Insight into the bioactivity and action mode of betulin, a candidate aphicide from plant metabolite, against aphids
- Key Laboratory of Agricultural Biosafety and Green Production of Upper Yangtze River (Ministry of Education), Chongqing Key Laboratory of Forest Ecological Restoration and Utilization in the Three Gorges Reservoir Area, College of Horticulture and Landscape Architecture, Southwest University, China
- College of Plant Protection, Southwest University, China
- Zhengzhou Fruit Research Institute, Chinese Academy of Agricultural Sciences, China
- Yibin Academy of Southwest University, China
Figures
Figure 1
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 hr 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 days. The error bars represent standard deviation (SD) with n=4 (greenhouse test) and 12 (field test), respectively. **p<0.01. ns, not significant.
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Figure 1—source data 1
LC50 values of betulin and pymetrozine against M. persicae at 48 hr, corresponding to Figure 1, panels D and F.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig1-data1-v1.docx
Figure 2
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% (vol/vol) Tween-80 and 3% (vol/vol) acetone was used as the control treatment (CK). (B) Principal coordinate analysis (PCA) of differentially expressed genes (DEGs) in RNA-seq. (C–D) Distribution (C) and heatmap (D) of the significantly DEGs in M. persicae. (E) qPCR validation of 15 DEGs identified by RNA-seq. The error bars represent SD with n=3. (F–G) Top 10 enriched Gene Ontology (GO) (F) and Kyoto Encyclopedia of Genes and Genomes (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.
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Figure 2—source data 1
Screening of genes significantly differentially expressed in M. persicae in response to betulin at 48 hr post treatment, corresponding to Figure 2, panel C.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig2-data1-v1.docx
Figure 3
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 hr. The bars represent the average (± SD). Different letters above the error bars indicate significant difference (analysis of variance [ANOVA], Tukey’s test, p<0.05). (E, F) Western blotting analysis of MpGABR protein after betulin treatment for 48 hr at three different concentrations (LC30, LC50, and LC70). The error bars represent SD. Different letters above the error bars indicate significant difference (ANOVA, Tukey’s test, p<0.05).
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Figure 3—source data 1
Complete sequence information for the GABAA receptor gene of M. persicae corresponding to Figure 3, panel A.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig3-data1-v1.docx
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Figure 3—source data 2
PDF file containing original membranes corresponding to Figure 3, panel E. GAPDH was used as a reference protein.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig3-data2-v1.zip
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Figure 3—source data 3
Original membranes without labels corresponding to Figure 3, panel E.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig3-data3-v1.zip
Figure 4 with 1 supplement
Silencing the expression of MpGABR, MpGABRAP, and MpGABRB via RNAi.
(A–C) qPCR expression analysis of MpGABR (A), MpGABRAP (B), and MpGABRB (C) after RNAi at 48 hr post-dsRNA feeding relative to the expression levels after DEPC-water treatment. (D) Schematic drawing of the RNAi assay in M. persicae. (E) Mortality of aphids exposed to the LC50 of betulin for 48 hr after RNAi. The error bars represent SD. 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.
Figure 4—figure supplement 1
Protein expression of MpGABR after RNAi.
Western blotting analysis (A) of MpGABR expression after RNAi at 48 hr post-dsRNA feeding, relative (B) to expression level after DEPC-water treatment. The error bars represent SD with n = 3.
Figure 5
Phylogenetic analysis of GABR in insects.
Neighbor-joining method with 1000 replicates based phylogenetic tree of GABR from six orders: Hemiptera, Diptera, Lepidoptera, Thysanoptera, Hymenoptera, and Coleoptera. Protein sequence alignment was performed using ClustalW in MEGA 7.
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Figure 5—source data 1
Sequences and relevant information for phylogenetic analysis of GABAA receptor.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig5-data1-v1.docx
Figure 6
The interaction of betulin with MpGABR protein.
(A) Expression of recombinant wild-type-aphid MpGABR (1, WT) protein by E. coli. (B) Quantification of the binding affinity of betulin with WT-aphid MpGABR using microscale thermophoresis (MST). Current responses (C) and inhibition activity (D) of MpGABR induced by different concentrations of betulin in the presence of GABA. 0 μM MpGABR indicates the presence of only GABA. The error bars represent SD with n=3.
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Figure 6—source data 1
PDF file containing original gel corresponding to Figure 6, panel A.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig6-data1-v1.zip
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Figure 6—source data 2
Original gel without labels corresponding to Figure 6, panel A.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig6-data2-v1.zip
Figure 7 with 1 supplement
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. The numbers next to the amino acid indicate the site of the last residue of the key amino acids. (C) Expression of the recombinant mutation-type aphid MpGABR by E. coli. Lane 1: R224A, 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 microscale thermophoresis (MST). The error bars represent SD with n=3. * indicates a significant difference (Student’s t test, ****p<0.0001).
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Figure 7—source data 1
Binding energy and nonbonding interactions between betulin and GABAA receptor, corresponding to Figure 7, panel A.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig7-data1-v1.docx
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Figure 7—source data 2
PDF file containing original gel corresponding to Figure 7, panel C.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig7-data2-v1.zip
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Figure 7—source data 3
Original gel without labels corresponding to Figure 7, panel C.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig7-data3-v1.zip
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Figure 7—source data 4
Parameters of dose-response curves from microscale thermophoresis experiments, corresponding to Figure 7, panel D.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig7-data4-v1.docx
Figure 7—figure supplement 1
Betulin binding site on the domain of MpGABR.
The structure of MpGABR protein was predicted on UniProt website. Pink bars indicate basic residue sequences and brown bars mean disordered regions.
Figure 8
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 (D. melanogaster) by E. coli. Lane 1: WT, Lane 2: R122T. (B, C) Quantification of the binding affinity of betulin (B) and pymetrozine (C) with WT and R122T of DmGABR using microscale thermophoresis (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. The error bars represent SD with n=3.
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Figure 8—source data 1
PDF file containing original gel corresponding to Figure 8, panel A.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig8-data1-v1.zip
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Figure 8—source data 2
Original gel without labels corresponding to Figure 8, panel A.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig8-data2-v1.zip
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Figure 8—source data 3
LD50 values of betulin and pymetrozine against D. melanogaster at 72 hr, corresponding to Figure 8, panels E and F.
- https://cdn.elifesciences.org/articles/107598/elife-107598-fig8-data3-v1.docx
Figure 9
Proposed model for the mechanism of action against M. persicae by targeting GABAA receptors (GABR).
After exposure to betulin, the expression of MpGABR was inhibited, and the level of MpGABR protein 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.
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Insight into the bioactivity and action mode of betulin, a candidate aphicide from plant metabolite, against aphids
eLife 14:RP107598.
https://doi.org/10.7554/eLife.107598.3
