Reduced toxin binding associated with resistance to Vip3Aa in the corn earworm (Helicoverpa zea)

Abstract

Helicoverpa zea is a major crop pest in the United States that is managed with transgenic corn and cotton that produce insecticidal proteins from the bacterium, Bacillus thuringiensis (Bt). However, H. zea has evolved widespread resistance to the Cry proteins produced in Bt corn and cotton, leaving Vip3Aa as the only plant-incorporated protectant in Bt crops that consistently provides excellent control of H. zea. The benefits provided by Bt crops will be substantially reduced if widespread Vip3Aa resistance develops in H. zea field populations. Therefore, it is important to identify resistance alleles and mechanisms that contribute to Vip3Aa resistance to ensure that informed resistance management strategies are implemented. This study is the first report of reduced binding of Vip3Aa to midgut receptors associated with resistance.

Keywords: Bacillus thuringiensis; Helicoverpa zea; Vip3Aa; altered binding; proteolytic processing; resistance.

Fig 1

Gut pH measurements from five gut sections of CEW-SS (A) and CEW-Vip-RR (B) fourth-instar H. zea larvae. Groups of nine larvae were fed a droplet of sucrose water containing Vip3Aa protoxin (Vip3Aa-Pro), Vip3Aa-activated toxin (Vip3Aa-Act), Cry1Ac protoxin (Cry1Ac-Pro), or with no treatment as a control (Control). Data plotted are the mean pH values and standard errors for each treatment in different gut regions.

Fig 2

Incubation of Vip3Aa or Cry1Ac protoxin with midgut fluids extracted from CEW-SS and CEW-Vip-RR H. zea larvae. Protoxin was incubated with midgut fluid extracts, and aliquots were taken at various timepoints. Vip3Aa (A) and Cry1Ac (B) samples were resolved using SDS-PAGE and stained with ProtoBlue safe; M, protein marker; I, protoxin input. Percent Vip3Aa (C) and Cry1Ac (D) protoxin processing over time by midgut fluid extracts from CEW-SS and CEW-Vip-RR larvae. Plotted is the percent intensity of the protoxin bands for each timepoint relative to the intensity of the input protoxin band as estimated by densitometry. Each point and standard error bar are representative of three independent experiments. No differences in the rate of processing were detected (generalized linear mixed model; Tukey post hoc; α = 0.05).

Fig 3

Binding of biotinylated Vip3Aa toxin bound to brush border membrane vesicles from CEW-SS and CEW-Vip-RR H. zea larvae. Brush border membrane vesicles were incubated with biotinylated Vip3Aa toxin with (non-specific binding) or without (total binding) a 100-fold molar excess of unlabeled Vip3Aa toxin. Bound biotinylated toxin was recovered by centrifugation, and final pellets were suspended in 1× sample buffer before visualizing by western blotting (A). V, 10 ng biotinylated Vip3Aa toxin; 1, CEW-SS total binding; 2, CEW-SS non-specific binding; 3, CEW-Vip-RR total binding; 4, CEW-Vip-RR non-specific binding. Densitometry was used to estimate band intensity, and specific binding was calculated by standardizing band intensity relative to the intensity of 10 ng of Vip3Aa toxin, then subtracting non-specific binding from total binding (B). Bars and standard errors are representative of the mean of two biologically independent experiments (different BBMV preparations) each with two technical replications. Specific binding of Vip3Aa was significantly reduced in CEW-Vip-RR relative to CEW-SS (independent t-test; α = 0.05).

Fig 4

Specific binding of radiolabeled Vip3Aa toxin (¹²⁵I-Vip3Aa) to brush border membrane vesicles from CEW-SS and CEW-Vip-RR H. zea larvae in saturation assays. Brush border membrane vesicles were incubated with increasing amounts of ¹²⁵I-Vip3Aa with (non-specific binding) or without (total binding) a 100-fold molar excess of unlabeled Vip3Aa. Bound ¹²⁵I-Vip3Aa was recovered by centrifugation and detected using a gamma counter. Specific binding was calculated by subtracting the amount of non-specifically bound ¹²⁵I-Vip3Aa from the total bound ¹²⁵I-Vip3Aa. Each point and standard error bar are representative of at least two biologically independent experiments (different BBMV preparations) each with two technical replications. Binding of ¹²⁵I-Vip3Aa was significantly reduced in CEW-Vip-RR (generalized linear model; Tukey-Kramer post hoc; α = 0.05).

Fig 5

Competition assays of ¹²⁵I-Vip3Aa (A) or ¹²⁵I-Cry1Ac (B) ligands binding to brush border membrane vesicles from CEW-SS and CEW-Vip-RR H. zea larvae. Brush border membrane vesicles were incubated with radiolabeled toxin and increasing amounts of unlabeled homologous competitor. Amounts of competitor tested were selected on the basis of labeled toxin input and consistency of competition. Bound toxin was recovered through centrifugation, and radiolabeled toxin was detected by a gamma counter. Each point and standard error bar are representative of at least two biologically independent experiments (different BBMV preparations), each technically replicated two times.

Fig 6

Alkaline phosphatase and aminopeptidase N activity from midgut homogenates and BBMV of two H. zea strains (black stripes: homogenate, CEW-SS; black solid: homogenate, CEW-Vip-RR; gray stripes: BBMV, CEW-SS; gray solid: BBMV, CEW-Vip-RR). Enzyme activity was measured in samples using leucine p-nitroanilide and p-nitrophenyl phosphate as substrates. Bars and standard errors represent three technical replications of two different BBMV preparations or their corresponding midgut homogenates. No significant differences in enzymatic activity were detected between CEW-SS and CEW-Vip-RR homogenates (independent t-test; α = 0.05).