Fig 1.
Time-series transcriptomic analysis of carabrone-treated Gaeumannomyces tritici.
(A) The structure of carabrone. (B) Schematic illustration of time-series transcriptomic analysis. (C) Clustering and expression trend analysis of differentially expressed genes (DEGs). (D) Kyoto encyclopedia of genes and genomes (KEGG) enrichment analysis of gene cluster 2. (E) Heatmap of gene expression clusters in the oxidative phosphorylation (OXPHOS) pathway. (F) Gene assignment of cluster 3 within the OXPHOS pathway. (G) The ATP level of G. tritici treatment by carabrone (100 and 200 μM), rotenone (20 μM) and oligomycin A (5 nM). (H) The O2.- level of G. tritici treatment by carabrone (100 and 200 μM) and rotenone (10 μM). Data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05).
Fig 2.
Analysis of the correlation between NAD and the antifungal activity of carabrone.
(A) The KEGG enrichment analysis of gene cluster 4 in total DEGs. (B) Heatmap of DEGs in the nicotinate and nicotinamide metabolism pathway. (C) RT-qPCR analysis of Gtnrk1 and Gtsir2 in G. tritici after carabrone (200 μM) treatment. (D) NAD⁺/NADH ratio in G. tritici after carabrone (200 μM) treatment at different time points. (E) NAD⁺/NADH ratio in G. tritici after carabrone treatment with different concentration (treatment at 4 h). (F) Schematic illustration of nicotinamide mononucleotide (NMN) supplementation can increase NAD+ synthesis by nicotinamide mononucleotide adenylyltransferase (NMNAT) activity. (G) Modulation of carabrone’s (200 and 400 μM) antifungal activity by NMN (400 μM) supplementation. (H) Schematic illustration of duroquinone can oxidize NADH to yield NAD+ and durohydroquinone by the activity of NAD(P)H dehydrogenase, quinone 1 (NQO1). (I) Modulation of carabrone’s (400 μM) antifungal activity by duroquinone supplementation. Data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05).
Table 1.
The inhibitory activities of carabrone and CAR-Y against G. tritici.
Fig 3.
Identification of the antifungal target of carabrone by activity-based protein profiling (ABPP).
(A) Schematic illustration of carabrone target protein identification in G. tritici by ABPP. (B) The structure of alkyne-tagged carabrone probe (CAR-Y). (C) In vivo labeling of CAR-Y in G. tritici, CAR-Y treatment for 4 hours. (D) In vivo competitive labeling with CAR-Y in G. tritici, carabrone pretreatment (4 h) followed by CAR-Y labeling in vitro. (E) Colocalization analysis of CAR-Y and mitochondria in G. tritici, Bar = 10 μm. (F) In vitro labeling of CAR-Y in mitochondria protein of G. tritici. (G) Enrichment and screening of CAR-Y-binding proteins in G. tritici. (H) Gene ontology (GO) enrichment analysis of potential carabrone target proteins (56 proteins) in G. tritici. (I) KEGG enrichment analysis of potential carabrone target proteins (56 proteins) in G. tritici. (J) Cofactor analysis of potential carabrone target proteins (56 proteins) in G. tritici. CCB: Coomassie brilliant blue. Data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05).
Fig 4.
Screening of carabrone target proteins.
(A) Schematic illustration of mitochondrial NAD⁺/NADH balance maintenance. (B) Sensitivity of ΔGtnuo49 silenced strains to carabrone. (C) Sensitivity of ΔGtndufv1 silenced strains to carabrone. (D) Sensitivity of ΔGtnuo78 silenced strains to carabrone. (E) Sensitivity of ΔGtatp3 silenced strains to carabrone. (F) Sensitivity of ΔGtdld2 silenced strains to carabrone. (G) Sensitivity of ΔGtfh silenced strains to carabrone. The concentration of carabrone is 200 μM. Data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05).
Fig 5.
Effect of carabrone on mitochondrial respiratory chain complex V (ATP synthase).
(A) Schematic illustration of OXPHOS and its uncoupling mechanism from the electron transport chain (ETC). (B) Measurement of mitochondrial membrane potential (MMP, ΔΨm) in G. tritici, treatment by carabrone (50 and 100 μM), FCCP (250 nM) and oligomycin A (5 nM), JC-1 monomers (green) indicate decreased MMP, JC-1 aggregates (red) indicate increased MMP, Bar = 100 μm. (C) Effect of FCCP (250 nM) on the antifungal activity of oligomycin A (5 nM). (D) NAD+/NADH ratio in G. tritici after FCCP (250 nM) and oligomycin A (5 nM) treatment. (E) Effect of FCCP (250 nM) on the antifungal activity of carabrone (200 and 400 μM). (F) NAD+/NADH ratio in G. tritici after FCCP (250 nM) and carabrone (200 and 400 μM) treatment. Data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05).
Fig 6.
Validation of carabrone targeting mitochondrial respiratory chain complex I in G. tritici.
(A) Schematic illustration of the mechanism by which pyruvate converts NADH to NAD⁺ via D-lactate dehydrogenase to support complex I function. (B) Modulation of carabrone’s (200 and 400 μM) antifungal activity by pyruvate (5 mM) supplementation. (C) Modulation of rotenone’s (20 μM) antifungal activity by pyruvate (5 mM) supplementation. (D) NAD+/NADH ratio in G. tritici after carabrone (200 and 400 μM) and pyruvate (5 mM) treatment. (E) Schematic illustration of the mechanism by which ndi1 overexpression supports mitochondrial complex I function. (F) Sensitivity of ndi1 overexpression strain to rotenone (20 μM). (G) NAD+/NADH ratio in ndi1 overexpression strain of G. tritici. (H) Sensitivity of ndi1 overexpression strain to carabrone (200 and 400 μM). (I) and (J) Enzymatic activity assay of mitochondrial respiratory chain complex I in G. tritici treated with carabrone. Data are mean ± SD of n = 3 biologically independent experiments. Statistical significance was determined by one-way ANOVA with Tukey’s post hoc test (P < 0.05).