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-R1 is a binding protein for Bt Cry1A toxins in midgut epithelia of tobacco hornworm (Manduca sexta). We previously identified the Bt-R1 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 ␤-strands and random coils with unfolded structure. CR12-MPED peptide bound brush border membrane vesicles with high affinity (Kd ‫؍‬ 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 M)-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.Cry insecticidal protein ͉ synergist B acillus thuringiensis (Bt) Cry1A proteins are pore-forming toxins that are specifically toxic to insect larvae in the order Lepidoptera. This family of proteins is widely used for insect control with Bt-transgenic crops, particularly cotton, and Bt microbial pesticides. Two issues on the deployment of Bt crops are the evolution of resistance in target pests and the lower level of control of specific target pests. Although insect resistance to Bt cotton has not caused a control failure in the field, there is a natural difference in lepidopteran susceptibility that effects insect control. For example, Bt cotton expressing only a Cry1A protein is highly effective in controlling tobacco budworm (Heliothis virescens) populations, whereas control of cotton bollworm (Helicoverpa zea) larvae is only achieved after additional insecticide treatment (1).The mode of action for Cry1A toxins includes sequential steps that determine their specificity. After ingestion by the lepidopteran larvae, Cry1A proteins are solubilized and activated to a toxic form by the insect digestive fluids. After crossing the peritrophic matrix, activated toxins bind to specific proteins on the midgut microvilli. According to a current model (2), monomeric toxin binds a cadherin, facilitating further processing necessary for toxin oligomerization. Toxin oligomers display high-affinity binding to proteins that are attached to the cell membrane by a glycosylphosphatidylinositol anchor, such as aminopeptidase or alkaline phosphatase. This binding and t...

Anopheles gambiae Cadherin AgCad1 Binds the Cry4Ba Toxin of Bacillus thuringiensis israelensis and a Fragment of AgCad1 Synergizes Toxicity

“…In contrast, the comparable peptide, CR12-MPED from Bt-R 1 , gave a strong signal on dot blots and bound toxin at a high-affinity site (K d = 9 nM) and low-affinity sites (K d = 1 μM) (23). Although we were unable to quantify the dot blot binding data shown in Figure 7 and calculate the affinity value, qualitatively the data suggest that the affinity of Cry4Ba for the CR11-MPED peptide is much lower than the affinity of Cry1Ab for CR12-MPED from Bt-R 1 .…”

“…We (23) also reported that Cry1Ab binding to CR12-MPED was necessary for toxin enhancement properties. Based on the data in Chen et al (23) and evidence that Cry4Ba bound a C-terminal fragment of AgCad1 ( Figure 6, lanes 2 -8), we expressed the comparable CR11-MPED region from AgCad1 in E. coli and tested the purified 24 kDa peptide with Cry4Ba in bioassays of mosquito larvae. As shown in Figure 7, The CR11-MPED peptide increased the toxicity of Cry4Ba against A. gambiae fourth instar larvae.…”

“…The CR11-MPED region was overexpressed by induction with 1 mM isopropyl β-Dthiogalactopyranoside (IPTG) when the culture OD 600 reached 0.5-0.6. The expression and purification protocols are described in a previous paper (23). Polyclonal antiserum against purified CR11-MPED, referred to as α-AgCad1 serum, was produced in New Zealand White rabbits at the Animal Resources Facility at the University of Georgia.…”

“…The truncated cadherin peptides, CR11-MPED and TM-Cyto, were expressed in E. coli and purified as previously described (23). Various amounts of purified peptides were dotted on PVDF filters and probed with 125 I-labeled Cry4Ba or with 125 I-labeled Cry4Ba plus unlabeled Cry4Ba (1000-fold) and exposed to X-ray film as above.…”

“…Typically, a peptide fragment of the receptor (21) or a phage mimic of the receptor (20) attenuates Cry in vivo toxicity to larvae. Recently, we reported an opposite effect in which a fragment of Bt-R 1 cadherin, the Cry1A receptor from Manduca sexta (21,22), not only bound toxin but enhanced Cry1A toxicity against lepidopteran larvae (23). If the binding residues within cadherin repeat 12 (CR) were removed, the resulting peptide lost the ability to bind toxin and lost its function as a toxin synergist.…”