An aphid-resistant plant metabolite as a candidate aphicide: Insight into the bioactivity and action mode of betulin against aphids

ABSTRACT 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, qRT‒PCR, and western blotting revealed that betulin significantly inhibited the expression of MpGABR (encoding a GABAA receptor). Besides, RNAi-mediated silencing of MpGABR markedly increased aphid sensitivity to betulin. Furthermore, MST (microscale thermophoresis) and voltage-clamp assays indicated that betulin bound to MpGABR (Kd = 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. Competing Interest Statement The authors have declared no competing interest. Footnotes The authors declare no competing financial interest. According to Reviewers' comments, an improved manuscript has been provided, which was mainly revised as follows (1)In Introduction section, the evolutionary conservation of GABA receptor and potential impact of betulin on a wider biological population were added. (2)In Discussion section, the discussion of the sensitivity of GABA receptor to betulin, potential ecological risks of deploying betulin as a bioinsecticide and possible reasons for the effect of betulin on MpGABR expression were re-discussed. (3)Phylogenetic analysis, using more represent species from each Order, has been re-performed (Fig. 5 and Fig. 7B). (4)The original Figure 5A and B, to improve clarity and logical flow, have been inserted into Figure 3, following Figure 3D. The original Figure 3E-I, to illustrate the RNAi assay, has been saved as a new figure. (5)The Materials and Methods section has been thoroughly reviewed. Especially, the additional details have been added. ABBREVIATIONS - GABA - Gamma-aminobutyric acid - GABR - GABAA receptor - GABRB - GABAA receptor β subunit - GABRAP - GABAA receptor associated protein - MST - microscale thermophoresis - PCA - principal coordinates analysis - DEGs - differentially expressed genes - GO - Gene Ontology - KEGG - Kyoto Encyclopedia of Genes and Genomes - Kd - dissociation constant - RDL - resistance to dieldrin - NCA - noncompetitive antagonists - CA - competitive antagonists.