Binding site concentration explains the differential susceptibility of Chilo suppressalis and Sesamia inferens to Cry1A-producing rice

Abstract

Chilo suppressalis and Sesamia inferens are two important lepidopteran rice pests that occur concurrently during outbreaks in paddy fields in the main rice-growing areas of China. Previous and current field tests demonstrate that the transgenic rice line Huahui 1 (HH1) producing a Cry1Ab-Cry1Ac hybrid toxin from the bacterium Bacillus thuringiensis reduces egg and larval densities of C. suppressalis but not of S. inferens. This differential susceptibility to HH1 rice correlates with the reduced susceptibility to Cry1Ab and Cry1Ac toxins in S. inferens larvae compared to C. suppressalis larvae. The goal of this study was to identify the mechanism responsible for this differential susceptibility. In saturation binding assays, both Cry1Ab and Cry1Ac toxins bound with high affinity and in a saturable manner to midgut brush border membrane vesicles (BBMV) from C. suppressalis and S. inferens larvae. While binding affinities were similar, a dramatically lower concentration of Cry1A toxin binding sites was detected for S. inferens BBMV than for C. suppressalis BBMV. In contrast, no significant differences between species were detected for Cry1Ca toxin binding to BBMV. Ligand blotting detected BBMV proteins binding Cry1Ac or Cry1Ca toxins, some of them unique to C. suppressalis or S. inferens. These data support that reduced Cry1A binding site concentration is associated with a lower susceptibility to Cry1A toxins and HH1 rice in S. inferens larvae than in C. suppressalis larvae. Moreover, our data support Cry1Ca as a candidate for pyramiding efforts with Cry1A-producing rice to extend the activity range and durability of this technology against rice stem borers.

FIG 1

Saturation binding experiments with BBMV proteins from C. suppressalis (striped stem borer [SSB]) and S. inferens (pink stem borer [PSB]) and increasing amounts of ¹²⁵I-Cry proteins. Specific binding of ¹²⁵I-Cry1Ab (A), ¹²⁵I-Cry1Ac (B), or ¹²⁵I-Cry1Ca (C) was calculated by subtracting nonspecific from total binding in the absence of unlabeled competitor. Data shown are the means and standard errors from at least two independent experiments performed in duplicate for each concentration of radiolabeled Cry toxin. The curves shown are derived from fitting the binding data to a model that considers a single population of binding sites as the best-fitting model.

FIG 2

Ligand blot analysis of ¹²⁵I-Cry1Ac (A) and ¹²⁵I-Cry1Ca (B) toxin binding to BBMV proteins from C. suppressalis (SSB) and S. inferens (PSB). Proteins (20 μg) were separated by SDS–8% PAGE, transferred to a PVDF filter, and then probed with ¹²⁵I-Cry1Ac or ¹²⁵I-Cry1Ca toxins. Bound toxin was detected by autoradiography at −80°C. Arrows indicate main BBMV proteins recognized by each toxin in each BBMV sample.