Peptide-mediated enhancement of bacterial pesticidal protein toxicity against Diaphorina citri

The Asian citrus psyllid, Diaphorina citri (Hemiptera; Liviidae) is the primary vector of Candidatus Liberibacter asiaticus, the causative agent of citrus greening (Huanglongbing). This disease has decimated global citrus production. D. citri resistance to traditional chemical insecticides highlights the need for alternative control strategies. Several native bacterial pesticidal proteins (BPPs) are toxic to D. citri but with relatively low toxicity. We explored the use of protein engineering to enhance the insecticidal activity of a lead candidate, Tpp78Aa1, by substituting native amino acid sequences with those of a D. citri gut-binding peptide (GBP). The gut binding peptide DcNy6, was identified through screening of a phage display library for peptides that bind to the D. citri gut. DcNy6 binding was confirmed by visualization of fluorescence in the gut following feeding on DcNy6-GFP. Candidate substitution sites were selected based on predicted structural flexibility and tolerance to mutation and five Tpp78Aa1 variants were designed with DcNy6 inserted at different positions. All variants were cloned into pET30(a), expressed in BL21(DE3) cells, purified via His-tag affinity chromatography, and evaluated for toxicity against first instar nymphs using a standardized sandwich feeding assay. Toxicity was assessed using both single-dose (10 µg/mL) and LC₅₀ assays, with significant increases in toxicity relative to the native protein observed in some cases. This result confirms that targeted modification by use of gut binding peptides for increased gut binding can significantly enhance BPP toxicity, further highlighting their potential for use in D. citri control within an integrated management framework.