the binding reaction, 1,000 nM unlabeled Cry1Ac was added to the mixture. The time on the x axis represents postincubation time after addition of the unlabeled competitor. Binding was expressed as a percentage of the amount of toxin bound before addition of competitor. Each data point is a mean based on data from two independent trials done in dupliacte. Error bars depict standard deviation of the mean values.

FIG. 4.

Binding of biotinylated Cry1Ac (A) and Cry1Fa (B) toxins to BBMV from YDK, YHD2, and KCBhyb larvae. Toxins (12 nM) were incubated with BBMV proteins (20 μg) for 1 h. Binding reactions were stopped by centrifugation, and washed pellets were separated by SDS-10% PAGE and transferred to polyvinylidene difluoride filters. Biotinylated toxins were detected with streptavidin-peroxidase conjugate and enhanced chemiluminescence.