Gluconeogenic compensation renders glycogen phosphorylase non-essential as an insecticidal target in Plutella xylostella
- Key Laboratory of Plant Protection Resources and Pest Management of Ministry of Education, College of Plant Protection, Northwest A&F University, China
- Northwest A&F University Shenzhen Research Institute, China
Figures
Figure 1
Structural comparison of benzoylphenylurea (BPU) insecticides and a mammalian glycogen phosphorylase inhibitor (GPI) reveals a shared acylurea scaffold.
Chemical structures of four benzoylphenylurea (BPU) insecticides (Diflubenzuron, Teflubenzuron, Novaluron, and Flufenoxuron) and a human GPI are shown. All share a central N,N'-diphenylurea (acylurea) core. BPUs are characterized by halogenated (F, Cl) aromatic substituents and in some cases by additional ether or alkyl side chains. The GPI contains a methoxyphenyl group bearing an additional methylurea side chain.
Figure 2 with 1 supplement
Recombinant Plutella xylostella GP (PxGP) is inhibited by a mammalian glycogen phosphorylase (GP) inhibitor (GPI) but not by the benzoylphenylurea Diflubenzuron.
(A) Purification of 6× His-tagged PxGP from Sf9 cells. Left panel: Coomassie-stained SDS-PAGE. Right: Western blot with anti-His antibody. The expected ~100 kDa band is indicated (red box). M, marker; S, soluble fraction; P, pellet; F, flow-through; lanes 1–4, imidazole elution fractions. (B) Dose-response inhibition by GPI. Activated PxGP-a activity was measured after pre-incubation with GPI (0.16–500 nM). Data (mean ± SEM, n=3) were fit to a four-parameter logistic curve. The calculated IC50 is 2.96 nM. Inset: GPI structure with its characteristic asymmetric side chains (orange). (C) Inhibition assay with diflubenzuron (DFB) (0.54–1,700 nM). DFB did not yield a complete sigmoidal inhibition curve; maximum inhibition did not exceed 55.07±7.05% at the highest concentration. Inset: DFB structure with its characteristic halogenated benzoyl groups (orange). This rules out PxGP as a direct target of DFB.
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Figure 2—source data 1
Original files for SDS-PAGE and Western blot to Figure 2A.
- https://cdn.elifesciences.org/articles/111144/elife-111144-fig2-data1-v1.zip
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Figure 2—source data 2
Labelled gels and blots for Figure 2A, with relevant bands clearly indicated.
- https://cdn.elifesciences.org/articles/111144/elife-111144-fig2-data2-v1.zip
Figure 2—figure supplement 1
Enzymatic activity of purified Plutella xylostella GP (PxGP) and inhibition assays using crude larval lysate.
(A) Reaction kinetics of purified, activated PxGP-a (red line) compared to a no-enzyme control (CK, blue line). The specific activity was calculated to be 294.25±3.45 U/mg Prot (mean ± SEM). (B) Inhibition of native glycogen phosphorylase (GP) activity in a crude larval lysate by GP inhibitor (GPI). Activity was measured at 5, 15, 25, and 45 min post-incubation. (C) Effect of diflubenzuron (DFB) on native GP activity in a crude larval lysate. DFB showed no significant inhibition at any concentration or time point. Data in (B) and (C) are presented as mean ± SEM (n=3).
Figure 3
The GP inhibitor (GPI) is non-toxic to Plutella xylostella, unlike the benzoylphenylurea diflubenzuron (DFB).
(A) Developmental stage distribution over time for control, GPI (250, 500 mg/L), and DFB (125, 250 mg/L) treated larvae. DFB causes developmental arrest. (B) Quantitative effects on mortality (top), pupation (middle), and adult emergence (bottom). GPI groups did not differ from control (p>0.05), whereas DFB caused significant, dose-dependent mortality and severely impaired development (p<0.05). Data are mean ± SEM (n=3). Different letters denote statistical significance. (C) Larval phenotypes at 96 hr. Control and GPI-treated larvae are normal. DFB-treated larvae show catastrophic molting failure and ‘double cuticle’ (red arrows). Scale bar, 1 mm. (D) Pupal and adult phenotypes. GPI-treated individuals develop normally. DFB treatment results in deformed pupae and trapped pharate adults (red arrows). Scale bar, 1 mm.
Figure 4
Ingestion of glycogen phosphorylase inhibitor (GPI) induces compensatory upregulation of Plutella xylostella GP (PxGP) transcription.
Relative PxGP mRNA levels in larvae after continuous exposure to 500 mg/L GPI or control (CK). Expression was normalized to PxRPS13 and calibrated to the 24 hr control sample (set to 1.0) using the 2-ΔΔCt method. Data are mean ± SEM (n=3). GPI triggered a rapid and sustained transcriptional increase, peaking at 48 hr (3.48-fold). Significance vs. control: *p<0.05, **p<0.01, ***p<0.001 (two-way ANOVA with Sidak’s test).
Figure 5 with 1 supplement
Plutella xylostella GP (PxGP) sequence conservation and domain architecture.
(A) Schematic of PxGP functional domains: pyridoxal phosphate (PLP) binding site (blue boxes), AMP allosteric site (yellow boxes), and glycogen-binding/active site pocket (blue shading). (B) Multiple sequence alignment of glycogen phosphorylase (GP) from P. xylostella (PxGP), other lepidopterans (Helicoverpa armigera HaGP, Spodoptera exigua SeGP, Ostrinia furnacalis OfGP), human (HsGP), and rabbit (OcGP). Identical residues are shaded red. The gray box indicates the 606 bp region (aa 22–224) targeted for RNAi, which overlaps the conserved AMP-binding domain. PxGP shares 82.29% similarity with human GP.
Figure 5—figure supplement 1
Heatmap depicting pairwise sequence similarity of glycogen phosphorylase among six species.
The heatmap displays the percentage of global sequence similarity calculated from pairwise alignments using TBtools. The six species (Plutella xylostella, Homo sapiens, Oryctolagus cuniculus, Helicoverpa armigera, Ostrinia furnacalis, and Spodoptera exigua) are arranged in the same order along both axes. The color intensity in each cell corresponds to the similarity percentage, as shown in the color key (from blue (low) to red (high)). Numerical values are shown within the cells.
Figure 6
Developmental expression profile of Plutella xylostella GP (PxGP).
Relative PxGP mRNA levels across life stages (Egg, larval instars L1-L4, Pupa, Adult), normalized to PxRPS13 (Egg set to 1.0). Data are mean ± SEM (n=3). Expression peaks in adults. Different letters indicate significant differences (p<0.05, one-way ANOVA with Tukey’s test).
Figure 7
RNA interference (RNAi)-mediated knockdown of Plutella xylostella GP PxGP is dose- and time-dependent.
(A) Experimental workflow. Third-instar larvae were injected with dsRNA targeting PxGP (dsGP) at three concentrations or a control dsRNA (dsGFP), then sampled for analysis. (B) Time-course of PxGP mRNA levels post-injection, relative to dsGFP control (set to 1.0). The highest dsGP dose (14,000 ng/µL) caused maximal suppression (87.6%) at 48 hr. Data are mean ± SEM (n=3). Significance vs. dsGFP at each time point: *p<0.05, **p<0.01, ***p<0.001 (one-way ANOVA with Tukey’s test).
Figure 8
Plutella xylostella GP (PxGP) knockdown does not impair development or survival.
(A) Developmental stage distribution post-injection shows similar progression across all groups. (B) Cumulative mortality (top), pupation (middle), and adult emergence (bottom) rates were not significantly different between dsGP and dsGFP control groups (p>0.05). Data are mean ± SEM (n=3). (C) Representative larval phenotypes at 48 hr and 96 hr post-injection are normal in both dsGFP and high-dose dsGP groups. Scale bar, 1 cm. (D) Pupae and adults from both groups are morphologically normal. Scale bar, 1 mm.
Figure 9
Plutella xylostella GP (PxGP) knockdown triggers biphasic metabolic reprogramming, culminating in gluconeogenic compensation.
(A, B) Concurrent suppression of downstream genes Trehalase (PxTre) and Hexokinase (PxHex) post-knockdown. (C) Upregulation of gluconeogenic genes PxPEPCK and PxG-6-Pase at 96 h. Data in A-C are mean ± SEM (n=3); *p<0.05, **p<0.01, ***p<0.001 vs. dsGFP control. (D–H) Metabolite levels post-knockdown. (D) Total protein decreased at 72 hr (p<0.05). (E) Glycogen remained stable. (F) Trehalose levels were elevated 7.44-fold relative to control at 96 hr (p<0.05). (G) Glucose-6-phosphate (G6P) increased 6.49-fold at 96 hr (p<0.05). (H) Free glucose showed a transient dip at 72 hr (p<0.05) before recovery. Data are mean ± SEM (n=3); *p<0.05 vs. control at each time point.
Figure 10 with 2 supplements
Effects of Plutella xylostella GP (PxGP) knockdown on glycogen phosphorylase (GP) enzyme activity and the expression of glycogen metabolism-related genes.
(A) GP enzyme activity normalized to sample protein concentration. A significant decrease in activity was detected only at 48 hr (p<0.01). (B) GP enzyme activity per insect. Significant decreases were detected at 24 and 48 hr (*p<0.05, **p<0.01). (C) Expression levels of the glycogen branching enzyme (GBE) gene at different time points after RNA interference (RNAi) treatment. (D) Expression levels of the α-Amylase gene at different time points after RNAi treatment. All data are presented as mean ± SEM (n=3); *p<0.05, **p<0.01, ***p<0.001, dsGFP vs. dsGP treatment groups. An independent samples t-test was used in (A) and (B), and two-way ANOVA with Sidak’s test was used in (C) and (D).
Figure 10—figure supplement 1
Expression levels of the Plutella xylostella GP (PxGP) gene after double-stranded RNA injection.
Data are presented as mean ± SEM (n=3); ***p<0.001, dsGP vs. dsGFP treatment group (independent samples t-test).
Figure 10—figure supplement 2
Total protein concentration in test insects subjected to glycogen phosphorylase activity determination, analysis of alternative glycogen-metabolizing enzymes, and fitness cost evaluation.
Data are mean ± SEM (n=3). Significance vs. control: **p<0.01 (two-way ANOVA with Sidak’s test).
Figure 11 with 2 supplements
Assessment of fitness cost of Plutella xylostella GP (PxGP) gene silencing by RNA interference (RNAi) in P. xylostella.
(A) Schematic diagram of fitness cost evaluation at different developmental stages of P. xylostella after dsRNA injection. (B) Food consumption per 10 larvae at different time points. (C) Average body weight per larva at different time points. (D) Pupal weight on day 3 of pupation (n=21). (E) Adult wing area (n=20–21). (F) Number of eggs laid by females within 3 days (n=10–11). Data in (B) and (C) are mean ± SEM of three biological replicates. Data in (D–F) are shown as box plots (box, interquartile range; horizontal line, median; individual points, single insects), with sample sizes as given above for each panel. *p<0.05, dsGP vs. dsGFP treatment group (independent samples t-test).
Figure 11—figure supplement 1
Wing phenotype of adult insects emerged from the dsGFP-treated group.
Figure 11—figure supplement 2
Wing phenotype of adult insects emerged from the dsGP-treated group.
Figure 12
Binding mode analysis of GP inhibitor (GPI) and diflubenzuron (DFB) with Plutella xylostella GP (PxGP).
(A) 3D binding model of PxGP with GPI. (B, C) The key intermolecular interactions for GPI (B) and DFB (C). 2D interaction profile analysis of GPI (D) and DFB (E) with PxGP.
Figure 13
Schematic of glycogen metabolism and compensatory glucose homeostasis.
Red arrows: blocked flux from glycogen to glucose-6-phosphate (G6P); Green arrows: activated gluconeogenesis. Solid arrows represent single enzymatic steps, while dashed arrows indicate multi-step processes.
Tables
Table 1
MM/GBSA binding free energy analysis and interaction profile of GP inhibitor (GPI) and diflubenzuron (DFB) with Plutella xylostella GP (PxGP).
| GPI | DFB | ΔΔG (GPI − DFB) | Structural interpretation | |
|---|---|---|---|---|
| Binding free energy (kcal/mol) | ||||
| Total ΔG | −34.63 | −29.29 | −5.34 | GPI binds PxGP with substantially higher affinity |
| Energy decomposition (kcal/mol) | ||||
| Van der Waals | −53.10 | −41.61 | −11.49 | GPI: superior shape complementarity across dimer interface |
| Electrostatic | −22.51 | −25.89 | +3.38 | DFB: slightly stronger polar interactions via Arg193 |
| Polar solvation (EGB) | +47.23 | +43.44 | +3.79 | GPI: higher desolvation penalty (deeper burial) |
| Nonpolar solvation (ESURF) | −6.25 | −5.23 | −1.02 | GPI: marginally more buried nonpolar surface |
| Interaction profile | ||||
| Contacting residues | 7 | 6 | — | |
| Cross-subunit contacts | Yes (2 from chain A) | Weak (1 from chain A) | — | GPI anchors across dimer interface |
| Strong H-bonds (≤2.8 Å) | 3 (Asp227) | 2 (Arg193) | — | Different H-bond anchor residues |
| Solvent-exposed substituent | None | Difluorobenzoyl | — | DFB side chain lacks protein contacts |
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Note: MM/GBSA binding free energy analysis and interaction profile of GPI and DFB with PxGP. Binding free energies were estimated by MM/GBSA (Molecular Mechanics with Generalized Born and Surface Area solvation) single-point calculations using Cresset Flare V11. The PxGP homodimer was modeled using SWISS-MODEL with PDB: 2ATI as the template. ΔΔG = ΔG(GPI) − ΔG(DFB); negative values indicate more favorable binding for GPI. Van der Waals, intermolecular van der Waals interactions reflecting shape complementarity; Electrostatic, intermolecular Coulombic interactions; EGB, polar component of solvation free energy estimated by the Generalized Born model; ESURF, nonpolar component of solvation free energy proportional to solvent-accessible surface area. Internal energy terms (bond, angle, dihedral, 1–4 van der Waals, 1–4 electrostatic) were zero for both compounds in this single-point calculation and are omitted. Interaction profiles were derived from protein-ligand interaction analysis in Flare V11. GPI, glycogen phosphorylase inhibitor (PubChem CID: 10070301); DFB, diflubenzuron (PubChem CID: 37123).
Key resources table
| Reagent type (species) or resource | Designation | Source or reference | Identifiers | Additional information |
|---|---|---|---|---|
| Gene (Plutella xylostella) | PxGP | GenBank | NCBI:XM_038119125.2 | |
| Strain, strain background (Escherichia coli) | DH10Bac | Invitrogen | Cat# EC0113 | |
| Cell line (Spodoptera frugiperda) | Sf9 | Gibco (Thermo Fisher Scientific) | Cat# 11496015 | |
| Biological sample (P. xylostella) | Susceptible laboratory strain | Northwest A&F University colony | Maintained without insecticide exposure | |
| Antibody | Anti-His (mouse monoclonal) | Abbkine | Cat# ABT2050 | WB (1:1500) |
| Antibody | AP, Goat Anti-Mouse IgG (goat) | Abbkine | Cat# A21110 | WB (1:4000) |
| Recombinant DNA reagent | pFastBac1 vector (plasmid) | Invitrogen | Cat# 10359016 | |
| Peptide, recombinant protein | immobilized Phosphorylase Kinase | MedChemExpress | Cat# HY-P2757 | |
| Commercial assay or kit | Glycogen Phosphorylase Activity Assay Kit | Solarbio Life Science | Cat# BC3345 | |
| Commercial assay or kit | Glycogen and Glucose Assay Kit | Grace Biotechnology | Cat# G0590W96 | |
| Commercial assay or kit | Trehalose Assay Kit | Grace Biotechnology | Cat# G0553W96 | |
| Commercial assay or kit | Glucose-6-phosphate (G6P) Assay Kit | Beyotime Biotechnology | Cat# S0185 | |
| Commercial assay or kit | Protein Quantification Kit (BCA Assay) | Abbkine | Cat# KTD3001 | |
| Commercial assay or kit | T7 RiboMAX Express RNAi System | Promega | Cat# P1700 | |
| Commercial assay or kit | TB Green Premix Ex Taq II (Tli RNaseH Plus) | TaKaRa | Cat# RR820A | |
| Commercial assay or kit | ClonExpress II One Step Cloning Kit | Vazyme | Cat# C112-01 | |
| Chemical compound, drug | Diflubenzuron (DFB) | Shanghai Topscience | Cat# T7088 | ≥98% purity |
| Chemical compound, drug | Glycogen phosphorylase-IN-1 (GPI) | TargetMol | Cat# T37577 | ≥98% purity |
| Software, algorithm | GraphPad Prism 9.0 | GraphPad Software | RRID:SCR_002798 | IC50, statistics |
| Software, algorithm | Cresset Flare V11 | Cresset https://www.cresset-group.com/flare/ | Docking, MM/GBSA | |
| Software, algorithm | SWISS-MODEL | Biozentrum, University of Basel https://swissmodel.expasy.org | Homology modeling | |
| Software, algorithm | POLO-Plus | LeOra Software | Probit analysis | |
| Software, algorithm | ImageJ | NIH | RRID:SCR_003070 | Leaf area, wing area |
| Other | BM Purple AP Substrate | Roche | Cat# 11442074001 | WB detection |
Additional files
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Supplementary file 1
Primer sequences used in this study.
- https://cdn.elifesciences.org/articles/111144/elife-111144-supp1-v1.docx
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Supplementary file 2
Toxicity of diflubenzuron (DFB) against third-instar Plutella xylostella larvae at 120 hr.
- https://cdn.elifesciences.org/articles/111144/elife-111144-supp2-v1.docx
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MDAR checklist
- https://cdn.elifesciences.org/articles/111144/elife-111144-mdarchecklist1-v1.docx
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Source data 1
Contains values used for data presentation of Figures 2—4, 6—11, and Figure 2—figure supplement 1, Figure 10—figure supplements 1 and 2.
- https://cdn.elifesciences.org/articles/111144/elife-111144-data1-v1.xlsx
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Gluconeogenic compensation renders glycogen phosphorylase non-essential as an insecticidal target in Plutella xylostella
eLife 15:RP111144.
https://doi.org/10.7554/eLife.111144.4
