BACKGROUND: Myzus persicae (Hemiptera: Aphididae) is one of the most notorious pests of many crops worldwide. Most Cry toxins produced by Bacillus thuringiensis show very low toxicity to M. persicae; however, a study showed that Cry41-related toxin had moderate toxic activity against M. persicae. In our previous work, potential Cry41-related toxin-binding proteins in M. persicae were identified, including cathepsin B, calcium-transporting ATPase, and Buchnera-derived ATP-dependent 6-phosphofructokinase (PFKA). Buchnera is an endosymbiont present in almost all aphids and it provides necessary nutrients for aphid growth. This study investigated the role of Buchnera-derived PFKA in Cry41-related toxicity against M. persicae.RESULTS: In this study, recombinant PFKA was expressed and purified, and in vitro assays revealed that PFKA bound to Cry41-related toxin, and Cry41-related toxin at 25 ∼g ml −1 significantly inhibited the activity of PFKA. In addition, when M. persicae was treated with 30 ∼g ml −1 of Cry41-related toxin for 24 h, the expression of dnak, a single-copy gene in Buchnera, was significantly decreased, indicating a decrease in the number of Buchnera.CONCLUSION: Our results suggest that Cry41-related toxin interacts with Buchnera-derived PFKA to inhibit its enzymatic activity and likely impair cell viability, resulting in a decrease in the number of Buchnera, and finally leading to M. persicae death. These findings open up new perspectives in our understanding of the mode of action of Cry toxins and are useful in helping improve Cry toxicity for aphid control.

Cry toxins produced by Bacillus thuringiensis (Bt) are well known for their insecticidal activities against Lepidopteran, Dipteran, and Coleopteran species. In our previous work, we showed that trypsin-digested full-length Cry7Ab4 protoxin did not have insecticidal activity against Plutella xylostella larvae but strongly inhibited their growth. In this paper, we expressed and purified recombinant active Cry7Ab4 toxic core from Escherichia coli for bioassay and identified its binding proteins. Interestingly, Cry7Ab4 toxic core exhibited activity to delay the pupation of P. xylostella larvae. Using protein pull-down assay, several proteins, including basic juvenile hormone-suppressible protein 1-like (BJSP-1), were identified from the midgut juice of P. xylostella larvae as putative Cry7Ab4-binding proteins. We showed that feeding P. xylostella larval Cry7Ab4 toxic core upregulated the level of BJSP-1 mRNA in the hemocytes and fat body and decreased the free juvenile hormone (JH) level in larvae. BJSP-1 interacted with Cry7Ab4 and bound to free JH in vitro. A possible mechanism of Cry7Ab4 in delaying the pupation of P. xylostella larvae was proposed.

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.