Cry41-related mutants against Myzus persicae based on its interaction with Cathepsin B

Abstract

Cry toxins produced by Bacillus thuringiensis (Bt) are toxic to Lepidoptera, Coleoptera, and Diptera, but display very low activity against aphids. Recently, Cry41-related toxin was found with moderate toxicity against Myzus persicae. This study focused on constructing Cry41-related mutants against M. persicae based on its interaction with Cathepsin B. First, eight key interacting residues in Cry41-related toxin were identified using alanine scanning and site-directed saturation mutagenesis. Subsequently, the positive mutant Cry41-7M protein (mutation of Gly48, Ile59, Lys364, Gln367, Gln377, Tyr378, and Ser400 to Tyr, Ala, Arg, Lys, Lys, Lys, and Ala in Cry41-related toxin, respectively) and the negative mutant Cry41-6A protein (mutation of Gly48, Lys364, Gln367, Gln377, Tyr378 to Ala and mutation of Pro453 to Glu in Cry41-related toxin) were constructed, expressed in vitro and purified. We then found that Cry41-7M protein performed slightly stronger than Cry41-related toxin in enhancing the enzymatic activity of Cathepsin B, whereas Cry41-6A protein did not affect Cathepsin B activity. A further bioassay showed that, in contrast to a decreased mortality caused by Cry41-6A protein, the mortality caused by Cry41-7M protein was marginally higher than that of Cry41-related toxin (1.7-fold). These results are expected to open new avenues for improving Cry aphidicidal activity.