A Bacillus thuringiensis Cry protein controls soybean cyst nematode in transgenic soybean plants.

Plant-parasitic nematodes (PPNs) are economically important pests of agricultural crops, and soybean cyst nematode (SCN) in particular is responsible for a large amount of damage to soybean. The need for new solutions for controlling SCN is becoming increasingly urgent, due to the slow decline in effectiveness of the widely used native soybean resistance derived from genetic line PI 88788. Thus, developing transgenic traits for controlling SCN is of great interest. Here, we report a Bacillus thuringiensis delta-endotoxin, Cry14Ab, that controls SCN in transgenic soybean. Experiments in C. elegans suggest the mechanism by which the protein controls nematodes involves damaging the intestine, similar to the mechanism of Cry proteins used to control insects. Plants expressing Cry14Ab show a significant reduction in cyst numbers compared to control plants 30 days after infestation. Field trials also show a reduction in SCN egg counts compared with control plants, demonstrating that this protein has excellent potential to control PPNs in soybean.

Enhanced insecticidal activity of Bacillus thuringiensis using a late embryogenesis abundant peptide co-expression system.

Bacillus thuringiensis (Bt) is a ubiquitous, gram positive, spore-forming bacterium that synthesizes parasporal crystalline inclusions containing crystal protein, some of which are toxic against a wide range of insect orders like caterpillars, beetles, and flies, including mosquitoes. Regarding the biological control of insects, Bt is the mostly used microorganism worldwide and also alternatives to chemical insecticides for environmental conservation. Some strains of Bt are showing a promising activity against a wide variety of mosquito like Aedes, Culex, and Anopheles and so on with extremely damages in the larval midgut and ultimate death. Here, we introduced a late embryogenesis abundant (LEA) peptide co-expression system based on the expression vector pHT01 with a strong σA-dependent promoter to enhance the expression of insecticidal crystal proteins in native Bt. Two types of LEA peptide (LEA-II and LEA-K) were designed based on the sequence of group-3 LEA protein, which consists of a repetitive sequence of 11 amino acids. The LEA-II mediated co-expression system enhanced the production of crystal protein 3-fold after 12 h of induction of the peptide with 0.5 mM IPTG. Enhanced expression of crystal protein was confirmed by bioassay using 4th instar Aedes albopictus larvae. This unique approach has great potential to produce bio-pesticides by enhanced crystal protein expression not only for mosquitoes but also for other insects.

Bacillus thuringiensis Cry1Ab Domain III ß-16 Is Involved in Binding to Prohibitin, Which Correlates with Toxicity against Helicoverpa armigera (Lepidoptera: Noctuidae).

Helicoverpa armigera is a major insect pest of several crops worldwide. This insect is susceptible to some Bacillus thuringiensis (Bt) Cry insecticidal proteins expressed in transgenic crops or used in biopesticides. Previously, we identified H. armigera prohibitin (HaPHB) as a Cry1Ac-binding protein. Here, we further analyzed the potential role of PHB as a Cry toxin receptor in comparison to cadherin (CAD), well recognized as a Cry1Ac receptor. HaPHB-2 midgut protein and HaCAD toxin-binding region (TBR) fragment from H. armigera were expressed in Escherichia coli cells, and binding assays with different Cry1 toxins were performed. We demonstrated that Cry1Ab, Cry1Ac, and Cry1Fa toxins bound to HaPHB-2 in a manner similar to that seen with HaCAD-TBR. Different Cry1Ab mutant toxins located in domain II (Cry1AbF371A and Cry1AbG439D) or domain III (Cry1AbL511A and Cry1AbN514A), which were previously characterized and found to be affected in receptor binding, were analyzed regarding their binding interaction with HaPHB-2 and toxicity against H. armigera One ß-16 mutant (Cry1AbN514A) showed increased binding to HaPHB-2 that correlated with 6-fold-higher toxicity against H. armigera, whereas the other ß-16 mutant (Cry1AbL511A) was affected in binding to HaPHB-2 and lost toxicity against H. armigera Our data indicate that ß-16 from domain III of Cry1Ab is involved in interactions with HaPHB-2 and in toxicity. This report identifies a region of Cry1Ab involved in binding to HaPHB-2 from a Lepidoptera insect, suggesting that this protein may participate as a novel receptor in the mechanism of action of the Cry1 toxins in H. armigeraIMPORTANCEHelicoverpa armigera is a polyphagous pest that feeds on important crops worldwide. This insect pest is sensitive to different Cry1 toxins from Bacillus thuringiensis In this study, we analyzed the potential role of PHB-2 as a Cry1 toxin receptor in comparison to CAD. We show that different Cry1 toxins bound to HaPHB-2 and HaCAD-TBR similarly and identify ß-16 from domain III of Cry1Ab as a binding region involved in the interaction with HaPHB-2 and in toxicity. This report characterized HaPHB-Cry1 binding interaction, providing novel insights into potential target sites for improving Cry1 toxicity against H. armigera.

Rearrangement of N-Terminal α-Helices of Bacillus thuringiensis Cry1Ab Toxin Essential for Oligomer Assembly and Toxicity.

Cry proteins produced by Bacillus thuringiensis are pore-forming toxins that disrupt the membrane integrity of insect midgut cells. The structure of such pore is unknown, but it has been shown that domain I is responsible for oligomerization, membrane insertion and pore formation activity. Specifically, it was proposed that some N-terminal α-helices are lost, leading to conformational changes that trigger oligomerization. We designed a series of mutants to further analyze the molecular rearrangements at the N-terminal region of Cry1Ab toxin that lead to oligomer assembly. For this purpose, we introduced Cys residues at specific positions within α-helices of domain I for their specific labeling with extrinsic fluorophores to perform Föster resonance energy transfer analysis to fluorescent labeled Lys residues located in Domains II-III, or for disulfide bridges formation to restrict mobility of conformational changes. Our data support that helix α-1 of domain I is cleaved out and swings away from the toxin core upon binding with Manduca sexta brush border membrane vesicles. That movement of helix α-2b is also required for the conformational changes involved in oligomerization. These observations are consistent with a model proposing that helices α-2b and α-3 form an extended helix α-3 necessary for oligomer assembly of Cry toxins.