Toxicity and mode of action of Bacillus thuringiensis Cry proteins in the Mediterranean corn borer, Sesamia nonagrioides (Lefebvre)

Abstract

Sesamia nonagrioides is one of the most damaging pests of corn in Spain and other Mediterranean countries. Bt corn expressing the Bacillus thuringiensis Cry1Ab toxin is being grown on about 58,000 ha in Spain. Here we studied the mode of action of this Cry protein on S. nonagrioides (binding to specific receptors, stability of binding, and pore formation) and the modes of action of other Cry proteins that were found to be active in this work (Cry1Ac, Cry1Ca, and Cry1Fa). Binding assays were performed with (125)I- or biotin-labeled toxins and larval brush border membrane vesicles (BBMV). Competition experiments indicated that these toxins bind specifically and that Cry1Aa, Cry1Ab, and Cry1Ac share a binding site. Cry1Ca and Cry1Fa bind to different sites. In addition, Cry1Fa binds to Cry1A's binding site with very low affinity and vice versa. Binding of Cry1Ab and Cry1Ac was found to be stable over time, which indicates that the observed binding is irreversible. The pore-forming activity of Cry proteins on BBMV was determined using the voltage-sensitive fluorescent dye DiSC(3)(5). Membrane permeability increased in the presence of the active toxins Cry1Ab and Cry1Fa but not in the presence of the nonactive toxin Cry1Da. In terms of resistance management, based on our results and the fact that Cry1Ca is not toxic to Ostrinia nubilalis, we recommend pyramiding of Cry1Ab with Cry1Fa in the same Bt corn plant for better long-term control of corn borers.

FIG. 1.

Binding of ¹²⁵I-Cry1Ab (A), ¹²⁵I-Cry1Ac (B), and ¹²⁵I-Cry1Ca (C) to BBMV from S. nonagrioides with different concentrations of unlabeled competitor. ▴ and solid line, Cry1Aa; • and dashed line, Cry1Ab; ▾ and dotted line, Cry1Ac; ⧫ and dashed and dotted line, Cry1Ca; ▪, Cry1Fa.

FIG. 2.

Dissociation kinetics of the binding of ¹²⁵I-Cry1Ab (• and dashed line) and ¹²⁵I-Cry1Ac (▾ and dotted line) to S. nonagrioides BBMV. After 1 h of incubation of the BBMV with the labeled toxins, the assay was initiated by adding an excess of unlabeled homologous competitor.

FIG. 3.

Binding of biotin-labeled Cry1Fa (A) or Cry1Ab (B) to BBMV from S. nonagrioides in competition assays. (A) Lane MW, molecular weight markers (Precision Plus protein dual-color standard; Bio-Rad Laboratories GmbH, Munich, Germany); lane BBMV, BBMV without toxin; lane 1Fa*, BBMV with biotin-labeled Cry1Fa (Cry1Fa*); lane 1Fa, competition assay with Cry1Fa* and Cry1Fa (1,000×); lane 1Ab, competition assay with Cry1Fa* and Cry1Ab (1,000×); lane 1Ac, competition assay with Cry1Fa* and Cry1Ac (1,000×). (B) Lane MW, molecular weight markers; lane 1Ab*, BBMV with biotin-labeled Cry1Ab (Cry1Ab*); lane 1Ab 2×, competition assay with Cry1Ab* and Cry1Ab (2×); lane 1Ab 5×, competition assay with Cry1Ab* and Cry1Ab (5×); lane 1Ac 2×, competition assay with Cry1Ab* and Cry1Ac (2×); lane 1Ac 5×, competition assay with Cry1Ab* and Cry1Ac (5×); lane 1Fa 2×, competition assay with Cry1Ab* and Cry1Fa (2×); lane 1Fa 5×, competition assay with Cry1Ab* and Cry1Fa (5×); lane 1Ab*, BBMV with Cry1Ab*.

FIG. 4.

Effects of different Cry proteins on K⁺ permeability in BBMV from S. nonagrioides. BBMV that were resuspended in 300 mM mannitol-1 mM KCl-10 mM HEPES-Tris (pH 7.2) were preincubated for 30 min with just the buffer in which the toxins were dissolved (control) or with Cry1Ab (15 μg/mg BBMV protein) (B), with Cry1Fa (25 μg/mg BBMV protein) (C), or with Cry1Da (40 μg/mg BBMV protein) (D). BBMV were then diluted in a spectofluorometer cuvette with the resuspension buffer supplemented with 6 μM DiSC₃(5). For panel A, 23 μM valinomycin was added to the diluting buffer. KCl was added at the times indicated by the arrows to obtain final concentrations of 20, 40, 60, and 80 mM. Each trace represents the means ± standard errors for different experiments performed six times (control) or three times. AU, arbitrary units.