Identification and Analysis of the Mpp5Ab1-Interacting Protein in the Midgut of the Colaphellus bowringi Baly

To elucidate the mode of action of Mpp5Ab1 against Colaphellus bowringi Baly larvae, this study aimed to identify midgut proteins interacting with the toxin. A validated bait plasmid, pBT3-SUC-mpp5Ab1, was used to screen a larval midgut cDNA library via the split-ubiquitin yeast two-hybrid system. A total of 33 positive clones representing five distinct proteins were obtained, among which bioinformatic analyses prioritized three candidates: Cb-RP-L23e, Cb-CTSL, and Cb-TsetseEP. Subsequent bimolecular fluorescence complementation (BiFC) assays in Sf9 cells specifically confirmed interactions between Mpp5Ab1 and both Cb-CTSL and Cb-TsetseEP, whereas no fluorescence signal was observed for Cb-RP-L23e. Molecular docking further supported stable interactions between Mpp5Ab1 and the validated candidate proteins through hydrogen bonds, salt bridges, and hydrophobic interactions. These findings suggest that Cb-CTSL and Cb-TsetseEP may function as candidate interacting proteins associated with the activity of Mpp5Ab1 in the larval midgut of C. bowringi. Overall, this study provides new insight into the molecular interactions of Mpp5Ab1 and establishes a foundation for future functional studies on its insecticidal mechanism and receptor validation.