The aphicidal activity of plant-derived derivative betulin against M. persicae.

(A-G) Representative images of the control effects of betulin (A, 0.1641 mg·mL-1, 48 h LC50 value of betulin), pymetrozine (B, 1.0612 mg·mL-1, a positive control), and CK (C, a negative control) against M. persicae in greenhouse (A-E) and field (F, G) tests after treatment for 14 d. The data are shown as the mean ± SD from twelve independent experiments. **p < 0.01. ns, not significant.

RNA-Seq analysis revealed candidate targets for betulin against M. persicae.

(A) Diagram of the candidate targets for betulin against aphids as determined by RNA-Seq. Water containing 0.1% (v/v) Tween-80 and 3% (v/v) acetone was used as the control treatment (CK). (B) Principal coordinates analysis (PCA) analysis differentially expressed genes in RNA-Seq. (C-D) Distribution (C) and heatmap (D) of the significantly differentially expressed genes (DEGs) in M. persicae. (E) qPCR validation of 15 DEGs identified by RNA-Seq. (F-G) Top 10 enriched GO (F) and KEGG (G) pathways of the DEGs. The “rich ratio” was defined as the ratio of the number of DEGs enriched in the pathway to the total number of genes enriched in the same pathway. (H) Heatmap of the expression level of the GABR genes identified by RNA-Seq. MpGABR, encoding GABAA receptor; MpGABRAP, encoding GABAA receptor associated protein; MpGABRB, encoding GABAA receptor β subunit.

MpGABR expression in aphids was significantly inhibited by treatment with betulin.

(A) Schematic drawing of MpGABR, MpGABRAP and MpGABRB. TM, transmembrane helices. (B-D) qPCR expression analysis of MpGABR (B), MpGABRAP (C) and MpGABRB (D) transcripts in M. persicae exposed to LC30, LC50, and LC70 of betulin for 48 h. The bars represent the average (± SD). Different letters above the error bars indicate significant difference (ANOVA, Tukey’s test, P < 0.05). (E-G) qPCR expression analysis of MpGABR (E), MpGABRAP (F) and MpGABRB (G) after RNAi at 48 h post-dsRNA feeding relative to the expression levels after DEPC-water treatment. (H) Schematic drawing of the RNAi assay in M. persicae. (I) Mortality of aphids exposed to the LC50 of betulin for 48 h after RNAi. An asterisk (*) on the error bar indicates a significant difference between the treatment and group CK according to t tests, ***P < 0.001, ****P < 0.0001. ns, not significant.

Phylogenetic analysis of GABR in insects.

Maximum likelihood based phylogenetic tree of GABR from six orders: Hemiptera, Diptera, Lepidoptera, Thysanoptera, Hymenoptera, and Coleoptera. Protein sequence alignment was performed using MUSCLE in MEGA X. The sequences used for constructing the tree were listed in Table S6.

The interaction of betulin with MpGABR protein.

(A, B) Western blotting analysis of MpGABR protein after betulin treatment for 48 h at three different concentrations (LC30, LC50 and LC70). The bars represent the average (± SD). Different letters above the error bars indicate significant difference (ANOVA, Tukey’s test, P < 0.05). (C) Expression of recombinant wild type-aphid MpGABR (1, WT) protein by Escherichia coli. (D) Quantification of the binding affinity of betulin with wild type-aphid MpGABR using MST. Current responses (E) and inhibition activity (F) of MpGABR induced by different concentration of betulin in the presence of GABA. 0 µM MpGABR indicates the presence of only GABA.

Molecular docking, binding site and inhibitory effect of betulin on MpGABR.

(A) Best conformations of betulin docked to the binding pocket of MpGABR in aphids. An enlarged view of the betulin binding sites in MpGABR is indicated by a dashed frame. Potential Pi–alkyl (green), Alkyl (yellow), and hydrogen bond (red) interactions are indicated by dashed lines. (B) Sequence alignment of the key amino acids bound to betulin. The conserved residues among the different species in GABR are shown in orange, cyan, and light green. (C) Expression of the recombinant mutation type-aphid MpGABR by Escherichia coli. Lane 1: R244A, Lane 2: A226T, Lane 3: F227Y, Lane 4: T228R. (D, E) Quantification of the binding affinity of betulin with wild-type and mutant type-aphid MpGABR using MST. The bars represent the average (± SD) values. * indicates a significant difference (Student’s t test, ****P < 0.0001).

Gene editing in Drosophila validated the species-specific binding site of betulin.

(A) Expression of the recombinant wild type (WT) and mutation type (R122T) of DmGABR (Drosophila melanogaster) by E. coli. Lane 1: wild type, Lane 2: R122T. (B, C) Quantification of the binding affinity of betulin (B) and pymetrozine (C) with WT and R122T of DmGABR using MST. (D) Sanger sequencing of the DmGABR gene in flies. Direct sequencing chromatograms of PCR products amplified from a fragment of gDNA flanking the WT (a), and introduced R122T (b) mutant flies. (E-F) Toxicity curves of betulin (E), and pymetrozine (F) against WT, and the DmGABRR122T of the Cas9 Drosophila.

Proposed model for the mechanism of action against Myzus persicae by targeting GABAA receptors (GABR).

After exposure to betulin, the expression of MpGABR was inhibited the level of MpGABR protein is decreased, resulting in a decrease in the channel of chloride ion influx. Besides, betulin directly and specifically binds to the amino acid residue THR228 of MpGABR, thereby disabling it.