Insight into the bioactivity and action mode of betulin, a candidate aphicide from plant metabolite, against aphids

Pest-resistant plants usually utilize secondary metabolites to cope with insect infestation. Betulin, a key bioactive compound in aphid-resistant wild peach, possesses promising applications in crop protection. Here, betulin, in both greenhouse and field experiments, displayed excellent control efficacy against Myzus persicae. RNA-seq, quantitative real-time PCR (qRT-PCR), and western blotting revealed that betulin significantly inhibited the expression of MpGABR (encoding a GABA_A receptor). Besides, RNAi-mediated silencing of MpGABR markedly increased aphid sensitivity to betulin. Furthermore, microscale thermophoresis (MST) and voltage-clamp assays indicated that betulin bound to MpGABR (K_d = 2.24 µM) and acted as an inhibitor of MpGABR. Molecular docking, mutagenesis, and genome editing suggested that THR228 is a critical and highly conserved site in MpGABR that betulin binds to specifically, causing aphid death. Overall, the activity of betulin depends on specific targeting and inhibition of MpGABR. Elucidating the mechanism of action of this peach-derived insecticide may offer a sustainable green strategy for aphid control.