Egyptian Journal of Biological Pest Control (Aug 2023)

Site-directed mutagenesis in Cry proteins of Bacillus thuringiensis to demonstrate the role of domain II and domain III in toxicity enhancement toward Spodoptera litura

  • Huma Khurshid,
  • Hafsa Zaheer,
  • Fakhar-un-Nisa Yunus,
  • Farkhanda Manzoor,
  • Aasma Abdul Latif,
  • Farzana Rashid

DOI
https://doi.org/10.1186/s41938-023-00731-x
Journal volume & issue
Vol. 33, no. 1
pp. 1 – 9

Abstract

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Abstract Background Bacillus thuringiensis (Bt) is a gram-positive bacterium responsible for the production of a wide variety of insecticidal Cry, Cyt, and Vip proteins with distinct insect specificities. The bioinsecticides derived from Bt Cry proteins account for > 95% of the microbial biopesticide market to combat a variety of pest species belonging to the order Lepidoptera (including Spodoptera spp.), Coleoptera, Diptera, etc. Cry proteins are engineered by using different molecular techniques to control the development of multiple insecticide resistance problems in major insect pests using bio-toxicity assays. Main body It is common knowledge that the Cry proteins domain II and III are involved in pore formation or interaction between several insect larval receptors and the membranes of epithelial cells. In the present research, the PCR site-directed mutagenesis technique was used to introduce a total of four mutations into the cry genes (cry1 and cry2) near key regions of active proteins. The diet overlay bioassay was used to test the efficacy of expressed mutant Cry proteins against Spodoptera litura (Fabricius) (Lepidoptera: Noctuidae), one of the most damaging bollworms to cotton, causing severe output losses. Results Two amino acid replacements in the receptor binding domain of Cry1Ac toxins (S573, L588) showed decrease in activity for Cry1Ac mutants. However, amino acid substitutions in the receptor binding/pore formation domain of Cry2Aa (T325, S445) were to some extent proved more toxic than wild-type recombinant Cry2Aa protein, with an increase in mortality percentage from 3.33 to 6.66% after 24 to 72 h of treatment, respectively, against 2nd instar larvae of S. litura. Conclusion A comparison of activity demonstrated that larvae of S. litura were more susceptible to Cry2Aa toxins than those of Cry1Ac toxins after being treated with Bt toxins. The LC50 values of Cry2Aa mutants were slightly reduced for S. litura larvae than those of Cry1Ac mutants.

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