Synergism of Bacillus thuringiensis toxins by a fragment of a toxin-binding cadherin

Abstract

The insecticidal crystal proteins produced by Bacillus thuringiensis (Bt) are broadly used to control insect pests with agricultural importance. The cadherin Bt-R(1) is a binding protein for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R(1) region most proximal to the cell membrane (CR12-MPED) as the essential binding region required for Cry1Ab-mediated cytotoxicity. Here, we report that a peptide containing this region expressed in Escherichia coli functions as a synergist of Cry1A toxicity against lepidopteran larvae. Far-UV circular dichroism and (1)H-NMR spectroscopy confirmed that our purified CR12-MPED peptide mainly consisted of beta-strands and random coils with unfolded structure. CR12-MPED peptide bound brush border membrane vesicles with high affinity (K(d) = 32 nM) and insect midgut microvilli but did not alter Cry1Ab or Cry1Ac binding localization in the midgut. By BIAcore analysis we demonstrate that Cry1Ab binds CR12-MPED at high (9 nM)- and low (1 microM)-affinity sites. CR12-MPED-mediated Cry1A toxicity enhancement was significantly reduced when the high-affinity Cry1A-binding epitope ((1416)GVLTLNIQ(1423)) within the peptide was altered. Because the mixtures of low Bt toxin dose and CR12-MPED peptide effectively control target insect pests, our discovery has important implications related to the use of this peptide to enhance insecticidal activity of Bt toxin-based biopesticides and transgenic Bt crops.

Fig. 1.

Purified CR12-MPED has unfolded structure. (A) Structure of BtR₁ with numbers of ectodomains reported to contain Cry1Ab binding sites in black. (B) SDS/PAGE of lysates from E. coli transformed with pET-CR12-MPED before (IPTG-0h) and 6 h after induction (IPTG-6h) and purified CR12-MPED obtained from a Ni²⁺-column. (C) Far-UV CD spectrum (190–250 nm) of CR12-MPED peptide, indicative of a mixture of 15% helix, 37% β-strands, and 48% random coils. (D) Chemical shifts of amide protons (red boxed) on CR12-MPED backbone clustered within a narrow range of ≈7–8.5 ppm in 600 MHz ¹H-NMR spectroscopy, evidence of the unfolded state of this peptide.

Fig. 2.

CR12-MPED peptide enhances Cry1A toxicity toward lepidopteran larvae in diet surface bioassays. Cry1A toxins were mixed with purified CR12-MPED at toxin:peptide ratios of 1:0, 1:1, 1:10, and 1:100. Control treatments included buffer and CR12-MPED alone. (A) Cry1Ab plus CR12-MPED against M. sexta. (B) Cry1Ac plus CR12-MPED against M. sexta. (C) Cry1Ac plus CR12-MPED against H. virescens. (D) Cry1Ac plus CR12-MPED against H. zea. Each column presents data for the mean ± standard errors from three replicate bioassays with 32 larvae per treatment. For each species, an asterisk above the column indicates that the mortality of Cry1A plus CR12-MPED treatment showed significant difference from Cry1A alone treatment with the same Cry1A dosage (Tukey's test, α = 0.05).

Fig. 3.

Toxicity enhancement correlates with Cry1Ab binding to CR12-MPED. (A) Peptides were immobilized on a BIAcore chip via their C-terminal His tags. BIAcore response curves are shown for the indicated toxin concentrations. (Inset) CR12-MPED or CR12-MPED/Del (5 μg) were spotted in duplicate on a polyvinylidene difluoride filter and probed with ¹²⁵I-Cry1Ab alone or in the presence of a 1,000-fold excess of unlabeled Cry1Ab. (B) Cry1Ab toxicity against M. sexta larvae when combined with either CR12-MPED or CR12-MPED/Del at a toxin:peptide mass ratio of 1:100.

Fig. 4.

CR12-MPED and CR12-MPED/Del peptides bind to BBMV. (A and B) Dot blotted M. sexta or H. virescens BBMVs (5 μg) were probed with ¹²⁵I-CR12-MPED (3.3 ng/ml) alone or in the presence of 500-fold unlabeled peptide (A), or ¹²⁵I-Cry1Ab or ¹²⁵I-Cry1Ac (3.3 ng/ml) alone or in the presence of 100-fold unlabeled CR12-MPED (B). (C) Saturation assays of ¹²⁵I-labeled CR12-MPED or CR12-MPED/Del binding to BBMV prepared from M. sexta. Data are from three experiments conducted in duplicate. Individual points are the mean amounts of peptide specifically bound-calculated after subtraction of nonspecific binding in the presence of a 500-fold excess homologous competitor from total binding.

Fig. 5.

Addition of CR12-MPED does not alter localization of Cry1A toxin binding in midgut sections. (A–E) Midgut sections were treated with TAMRA-labeled Cry1Ab (A) or Cry1Ac (D) alone or in the presence of a 20-fold excess of CR12-MPED (B and E, respectively) or CR12-MPED/Del (C). (F) Control treatment with TAMRA-labeled BSA. (G) Binding of TAMRA-labeled CR12-MPED. (H) Antiserum against CR12-MPED specifically immunostained the apex and base of microvilli. ∗, TAMRA-labeled; AMv, apical tip of microvilli; BL, basal lamina; BMv, base of microvilli; Nu, nucleus; PM, peritrophic matrix. (Scale bar, 10 μm.)