Cry toxins produced by Bacillus thuringiensis are well known for their high insecticidal activities against Lepidoptera, Diptera, and Coleoptera; however, their activities against Aphididae are very low. Recently, it has been reported that a Cry41-related toxin exhibited moderate activity against the aphid Myzus persicae, and thus, it is highly desirable to uncover its unique mechanism. In this paper, we report that Cathepsin B, calcium-transporting ATPase, and symbiotic bacterial-associated protein ATP-dependent-6-phosphofructokinase were pulled down from the homogenate of M. persicae as unique proteins that possibly bound to Cry41related toxin. Cathepsin B has been reported to cleave and inactivate antiapoptotic proteins and plays a role in caspase-initiated apoptotic cascades. In this study, Cathepsin B was expressed in Escherichia coli and purified, and in vitro interaction between recombinant Cathepsin B and Cry41-related toxin was demonstrated. Interestingly, we found that addition of Cry41-related toxin obviously enhanced Cathepsin B activity. We propose a model for the mechanism of Cry41-related toxin as follows: Cry41-related toxin enters the aphid cells and enhances Cathepsin B activity, resulting in acceleration of apoptosis of aphid cells.

Heat shock proteins (HSPs) are essential for the survival and development of animals under various stresses. However, little is known about the function of HSPs in insects response to Bacillus thuringiensis (Bt) toxins treatment. Here, we investigated the role of HSP60 in Myzus persicae (M. persicae) treated with an active Cry7Ab4 toxic core. First, we demonstrated the insensitivity of M. persicae to the Cry7Ab4 toxic core through a membrane capsule method. Then, using protein pull-down assay, several putative Cry7Ab4-binding proteins, including HSP60, were identified in an M. persicae nymph. P-loop GTPase Obg-like ATPase-1 (OLA1) was also found to be a Cry2Ab12-binding protein (unpublished data). Subsequent enzymatic and RT-qPCR assays revealed that highly expressed HSP60 removed the enhanced OLA1 activity caused by Cry7Ab4. ELISA analysis confirmed the binding interactions between Cry7Ab4, HSP60, and OLA1. Interestingly, a combination of ELISA and molecular docking analysis further suggested that HSP60 could block the binding interaction between Cry7Ab4 and OLA1 via higher affinity with Cry7Ab4. Besides, the Jun N-terminal kinase (JNK) pathway was found to be activated. Overall, we proposed the model that HSP60 protects M. persicae from Cry7Ab4 toxin. The study implies that HSP60 can be a crucial factor in insect defense against Cry toxins.

Cry1A-type toxins are insecticidal proteins from Bacillus thuringiensis that are widely used to control Lepidoptera pests, and they are believed to be useless for the control of Hemiptera pests. In this paper, the purified Cry1Ab1 toxic core rarely killed peach aphids (Myzus persicae), as its Lethal Concentration 50 (LC50) was up to 1308.6 μg/ml, as measured using the membrane-encapsulation method. We then identified the proteins that bind to the Cry1Ab1 toxic core in M. persicae using pull-down assays and liquid chromatography−tandem mass spectrometry (LC-MS/MS), and then analyzed the associated functions of these proteins using the STRING database. GPN-loop GTPase 2, beta-actin, ATP synthase subunit alpha and an unknown and annotated protein, which are mainly involved in cell phagocytosis, RNA polymerase, cellular oxidative phosphorylation and other related functions, were the proteins that bound to the Cry1Ab1 toxic core. Indications from docking showed that Cry1Ab1 toxic core, ATP synthase subunit alpha and beta-actin or Cry1Ab1 toxic core, ATP synthase subunit alpha and GPN-loop GTPase 2 being a complex to exert effects. Based on the identification of Cry1Ab1-binding proteins from M.persicae, we presented here the molecular clues that Cry1A exert a negative effect on aphid growth and development, and we also proposed that Cry protein had the ability to disturb virus transmission by aphid. The two indications will result in new insights for aphid-effective Cry evaluation and improvement.

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.

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 to show moderate toxicity against Myzus persicae with the 50% lethal concentration (LC50) of 32.7 μg/mL, and its mode of action was proposed to be the acceleration of the apoptosis of aphid cells by enhancing Cathepsin B activity. 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 (mutations 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 (mutations of Gly48, Lys364, Gln367, Gln377, and Tyr378 to Ala and mutation of Pro453 to Glu in Cry41-related toxin) were constructed, expressed, 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 the mortality caused by Cry41-7M protein (LC50 = 19.144 μg/mL) was marginally higher than that of Cry41-related toxin, while Cry41-6A protein caused a decreased mortality (LC50 = 42.478 μg/mL). These results are expected to open new avenues for improving Cry aphidicidal activity.