Bacillus thuringiensisinsecticidal proteins (Bt toxins) have been widely used in crops for agricultural pest management and to reduce the use of chemical insecticides. Here, we have engineered Bt toxin Cry2Ab30 and bioconjugated it with 4”-O-succinyl avermectin (AVM) to synthesize Cry2Ab–AVM bioconjugate. It was found that Cry2Ab–AVM showed higher insecticidal activity againstPlutella xylostella, up to 154.4 times compared to Cry2Ab30. The binding results showed that Cry2Ab–AVM binds to the cadherin-like binding protein fragments, the 10th and 11th cadherin repeat domains in theP. xylostellacadherin (PxCR10–11), with a much higher affinity (dissociation equilibrium constantKD= 3.44 nM) than Cry2Ab30 (KD= 28.7 nM). Molecular docking suggested that the macrolide lactone group of Cry2Ab–AVM ligand docking into the PxCR10–11is a potential mechanism to enhance the binding affinity of Cry2Ab–AVM to PxCR10–11. These findings offer scope for the engineering of Bt toxins by bioconjugation for improved pest management.Keywords:Cry2Ab–AVM bioconjugate;binding affinity;molecular dockingKey Contribution:The study develops an engineered Cry2Ab by bioconjugation with 4”-O-succinyl avermectin to enhance insecticidal potency againstP. xylostella.This engineering also improves its affinity to the cadherin-like binding fragments. Homology modeling and docking study are also carried out to investigate the binding model.
Lian Xu,Zhi-Zhen Pan,Jing Zhang,Li-Yang Niu,Jie Li,Zheng Chen,Bo Liu,Yu-Jing Zhu,Qing-Xi Chen
Cry2Ab, a pore-forming toxin derived from Bacillus thuringiensis , is widely used as a bio-insecticide to control lepidopteran pests around the world. A previous study revealed that proteolytic activation of Cry2Ab by Plutella xylostella midgut juice was essential for its insecticidal activity against P. xylostella , although the exact molecular mechanism remained unknown. Here, we demonstrated for the first time that proteolysis of Cry2Ab uncovered an active region (the helices α4 and α5 in Domain I), which was required for the mode of action of Cry2Ab. Either the masking or the removal of helices α4 and α5 mediated the pesticidal activity of Cry2Ab. The exposure of helices α4 and α5 did not facilitate the binding of Cry2Ab to P. xylostella midgut receptors but did induce Cry2Ab monomer to aggregate and assemble a 250-kDa prepore oligomer. Site-directed mutagenesis assay was performed to generate Cry2Ab mutants site directed on the helices α4 and α5, and bioassays suggested that some Cry2Ab variants that could not form oligomers had significantly lowered their toxicities against P. xylostella . Taken together, our data highlight the importance of helices α4 and α5 in the mode of action of Cry2Ab and could lead to more detailed studies on the insecticidal activity of Cry2Ab.
J. B. Fan,S. van de Velde,W. B. Sun,H. Yu,Y. L. Zhang,D. Wang
The oriental armyworm, Mythimna separata Walker (Lepidoptera: Noctuidae), is a severe pest of graminaceous crops in Asia and Australia. In this study, we investigated the impacts of Cry2Ab, cantharidin and its analogues (cantharidin-23 and cantharidin-24) on M. separata growth, hydrolytic enzymes and detoxifying enzymes. Differences in weight gain and enzyme activities among all treatments were observed. Larval and pupal weight gain and hydrolytic enzyme activities declined when larvae were treated with Cry2Ab, cantharidin and its analogues, individually or in combination. The combination of Cry2Ab and cantharidin or cantharidin-24 had a markedly adverse effect on larval weight gain. Alkaline phosphatase and acid phosphatase were inhibited, whereas glutathione S-transferase was upregulated in all treatments with sublethal doses. The maximum reduction in alkaline phosphatase activity and increase in glutathione S-transferase activity occurred after the larvae were treated with a combination of Cry2Ab and cantharidin-24 over 48 h. The results suggest that the compositions of Cry2Ab, cantharidin and cantharidin-24 have a potential use in pest management.
Bollgard II cotton (which expresses two Bt insecticidal genes cry1Ac/cry2Ab) and conventional cotton, grown in the laboratory or field and sampled at different stages, was exposed to Helicoverpa armigera (Hübner) larvae of three genotypes: homozygous for resistance to Cry2Ab; homozygous for susceptibility to Cry2Ab, and heterozygous for resistance. Survival of all genotypes was limited on Bollgard II but increased as plants aged. This was particularly the case for homozygous resistant individuals, with 8.5% of this genotype surviving to pupation on mature cotton. The increasing survival is assumed to be caused by the decline in the titer of Cry1Ac toxin after flowering in Bollgard II because Cry2Ab homozygous resistant larvae can tolerate high levels of Cry2Ab toxin. Larvae heterozygous for resistance performed no better on Bollgard II than homozygous susceptible larvae. Survivors on Bollgard II grew more slowly and produced smaller pupae that yielded adults with reduced longevity and fecundity. When reared on conventional cotton, all genotypes generally performed equally, indicating an absence of fitness costs associated with Cry2Ab resistance under the conditions examined.
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