Developing eco-friendly, pest-resistant cotton by a heterologous multi-gene transformation system for caffeine synthesis

Cotton production faces significant challenges from insect pests, with chemical pesticide use becoming increasingly limited by resistance and environmental concerns. This study explores the potential use of caffeine, a natural plant alkaloid, as an environmentally friendly insect resistance strategy in cotton. Exogenous caffeine application demonstrated potent insecticidal effects against cotton bollworm (Helicoverpa armigera) larvae, with concentrations ≥ 2 mg mL⁻¹ causing near-complete feeding cessation and up to 70% larval mortality. Building on this, we engineered transgenic cotton (Gossypium hirsutum cv. Jin668) for heterologous caffeine biosynthesis by introducing three key N-methyltransferase genes (CaXMT1, CaMXMT1, CaDXMT1) by multiple gene transformation. Transgenic lines expressing all three genes showed remarkable caffeine accumulation (up to 3.59 mg g⁻¹ dry weight), whereas two-gene combinations exhibited wild-type-level production. Feeding preference assays revealed that caffeine-enriched cotton strongly deterred feeding by H. armigera. Non-choice feeding trials demonstrated reduced leaf consumption and reduced larval growth in H. armigera fed on caffeine-producing cotton. The study highlights the effectiveness of synthetic biology approaches using the TGSII-UNiE multigene stacking system, despite challenges in transgene stability. This work advances plant-derived insect resistance research and provides a sustainable framework for reducing chemical pesticide reliance in cotton production, while underscoring unique potential of cotton as a synthetic biology platform for secondary metabolite engineering.