Retargeting of the Bacillus thuringiensis toxin Cyt2Aa against hemipteran insect pests

Abstract

Although transgenic crops expressing Bacillus thuringiensis (Bt) toxins have been used successfully for management of lepidopteran and coleopteran pest species, the sap-sucking insects (Hemiptera) are not particularly susceptible to Bt toxins. To overcome this limitation, we demonstrate that addition of a short peptide sequence selected for binding to the gut of the targeted pest species serves to increase toxicity against said pest. Insertion of a 12-aa pea aphid gut-binding peptide by adding to or replacing amino acids in one of three loops of the Bt cytolytic toxin, Cyt2Aa, resulted in enhanced binding and toxicity against both the pea aphid, Acyrthosiphon pisum, and the green peach aphid, Myzus persicae. This strategy may allow for transgenic plant-mediated suppression of other hemipteran pests, which include some of the most important pests of global agriculture.

Keywords: aphid management; biotechnology; insect resistance.

Fig. 1.

Identification of APN as the GBP3.1 receptor. (A) A double derivatized synthetic GBP3.1, with a biotin residue at the N terminus and a UV-cross-linking residue (pbenzoyl-l- phenylalanine, BPa) replacing the tyrosine residue in the loop was used for receptor identification. (B) Coomassie Blue-stained SDS-polyacrylamide gel of proteins interacting with synthetic GBP3.1 in a pull-down experiment. The GBP3.1–receptor protein complex was pulled down by streptavidin agarose beads. Lane 1, GBP3.1 incubated with pea aphid gut and UV-cross-linked to interacting proteins. The ∼180-kDa protein (arrow) was consistently pulled down following UV-cross-linking to GBP3.1. Lane 2, GBP3.1 incubated with pea aphid gut, without UV-cross-linking. Lane 3, streptavidin beads with pea aphid gut only, showing the ∼140-kDa gut protein (arrow) pulled down by the streptavidin beads. Lane 4, streptavidin beads only. Molecular mass standards (M) are indicated. (C) Western blot analysis with anti-pea aphid APN antiserum confirmed the identity of the GBP3.1-interacting protein from pull-down experiments as APN. Lane 1, pea aphid gut proteins UV-cross-linked to GBP3.1. Lane 2, pea aphid gut proteins incubated with GBP3.1 without UV-cross-linking before pull-down. Lane 3, pea aphid gut proteins (positive control).

Fig. 2.

GBP3.1 specifically binds pea aphid BBMV. (A) Binding of GBP3.1-EGFP (GBP-EGFP; 50 nM) to pea aphid BBMV was out-competed by addition of synthetic GBP3.1 (50 µM). (B) GBP3.1 did not bind to BBMV proteins (10 µg) from H. virescens or L. decemlineata. Binding of EGFP and BBMV only were used as negative controls for both experiments. Positive control, GBP3.1-EGFP.

Fig. 3.

Ribbon structure of Cyt2Aa homology model showing the amino acid sequence of loops predicted to be on the surface of the molecule. The core Cyt2Aa structure is shown in green with loops of nonmodified Cyt2Aa shown in various colors. The aphid gut binding peptide GBP3.1 was inserted into each of these loops except for loop 6, which is implicated in Cyt toxin action (17). Arrows indicate sites of addition of GBP3.1 for CGALn toxins. Underlined amino acids were replaced with GBP3.1 in CGSLn toxins.

Fig. 4.

CGALn and CGSLn bind to pea aphid gut BBMV more strongly than Cyt2Aa. (A) Pull-down assays were conducted following incubation of activated Cyt2Aa, CGALn, or CGSLn and pea aphid gut BBMV. Membrane bound toxin was detected by Western blot with anti-Cyt2Aa antiserum. Western blot images (Lower) were scanned and processed using ImageJ to estimate the relative amount of activated toxin associated with pea aphid BBMV (histogram Upper). Relative toxin binding is shown for two pull-down assays with Western blot images shown for Replicate 1 in each case. (B) BIAcore surface plasmon resonance analysis of toxin binding to small unilamellar vesicles (SUV). Sensorgram showing the real-time interaction between 6 µM Cyt2Aa, CGAL1, CGAL3, CGAL4, CGSL1, CGSL4, and immobilized pea aphid gut membrane SUV. L1 chip surfaces were prepared with 4,000 RU of ligands. Data are shown for two independent experiments.

Fig. 5.

CGAL1 and CGSL4 cause extensive damage to second instar pea aphid midgut epithelium. (A) Light micrographs of midgut cross sections, and (B) transmission electron micrographs, showing the impact of CGAL1 and CGSL4 on the midgut epithelium relative to that of control and Cyt2Aa-fed aphids. Micrographs show the intact gut epithelial cell (EC), intact apical surface of the gut epithelial membrane with microvilli (M) projecting into the gut lumen (L) in aphids fed on control diet (Control). The microvilli of Cyt2Aa-fed aphids showed some damage, consistent with the low level of toxicity seen again A. pisum with this toxin. The integrity of the gut epithelia of aphids fed on CGAL1 and CGSL4 was severely compromised. LEC, lysed epithelial cells.