Domain III of Bacillus thuringiensis Cry1Ie Toxin Plays an Important Role in Binding to Peritrophic Membrane of Asian Corn Borer

Abstract

The insecticidal IE648 toxin is a truncated Cry1Ie protein with increased toxicity against Asian corn borer (ACB). Cry toxins are pore-forming toxins that disrupt insect midgut cells to kill the larvae. However, the peritrophic membrane (PM) is an important barrier that Cry toxins must cross before binding to midgut cells. Previously, it was shown that Cry toxins are able to bind and accumulate in the PM of several lepidopteran insects. Binding of IE648 toxin to PM of ACB was previously reported and the goal of the current work was the identification of the binding region between Cry1Ie and the PM of ACB. Homologous competition binding assays showed that this interaction was specific. Heterologous competition binding assays performed with different fragments corresponding to domain I, domain II and domain III allowed us to identify that domain III participates in the interaction of IE648 with the PM. Specifically, peptide D3-L8 (corresponding to Cry1Ie toxin residues 607 to 616), located in an exposed loop region of domain III is probably involved in this interaction. Ligand blot assays show that IE648 interact with chitin and PM proteins with sizes of 30, 32 and 80 kDa. The fact that domain III interacts with proteins of similar molecular masses supports that this region of the toxin might be involved in PM interaction. These data provide for the first time the identification of domain III as a putative binding region between PM and 3D-Cry toxin.

The C-Terminal Domain of the Bacillus thuringiensis Cry4Ba Mosquito-Specific Toxin Serves as a Potential Membrane Anchor

“…Accordingly, the lack of this exclusive β-strand might result in improper folding of the isolated Cry1Ab- and Cry1Ac-DIII truncates, leading to the formation of an insoluble aggregate of mis-folded proteins. This notion has been supported by another recent study for overproduction of the 28-kDa His-tagged Cry1Ie fragment which was also found to be in the form of a mis-folded insoluble aggregate [ 27 ].…”

“…Unlike DI and DII of Cry toxins, the role of DIII remains ambiguous although it has been suggested to be involved in maintaining the structural integrity of the active toxin [ 7 , 21 , 22 , 23 ] or in determining binding specificity [ 24 , 25 , 26 ]. Recently, DIII was also suggested to participate in the interaction of Cry1Ie with the larval peritrophic membrane of Ostrinia furnacalis (Asian corn borer) [ 27 ]. In our previous study, the 21-kDa isolated DIII fragment from the Cry4Ba mosquito-active toxin was shown to be capable of binding along the apical microvilli of A. aegypti larval midgut, conceivably participating in toxin interactions with either lipid membranes or target protein receptors [ 28 ].…”

Evolution of Asian Corn Borer Resistance to Bt Toxins Used Singly or in Pairs

“…In a review evaluating cross-resistance in seven pairs of Bt toxins, >100-fold cross-resistance was associated with >80% amino acid sequence similarity of domain II in nearly all cases [14]. However, the amino acid sequence similarity between Cry1Ie and Cry1F was 70% in domain III (Table 2), which also is involved in toxin binding [14,30]. It remains to be determined if the resistance and cross-resistance seen here are caused by reduced toxin binding or by other mechanisms.…”

Bacillus: A Biological Tool for Crop Improvement through Bio-Molecular Changes in Adverse Environments

“…colonize plant parts, including the phyllosphere, and larvae and/or adult pests ingest the Bacillus -containing plant tissues during feeding. A primary site of bacterial infection begins with extensive damage to the larval midgut epithelium by bacterial crystal proteins, which interact with chitin and peritrophic membranes (Vachon et al, 2012 ; Feng et al, 2015 ; Figure 1 ). During later stages of infection, Bacillus spp.…”