De novo design of modular insecticidal scaffold enhances cotton resistance to pink bollworm and whitefly

Abstract Background A variety of insect pests pose a serious threat to global cotton production. Among these, pink bollworm (PBW) and whiteflies are the most devastating, resulting in substantial yield losses and increased pesticide use. Conventional insect-resistance strategies, which primarily rely on single Bacillus thuringiensis (Bt) genes, often face limitations due to the development of resistance in target pests. To overcome these challenges, the fusion of multiple insecticidal proteins, including Bt toxins and plant-derived lectins, offers a synergistic approach to broaden the spectrum and durability of resistance. Results The modular insecticidal scaffold exhibited a distinct promoter-dependent expression pattern, highlighting its potential as a customisable platform for targeted cotton protection. Molecular docking revealed favorable interactions of Cry1Ac, Cry1Ab, Vip3A with the pink bollworm cadherin receptor, whereas ASAL showed favorable binding to mannose residues, representing its recognition of mannose-containing glycoconjugates on midgut cells, supporting the structural compatibility of the de novo assembled scaffold. The scaffold carrying these domains was synthesised and cloned into the pCAMBIA1302 vector under constitutive and fibre-specific promoters, and subsequently transformed into a local cotton variety. Transgenic lines harbouring the fibre-specific promoter ( GhSCFP ) displayed significantly elevated expression in bolls, reaching over a 100-fold increase compared with leaves, whereas the constitutive promoter ( CaMV35S ) drove higher expression in foliage, consistent with its broader activity profile. Promoter-guided expression patterns strongly influenced insecticidal outcomes: fibre-specific lines showed substantial mortality in PBW (up to 90–95%), whereas constitutive promoter-driven lines exhibited greater suppression of whiteflies (approximately 60–70%). The observed correlation between spatial expression and pest-specific lethality indicates that the scaffold functions as a cohesive insecticidal unit, effectively integrating multiple toxin domains while maintaining spatial regulation. This dual strategy of boll-targeted PBW protection and whole-plant whitefly resistance represents a significant advancement over single-gene Bt approaches, providing a more resilient and comprehensive resistance framework for cotton improvement. Conclusion Our findings indicate that promoter selection is a crucial factor influencing tissue-specific expression and insecticidal efficacy. The synergistic combination of fibre-specific and constitutive promoters establishes a dual framework for targeted boll protection and broader pest management. This modular, promoter-driven approach holds considerable promise for the development of next-generation, sustainable pest-resistant cotton suitable for integrated pest management strategies.