Indoloquinoline alkaloid neocryptolepine derivative inhibits Botrytis cinerea by targeting thiamine thiazole synthase

Abstract

The emergence and rapid spread of multidrug-resistant Botrytis cinerea strains pose a great challenge to the quality and safety of agricultural products and the efficient use of pesticides. Previously unidentified fungicides and targets are urgently needed to combat B. cinerea-associated infections as alternative therapeutic options. In this study, the promising compound Z24 demonstrated efficacy against all tested plant pathogenic fungi. Thiamine thiazole synthase (Bcthi4) was identified as a target protein of Z24 by drug affinity responsive target stability (DARTS), cellular thermal shift assay (CETSA), and surface plasmon resonance (SPR) assays. Molecular docking and enzyme activity experiments have demonstrated that Z24 can affect the function of Bcthi4. Last, mechanistic studies show that Z24 inhibits thiamine biosynthesis by binding to Bcthi4 and induces up-regulation of alternative splicing [alternative 5' splice site (A5SS)] of the Bcthi4 gene. In conclusion, by targeting Bcthi4, Z24 has the potential to be developed as a previously unidentified anti-B. cinerea candidate.

Fig. 1.. In vitro antifungal activity of Z24 against B. cinerea.

(A) Colony formation (n = 6) of phytopathogenic fungi after 3 to 7 days of growth on agar plates supplemented with Z1 and Z24 (5 μg/ml). (B) Compound Z1 moderately suppressed mycelial growth of B. cinerea Pers. and B. cinerea B05.10 at concentrations ranging from 5 to 50 μg/ml. (C) Compound Z24 effectively suppressed mycelial growth of B. cinerea Pers. and B. cinerea B05.10 at concentrations of 0.5 to 5 μg/ml. (D and E) Colony formation (n = 6) of B. cinerea Pers. after 4 days of growth on agar plates, supplemented with increasing concentrations of compound Z1 and Z24. The green dotted line indicates EC₅₀ concentration. (F) Representative images of the germination of B. cinerea B05.10 spores after treatment with Z1 and Z24. Scale bars, 100 μm. (G and H) Effects of (G) Z24 (n = 6 to 8) and (H) Z1 (n = 5 to 7) on the germination of B. cinerea B05.10 spores. (I) Representative images of the germination of B. cinerea Pers. spores after treatment with Z1 and Z24. Scale bars, 100 μm. (J and K) Effects of (J) Z1 (n = 6 to 10) and (K) Z24 (n = 7 to 8) on the germination of B. cinerea Pers. spores. (L) Transmission electron microscopy (TEM) images reveal the ultrastructure of B. cinerea Pers. after treatment with Z24 (1 μg/ml). Scale bars, 1.0 μm. (M) Intracellular ROS levels [2′,7′-dichlorodihydrofluorescein diacetate (DCFH-DA)] in B. cinerea Pers. spores after treatment with Z24 (20 μg/ml) were measured using DCFH-DA. Scale bars, 100 μm. (N) Nuclear dye propidium iodide (PI)/Hoechst 33342 was used to stain the mycelial cells treated with Z24 (0.5 μg/ml). Scale bars, 50 μm.

Fig. 2.. Z24 targets the Bcthi4 protein, which contributes to inhibit the growth of B. cinerea cells in vitro and in vivo.

(A) DARTS identifies a molecular target of Z24. (B) B. cinerea Pers. cell lysates were incubated with Z24 in vitro, followed by protease digestion and Coomassie bright blue (CBB) stain. (C) B. cinerea Pers. cell lysates were incubated with Z24 in vitro, followed by protease digestion and silver staining (n = 3 exp.). The red and black arrows indicate the locations of the different bands, B1 and B2. (D and E) Information on the top 10 proteins with the highest relative abundance in the B1 and B2 band. iBAQ, intensity-based absolute quantification. (F and G) Predicted binding mode of Z24 with Bcthi4. (H) Expression and purification of the Bcthi4^WT protein in a prokaryotic expression system. (I) Surface plasmon resonance (SPR) sensorgrams obtained from Bcthi4^WT-coated chips at different concentrations of Z24. (J) Dissociation constant (K_d) value of Z24 binding to the recombinant Bcthi4^WT protein. (K) Expression and purification of the Bcthi4^{MT(Met304/Thr232/Thr231)} mutant protein in a prokaryotic expression system. (L) Detection of the binding of Bcthi4^{MT(Met304/Thr232/Thr231)} to Z24 by SPR analysis. (M) K_d value of Z24 binding to the recombinant mutated Bcthi4^{MT(Met304/Thr232/Thr231)} protein. “NA” indicates that no binding activity was detected. K_a, acid constant. (N and O) Bcthi4 activity in B. cinerea Pers. and B. cinerea B05.10 after treatment with Z24 at 0.05 and 0.1 μg/ml, respectively. (P) Fold change in Bcthi4 activity in B. cinerea pers. and B. cinerea B05. (Q and R) The Bcthi4 rabbit polyclonal antibodies, G2206 and G2207, were successfully prepared. (S) Z24-enriched Bcthi4 protein was verified by DARTS (n = 3 exp.). (T) Thermal stability of Bcthi4 protein with or without Z24 treatment (n = 3 exp.).

Fig. 3.. DEGs and RASG analysis.

(A) Volcano plot analysis of differential gene expression, including 1907 up-regulated genes and 2366 down-regulated genes. FDR, false discovery rate. (B) Kyoto Encyclopedia of Genes and Genomes (KEGG) enrichment analysis of up-regulated differentially expressed genes (DEGs). (C) Overview of all detected AS events. (D) Types and numbers of AS events detected in up-regulated and down-regulated AS genes. (E) KEGG enrichment analysis of variable AS genes (RASGs). (F) KEGG enrichment analysis of the overlap between RASGs and DEGs. (G) Differential variable splicing events of the Bcthi4 gene.

Fig. 4.. Available thiamine promotes the growth of B. cinerea under Z24 stress by up-regulating genes and metabolites.

(A and B) Colony diameter measurement of B. cinerea Pers. and B. cinerea B05.10 following treatment with Z24 (2.5 and 5 μg/ml). (C) Colony diameter of B. cinerea Pers. after treatment with tebuconazole (2.5 μg/ml). d, days. (D and E) Reverse transcription quantitative polymerase chain reaction (RT-qPCR) analysis of Bcthi4 gene expression in B. cinerea Pers. and B. cinerea B05.10 after Z24 treatment. (F to H) Measurement of thiamine content in (F) B. cinerea Pers. and (G) B. cinerea B05.10, as well as the (H) fold change after Z24 treatment. (I to K) Measurement of TPP content in (I) B. cinerea Pers. and (J) B. cinerea B05.10, along with the (K) fold change after Z24 treatment. (L) Z24 specifically inhibits the growth of B. cinerea by targeting Bcthi4 in the thiamine biosynthesis pathway. HET-P, 4-methyl-5-(2-phosphooxyethyl)thiazole; NAD, nitric acid dihydrate.

Fig. 5.. Metabolic pathway blockade identifies fungicide Z24.

(A and B) The effect of exogenous thiamine (500 μg/ml) on the growth of B. cinerea Pers. and B. cinerea B05.10 after treatment with pyrimethanil (4 μg/ml) on PDA plates. (C and D) The growth inhibitory activity of B. cinerea Pers. and B. cinerea B05.10 in the presence of thiamine (500 μg/ml) after treatment with Z24 (0.25 μg/ml) on PDA plates. (E and F) The inhibitory activity of thiamine (at 50, 100, 250, 500 μg/ml) against B. cinerea Pers. and B. cinerea B05.10 on PDA plates. (G and H) The effect of exogenous thiamine (at 250 and 500 μg/ml) on the growth of B. cinerea Pers. after treatment with Z24 in liquid PDA medium. (I) The growth inhibitory activity of B. cinerea B05.10 in the presence of thiamine (250 and 500 μg/ml) after treatment with Z24 (0.1 μg/ml) in liquid PDA medium. (J and K) Inhibitory activity of thiamine (at 100, 250, and 500 μg/ml) against B. cinerea Pers. and B. cinerea B05.10 in liquid PDA medium. (L) The growth inhibitory activity of B. cinerea B05.10 in the presence of thiamine (500 μg/ml) after treatment with Z24 (10 μg/ml) on grapes.

Fig. 6.. The relative content of up-regulated and down-regulated metabolites shows a substantial correlation with amino acid metabolism in B. cinerea.

(A) Principal components analysis (PCA) score plots in electrospray ionization (ESI)⁺ mode. (B) Volcano plot analysis of differential metabolomics in ESI⁺ mode. FC, fold change. (C) Pathway enrichment analysis of differential metabolomics in ESI⁺ mode. (D to G) The fold change of metabolites in (D) alanine, aspartate, and glutamate metabolism; (E) biosynthesis of amino acids; (F) arginine biosynthesis; and (G) aminoacyl-tRNA biosynthesis in ESI⁺ mode. (H) PCA score plots in ESI⁻ mode. (I) Volcano plot analysis of differential metabolites in ESI⁻ mode. (J) Pathway enrichment analysis of differential metabolites in ESI⁻ mode. (K to N) The fold change of metabolites in (K) alanine, aspartate and glutamate metabolism; (L) biosynthesis of amino acids; (M) C5-branched dibasic acid metabolism; and (N) lipoic acid metabolism in ESI⁻ mode. The q value is obtained by correcting the P value using the FDR. BHcorrect, Benjamini-Hochberg correction.

Fig. 7.. In vitro and in vivo toxicity evaluation of compound Z24.

(A to C) Inhibitory activity of Z24 on (A) HIECs, (B) HL-7702 cells, and (C) GES-1 cells. HIECs, HL-7702 cells, and GES-1 cells were treated with Z24, Z1, and pyrimethanil for 48 hours, and cell viability was assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (D to H) Effects of Z24 (2000 mg/kg) on pathological changes in the (D) heart, (E) liver, (F) spleen, (G) lung, and (H) kidney tissues of Wistar rats. (I to M) Relative body weights of the (I) heart, (J) liver, (K) spleen, (L) lung, and (M) kidney in the control and Z24 (2000 mg/kg) groups. (N) Body weight changes in Wistar rats after Z24 administration. (O to R) Changes in blood biochemical markers: (O) GPT, (P) GOT, (Q) AKP/ALP, and (R) urea nitrogen in rats from the control and Z24 groups. (S and T) Representative images and quantitative statistical data for the Z24 and Triton X-100 hemolysis rates (n = 3).

Fig. 8.. Phytotoxicity and plant protection or therapy by Z24 in tomato and cucumber.

(A) Gray mold disease symptoms in tomatoes after treatment with various concentrations of Z24 (n = 5). (B) Gray mold disease symptoms on cucumber leaves; data are presented as means ± SD, n = 7 to 10 biologically independent samples. (C and D) Protective effects of Z24 and pyrimethanil on cucumber leaves after infection with B. cinerea Pers. plugs. (E and F) Therapeutic effects of Z24 and pyrimethanil on cucumber leaves after infection with B. cinerea Pers. plugs. h, hours. (G and H) Inhibitory activity of Z24 and carbendazim (a positive control against S. sclerotiorum strain) on sclerotia formation (n = 6). (I) Phytotoxicity of Z24 on radish seed (n = 12). (J) Effect of Z24 on the germination rate of radish seeds. (K) Effect of Z24 on the bud length of radish seeds. Note that control contains 0.25% DMSO. **P < 0.001, ***P = 0.0001, and ****P < 0.0001. n.s., not significant.