Binding and Synergizing Motif within Coleopteran Cadherin Enhances Cry3Bb Toxicity on the Colorado Potato Beetle and the Lesser Mealworm.

Youngjin Park,Milton Taylor,Suresh Ambati,Michael J Adang,Gang Hua

doi:10.3390/toxins11070386

Abstract

Cry3Bb toxin from Bacillus thuringiensis is an important insecticidal protein due to its potency against coleopteran pests, especially rootworms. Cadherin, a protein in the insect midgut epithelium, is a receptor of Cry toxins; in some insect species toxin-binding domains of cadherins-synergized Cry toxicity. Previously, we reported that the DvCad1-CR8-10 fragment of Diabrotica virgifera virgifera cadherin-like protein (GenBank Accession #EF531715) enhanced Cry3Bb toxicity to the Colorado Potato Beetle (CPB), Leptinotarsa decimlineata (L. decimlineata). We report that individual CR domains of the DvCad1-CR8-10 fragment were found to have strong binding affinities to α-chymotrypsin-treated Cry3Bb. The dissociation constant (Kd) of Cry3Bb binding to the CR8, CR9, and CR10 domain was 4.9 nM, 28.2 nM, and 4.6 nM, respectively. CR8 and CR10, but not CR9, enhanced Cry3Bb toxicity against L. decimlineata and the lesser mealworm Alphitobius diaperinus neonates. In-frame deletions of the DvCad1-CR10 open reading frame defined a high-affinity binding and synergistic site to a motif in residues I1226–D1278. A 26 amino acid peptide from the high affinity Cry3Bb-binding region of CR10 functioned as a Cry3Bb synergist against coleopteran larvae.

Highlights

The Cry3 insecticidal proteins of Bacillus thuringiensis (Bt) are toxic to larvae of beetles (Order: Coleoptera) that are some of the most important agricultural pests worldwide
We investigated the location of Cry3Bb sites in DvCad1-CR8-10 and demonstrated that CR8, CR9, and CR10 each had a high-affinity Cry3Bb binding site and that CR8 and CR10 enhanced that CR8, CR9, and CR10 each had a high-affinity Cry3Bb binding site and that CR8 and CR10
1A Cry3Bb shows the cadherin repeats (CR) with constructs made and synergized toxicity to the coleopteran larvae constructs made to to identify the regions of CR8-10 peptide involved in the binding and synergy of Cry3 toxins

Summary

Introduction

The Cry insecticidal proteins of Bacillus thuringiensis (Bt) are toxic to larvae of beetles (Order: Coleoptera) that are some of the most important agricultural pests worldwide. The Cry3Aa protein was shown to be produced in genetically modified potatoes for the control of the Colorado potato beetle (CPB) [1,2]. CPB is a serious pest of potato, due to development of resistance to many chemical insecticides. Alyokhin et al (2007) [3] reported increasing resistance in CPB against neonicotinoids, a chemical class that had been effective in controlling CPB populations. NovodorTM , a Bt biopesticide containing Cry3Aa crystal proteins and spores, is active against CPB and is reportedly effective against the first and second larval stages but not in the third instar and later stages [4,5]

Methods

Results

Discussion

Conclusion