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. 2026 Feb 5;27(2):e70215. doi: 10.1111/mpp.70215

A Coumarin Compound Derived From Zanthoxylum avicennae Reduces the Pathogenicity of Fusarium verticillioides by Directly Binding to and Inhibiting Glycoside Hydrolase 3 Activity

Duxuan Liu 1,2, Jing Hua 1,2, Haoyu Chen 2, Mingjie Wu 2, Zhiqing Mao 1,3, Zhen Yang 2, Xiubin Xu 2, Yanhong Hua 2, Chenwei Feng 2, Kun Zhang 2,✉, Jiahuan Chen 1,✉
PMCID: PMC12874491  PMID: 41641775

ABSTRACT

The cell wall serves as a critical barrier in plant defence against pathogen infection, whereas various Fusarium fungi secrete cell wall‐degrading enzymes (CWDEs) to facilitate hyphal infection. In this study, luvangetin, a coumarin compound isolated and identified from the root of Zanthoxylum avicennae, was found to affect the cell wall degradation capacity and pathogenicity of Fusarium verticillioides on maize. Enzymatic activity assays of secreted enzymes from F. verticillioides demonstrated that luvangetin significantly inhibited the activity of the fungal crude enzyme extract, with the highest inhibition (13.5%) observed on cellulase activity. It also impaired the enzymatic hydrolysis to straw, wheat bran and bagasse. Integrated transcriptomic, proteomic and in vitro activity analyses collectively revealed that luvangetin binds to three critical sites (Y193, D571 and E575) of the glycoside hydrolase 3 family (GH3) β‐glucosidase in Fusarium species. Gene knockout and overexpression mutants were generated to further demonstrate that FvBgls3 plays a critical role in the pathogenicity of F. verticillioides and that it is an important target of luvangetin. Luvangetin directly binds to the catalytic active centre of FvBgls3, thereby suppressing the activity of CWDEs in F. verticillioides and ultimately reducing its pathogenicity. This study is the first to report that a coumarin small molecule directly binds to and inhibit the activity of GH3 family enzymes, revealing the molecular mechanism by which luvangetin directly inhibits cell wall degradation capacity, providing novel targets and strategies for future control of F. verticillioides.

Keywords: β‐glucosidase, cell wall‐degrading enzymes, disease resistance, Fusarium verticillioides, glycoside hydrolase family 3, luvangetin

Luvangetin, a coumarin compound from Zanthoxylum avicennae roots, affects the cell wall degradation capacity and pathogenicity of Fusarium verticillioides on maize. Integrated transcriptomic, proteomic and in vitro activity analyses revealed that it binds to three critical sites of the Fusarium glycoside hydrolase 3 family (GH3) β‐glucosidase.

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1. Introduction

Zanthoxylum avicennae is a plant species belonging to the family Rutaceae (Li et al. 2022). It is primarily distributed in southern regions of China such as Yunnan, Fujian and Guangdong. Its roots, leaves and fruits are used in folk medicine for treating icterohepatitis, jaundice, nephritis, hepatitis B, hepatocirrhosis, colitis and stomatitis (Ji et al. 2022). Its active constituents include flavonoids, alkaloids and coumarins. Luvangetin, a coumarin derivative previously isolated from Z. avicennae in our laboratory (Chen et al. 2024), has been demonstrated to exhibit broad‐spectrum antifungal activity against multiple phytopathogenic fungi (Liu, Chen, et al. 2025). The structural uniqueness of luvangetin, characterised by its furanocoumarin framework, enables specific binding affinities to critical molecular targets, making it a promising candidate for drug development (Çelik et al. 2024; Tang, Wang, et al. 2024). Previous studies have established that luvangetin possesses both DNA‐binding and dsRNA‐binding capabilities (Chen et al. 2024; Islam et al. 2007; Liu, Chen, et al. 2025). However, whether it can interact with proteins and subsequently influence their structure and functional activity remains unknown.

Plant cell walls are dynamic structures that provide mechanical support, orchestrate cell growth and influence cell differentiation and fate, thereby shaping the overall architectural structure of the plant (Cosgrove 2024; Molina et al. 2024). When plants are attacked by pathogens (bacteria, fungi or viruses), the plant cell wall serves as a critical component of the active defence system (Zhang, Gao, et al. 2021). The hydrophobic cuticle and wax layers on the outer epidermal cell wall form a water‐repellent barrier, preventing pathogen spore adhesion, water infiltration and direct microbial infection. A thick, dense network of cellulose and hemicellulose provides mechanical resistance against hyphal penetration and enzymatic degradation by pathogens (Zhong et al. 2025). Concurrently, the cell wall releases chemical agents including reactive oxygen species (ROS), phenolic compounds (e.g., flavonoids, lignin monomers) and phytoalexins that directly suppress pathogen activity (Bacete et al. 2018; Wolf 2022).

Correspondingly, pathogen genomes encode an array of carbohydrate‐active enzymes (CAZymes), including cutinases and cell wall‐degrading enzymes (CWDEs), which facilitate attachment, infection, colonisation and nutrient acquisition in host plant cells by degrading cuticular and cell wall components (Dora et al. 2022; Juge 2006; Ma, Liu, et al. 2024; Wei et al. 2022; Wu et al. 2025). For example, Fusarium oxysporum secretes a broad repertoire of cellulolytic enzymes (glycosyl hydrolases [GHs] and lytic polysaccharide monooxygenases [AAs]), disrupting vascular systems in roots and stems to impair water flux and nutrient uptake (Gámez‐Arjona et al. 2022; Gordon 2017). Fusarium graminearum causes severe yield losses in cereals by secreting cutinases (CE5 family) and xylanases (GH10 family) that trigger scab disease (Tundo et al. 2015). As it also secretes the mycotoxin deoxynivalenol (DON), it causes secondary contamination of grains, posing a potential threat to human and animal health (Miao et al. 2024). The pectate lyase VdPEL1 from Verticillium dahliae and the cellulase MIB366 from Fusarium solani both play critical roles in the degradation of plant cell walls. Fusarium verticillioides causes yield‐limiting maize ear rot and stalk rot diseases (Tang, Ding, and Guo 2024). This pathogen produces carcinogenic mycotoxins called fumonisins in infected ears that pose critical health risks to animals and humans (Akanmu et al. 2020; Liu et al. 2023). Previous studies have demonstrated that the enzymes of F. verticillioides can function as auxiliary enzymes to enhance the saccharification and degradation of agricultural waste (Ravalason et al. 2012). F. verticillioides also possesses the capacity to secrete CWDEs, thereby facilitating cell wall disruption and promoting hyphal infection.

In this study, we observed that F. verticillioides‐infected maize leaves exhibited pronounced chlorosis and wilting, whereas luvangetin‐treated leaves retained a chlorosis‐free green hue with enhanced turgidity. We proposed that F. verticillioides, like other Fusarium fungi, facilitates infection by releasing CWDEs to depolymerise cellulose and pectin, thereby compromising the structural integrity of plant cell walls to enable hyphal penetration and colonisation. Critically, luvangetin directly binds to the catalytic active centre of FvBgls3, thereby suppressing the activity of CWDEs in F. verticillioides and ultimately reducing its pathogenicity. This study is the first to report that coumarin small molecules directly bind to and inhibit the activity of GH3 family enzymes, revealing the molecular mechanism by which luvangetin directly inhibits cell wall degradation capacity, providing novel targets and strategies for future control of F. verticillioides.

2. Results

2.1. Luvangetin Inhibits the Cell Wall Degradation Capability of F. verticillioides on Maize

We employed the ethanol cold immersion method to isolate and extract active substances from Z. avicennae in our preliminary studies (Chen et al. 2024), resulting in the obtainment of a small coumarin molecule known as luvangetin (Figure 1A). To demonstrate the safety and non‐toxicity of luvangetin to plants, we treated maize plants with a high concentration of luvangetin (1000 mg/L) in the absence of F. verticillioides infection. After 12 days of treatment, both the luvangetin‐treated and double‐distilled water (ddH2O)‐treated maize plants exhibited normal growth, with no significant differences in physiological parameters such as plant height, root length and number of adventitious roots (Figure S1). The results confirmed that luvangetin is safe and non‐toxic to plants, suggesting its potential application in the development of plant fungicides. To investigate the effect of luvangetin on F. verticillioides infection, we treated maize leaves with different components: ddH2O (Mock), 10% ethanol (negative control) and luvangetin (100, 200 and 300 mg/L), followed by inoculation with F. verticillioides. At 4 days post‐inoculation (dpi), maize leaves after luvangetin treatment appeared to exhibit slighter symptoms and exhibited more green colouration compared to the control groups (Figure 1B). Statistical analysis showed that the disease lesion areas caused by F. verticillioides with different concentrations of luvangetin treatments (1.01, 0.76 and 0.49 cm2) were significantly smaller than those in the 10% ethanol treatment (2.01 cm2, Figure 1C). Similarly, the relative biomass of F. verticillioides also showed a significant decrease (Figure 1D). The dry weight of healthy maize leaves was approximately 0.4 g. After infection with F. verticillioides, the dry weight significantly decreased to 0.29 g. However, the leaf dry weight reached 0.37 g after 300 mg/L of luvangetin treatment, showing no significant difference compared to healthy leaves (Figure S2). Subsequently, we quantified cellulose and pectin contents in maize leaves in different treatment groups. In healthy leaves, cellulose and pectin contents were 28% and 5.3%, respectively. Following F. verticillioides infection, these values significantly decreased to 5.5% and 0.8%. As the concentration of luvangetin increased, both cellulose and pectin contents gradually recovered, approaching the levels observed in healthy leaves (Figure 1E,F). We next investigated the relative expression levels of cellulose (ZmCesA1, ZmMYB92 and ZmNST3) and pectin (ZmPME5, ZmGAUT12) biosynthesis‐related genes (Appenzeller et al. 2004; Zhang et al. 2019) using reverse transcription‐quantitative PCR (RT‐qPCR). The experimental results showed that all of these genes were significantly up‐regulated after F. verticillioides infection. However, with increasing concentrations of luvangetin, the relative expression levels of these genes gradually decreased, approaching those observed in the healthy group. The same experiments were also conducted on maize leaves not infected with F. verticillioides. The results indicated that there were no significant differences in the relative expression levels of genes related to cellulose and pectin biosynthesis among the different treatment groups (Figure S3).

FIGURE 1.

FIGURE 1

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Luvangetin inhibits the cell wall degradation capability of Fusarium verticillioides on maize. (A) Flowchart for the isolation of compounds from Zanthoxylum avicennae. (B) Symptoms in maize leaves with F. verticillioides inoculation after different treatments: mock, double‐distilled water; 10% ethanol (Et), three concentrations (100, 200 and 300 mg/L in 10% ethanol) of luvangetin. Images were photographed at 4 days post‐inoculation. Bar, 1 cm. Each experiment was replicated three times, independently. (C) Lesions area of the maize leaves was analysed by ImageJ software. CK, mock treatment. Different letters indicate significant differences according to Tukey's test (p < 0.05). Error bars indicate the standard deviations (SD) of three samples. (D) Relative F. verticillioides biomass after different treatments. (E) Statistical analysis of cellulose content on maize after different treatments. (F) Statistical analysis of pectin content on maize after different treatments.

2.2. Luvangetin Inhibits the F. verticillioides Crude Enzyme Extract Activity

To investigate the effects of luvangetin on F. verticillioides secreted enzymes (secretome), crude enzyme extracts were prepared from cultures grown in enzyme‐production medium. The release of glucose, xylose, arabinose and reducing sugars was quantified during a 72‐h hydrolysis. Cellulose to glucose conversion yield reached 23.6% without luvangetin treatment, whereas after luvangetin treatment this value significantly decreased to 10.1% (Figure 2A). For xylose and arabinose release, as the addition of luvangetin to F. verticillioides crude enzyme extract led to a 3.9% decrease in xylan conversion (Figure 2B) and a 1.4% decrease in arabinan conversion (Figure 2C). Correspondingly, luvangetin significantly inhibited the release of reducing sugars (Figure 2D). The above results demonstrated that luvangetin exhibited the greatest inhibitory effect (13.5%) on cellulase activity in F. verticillioides. Cellulose constitutes the highest proportion of plant cell walls, providing mechanical support against pathogen infection (Khodayari et al. 2024; Zhou et al. 2024). Correspondingly, cellulases occupy a critical position among CWDEs (Zhang et al. 2022). We therefore investigated the effects of temperature and pH on the inhibition of F. verticillioides cellulase activity by luvangetin. At 50°C, F. verticillioides cellulase activity peaked (7.28 IU/mL). After luvangetin treatment, the value decreased to 3.95 IU/mL, yielding a 45.7% inhibition rate (Figure 2E). At pH 8.0, enzyme activity was 7.04 IU/mL, whereas the luvangetin‐treated group showed only 4.02 IU/mL (42.9% inhibition rate, Figure 2F). Subsequently, we constructed a three‐dimensional response surface diagram of temperature and pH effect on the cellulase activity of F. verticillioides without (Figure 2G) and with luvangetin treatment (Figure 2H). The results revealed that the optimal inhibition effect of luvangetin on F. verticillioides cellulase activity was at 50.65°C and pH 7.94 (Figure 2I), This was in agreement with the report of Ping et al. (2017).

FIGURE 2.

FIGURE 2

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Luvangetin inhibits the Fusarium verticillioides crude enzyme extract activity. Time course of (A) cellulose conversion, (B) xylan conversion, (C) arabinan conversion and (D) reducing sugars release by F. verticillioides crude enzyme extract with and without luvangetin treatment. Each experiment was replicated three times, independently. Error bars indicate the standard deviations (SD) of three samples. The symbols represent the following levels of significance: **p < 0.01, *p < 0.05, ‘ns’ indicates no significant difference between the compared groups. (E) Optimum temperature curves of F. verticillioides cellulase activity with and without luvangetin treatment. (F) Optimum pH curves of F. verticillioides cellulase activity with and without luvangetin treatment. (G) Three‐dimensional response surface diagram of temperature and pH effect on the F. verticillioides cellulase activity without luvangetin treatment. (H) Three‐dimensional response surface diagram of temperature and pH effect on the F. verticillioides cellulase activity with luvangetin treatment. (I) Three‐dimensional response surface diagram of temperature and pH effect on the inhibition rate of luvangetin on F. verticillioides cellulase activity.

We treated straw, wheat bran and bagasse with F. verticillioides crude enzyme extract and examined the effect of luvangetin on the enzymatic degradation capacity. As observed in Figure 3A, the content of straw, wheat bran and bagasse treated with F. verticillioides crude enzyme treatment (Mock) decreased compared to the uninoculated enzyme‐production medium treatment (CK), indicating the cellulose‐degrading capability of the crude enzyme extract. However, after adding luvangetin, the residual content of these substrates approached levels similar to CK, demonstrating that the degradation ability of the F. verticillioides crude enzyme extract was inhibited by luvangetin. Subsequently, we performed scanning electron microscopy (SEM) observations on the surface structures of straw, wheat bran and bagasse after different treatments. The results showed that F. verticillioides crude enzyme extract greatly changed the surface structure of straw (Figure 3B). Compared with the CK, tuberculate or warty protrusions were almost everywhere on the surface of straw treated with F. verticillioides crude enzyme extract. Some protrusions even covered and blocked the stomata of the straw surface with a characteristic dumbbell shape, which may be one of the reasons for the reduction in dissolving sugars and repression of cellulase hydrolysis of straw (Gu et al. 2021; Liu et al. 2014). In the luvangetin‐treated group, a significant reduction in surface irregularities and protrusions was observed. The SEM observations on the surface structure of wheat bran and bagasse were consistent with the results on straw. In the degradation experiment of straw, CK showed a surface area of 2.78 cm2 and a dry weight of 183 mg, whereas the luvangetin treatment exhibited values of 2.16 cm2 and 132 mg, both significantly higher than Mock (1.49 cm2 and 67.6 mg). A consistent trend was observed in the degradation experiments of wheat bran and bagasse (Figure 3C,D). These results demonstrated that the crude enzyme extract of F. verticillioides exerted strong hydrolytic effects on straw, wheat bran and bagasse, whereas luvangetin markedly suppressed this enzymatic degradation.

FIGURE 3.

FIGURE 3

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Effect of luvangetin on the degradation of straw, wheat bran and bagasse by Fusarium verticillioides crude enzyme extracts. (A) Assay of the degradation ability of F. verticillioides crude enzyme extract on straw, wheat bran and bagasse with and without luvangetin treatment. Images were taken at 10 days post‐incubation (dpi). Bar, 1 cm. Each experiment was replicated three times, independently. (B) Scanning electron microscopy observation of the surfaces of straw, wheat bran and bagasse after different treatments. (C) Statistical analysis of the surface area of straw, wheat bran and bagasse after different treatments. The symbols represent the following levels of significance: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns no significant difference between the compared groups. Error bars indicate the standard deviations (SD) of five samples. (D) Statistical analysis of the dry weight of straw, wheat bran and bagasse after different treatments.

2.3. Transcriptome Analyses of the Effects of Luvangetin on F. verticillioides Cell Wall Degradation Capability

To reveal the molecular mechanism by which luvangetin inhibits the CWDE activity of F. verticillioides, we prepared samples for sequencing by adding 0.5% luvangetin in the culture medium. Total proteins and RNAs were extracted and subjected to integrated omics analysis. In transcriptomics, fragments per kilobase per million mapped reads (FPKM) serve as a normalised expression metric accounting for gene length and sequencing depth biases. The boxplots of FPKM values demonstrated central expression trends clustered around the median, indicating high intragroup concordance among biological replicates (Figure 4A). Heatmap of correlation coefficients between samples revealed that the distances of the three samples of Fv‐T (treated with luvangetin) or Fv‐NT (not treated with luvangetin) were close, whereas the distance between each sample from Fv‐T to the sample from Fv‐NT was far, indicating high repeatability of the sequencing and significant difference between Fv‐T and Fv‐NT (Figure 4B). Hence, these two methods provide high sequencing quality. The two approaches validated the high quality of the sequencing data, supporting their suitability for subsequent analyses. There were 1837 differentially expressed genes (DEGs), of which 1010 genes were up‐regulated and 827 genes were down‐regulated in the F. verticillioides treated with luvangetin (Figure 4C). The transcription profile of genes related to cell wall degradation was selected and illustrated by a heatmap. The expression of most genes related to cell wall degradation was significantly suppressed after luvangetin treatment in F. verticillioides (Figure 4D). Cellulose‐degrading genes constituted the highest proportion (21.2%) of significantly down‐regulated transcripts (Figure 4E). We selected a few genes (Figure 4D, red framed) and validated their expression through RT‐qPCR. The results showed that the expression profile of the selected genes, including FvMro (FVEG_07688), FvRhiE (FVEG_04751), FvAmI (FVEG_13849), FvAgaA1 (FVEG_12289) and FvAga2 (FVEG_07518), was highly consistent with the transcriptome data (Figure 4F), indicating that the transcriptome data is credible and the luvangetin treatment inhibited the expression of genes relate to cell wall degradation in F. verticillioides.

FIGURE 4.

FIGURE 4

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Transcriptome analyses of Fusarium verticillioides with (Fv‐T) and without luvangetin treatment (Fv‐NT). (A) Box‐whisker plot of gene expression levels. Each box plot displays five statistical measures: Maximum, third quartile, median, first quartile and minimum (listed top to bottom). (B) Heatmap of correlation coefficients between samples. Colour represents the magnitude of correlation coefficients. (C) Bar plot of differentially expressed gene (DEG) counts. Up, significantly up‐regulated DEGs; Down, significantly down‐regulated DEGs. (D) Heat map shows the gene expression levels associated with cell wall degradation with and without luvangetin treatment. (E) Functional classification of DEGs associated with cell wall degradation. (F) Relative expression levels of five key genes: FvMro (FVEG_07688), FvRhiE (FVEG_04751), FvAmI (FVEG_13849), FvAgaA1 (FVEG_12289) and FvAga2 (FVEG_07518) using reverse transcription‐quantitative PCR analyses. The symbols represent the following levels of significance: **p < 0.01 between the compared groups. Error bars indicate the standard deviations (SD) of three samples.

2.4. Proteomic Analyses of the Effects of Luvangetin on F. verticillioides Cell Wall Degradation Capability

We performed proteomic analysis of F. verticillioides with and without luvangetin treatment. In proteomics analysis, principal component analysis (PCA) was employed to reduce the complexity of high‐dimensional protein expression data and reveal biological differences and underlying patterns among samples (Han et al. 2020). Samples treated with 0.5% luvangetin (Fv‐T‐1, Fv‐T‐2 and Fv‐T‐3) and untreated controls (Fv‐NT‐1, Fv‐NT‐2 and Fv‐NT‐3) formed tight intragroup clusters in PCA space (PC1: 58.06%, PC2: 12.18%; Figure 5A), demonstrating strong reproducibility and significant separation between treated and untreated groups. Normalised protein expression represents technically adjusted protein abundance values accounting for experimental biases. The results demonstrated high comparability among the three biological replicates within the same treatment (Figure 5B). In total, 68,215 peptides were identified through MS, and 5840 proteins were identified through alignment and annotation. The peptide length distribution of these identified proteins was mapped; proteins yielding peptides within the 9–12 amino acid length range demonstrated the highest enrichment (Figure 5C). The differentially expressed proteins relate to cell wall degradation, which included 118 up‐regulated and 130 down‐regulated proteins in Fv‐T samples compared with Fv‐NT samples, outlined using a volcano diagram (Figure 5D). Proteins exhibiting significant changes were subsequently selected for heatmap analysis (Figure 5E).

FIGURE 5.

FIGURE 5

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Proteomic analyses of Fusarium verticillioides with and without luvangetin treatment. (A) The principal component analysis of the proteomic sequencing data with and without luvangetin treatment. Fv‐T, F. verticillioides with luvangetin treatment. Fv‐NT, F. verticillioides without luvangetin treatment. (B) Box plots of expression levels for credible proteins. (C) Distribution of peptide lengths. (D) The volcano diagram represents the differentially expressed proteins between samples with and without luvangetin treatment (fold change > 2, p < 0.05). Red dots, up‐regulated proteins; blue dots, down‐regulated proteins. (E) Heat map of the protein expression levels associated with cell wall degradation with and without luvangetin treatment.

2.5. Effect of Luvangetin on the Activity of FvBgls3 and Its Mutant Protein

To investigate genes and amino acid sequences associated with cell wall degradation that exhibited significant changes in transcriptomics and proteomics analyses, we constructed phylogenetic trees separately. As shown in Figure 6A, the transcriptional levels of five glycoside hydrolase family proteins (GH3, GH15, GH27, GH32 and GH43 families) were significantly down‐regulated. In Figure 6B, the protein expression levels of three glycoside hydrolase families (GH3, GH29 and GH93 families) were also markedly down‐regulated. Integrated omics analyses revealed that only the GH3 family proteins showed significant down‐regulation at both transcriptional and translational levels. We hypothesised that GH3 family proteins may play a crucial role in F. verticillioides pathogenicity. In addition to affecting the transcription and translation of GH3 family proteins, luvangetin might also directly bind to and inhibit the active sites of key GH3 family enzymes, thereby reducing the activity of CWDEs in F. verticillioides. To explore the role of GH3 family proteins in F. verticillioides pathogenicity and the impact of luvangetin on these proteins, we selected β‐glucosidases (FVEG_09248), the core catalytic components of GH3 enzymes (de Andrades et al. 2024), for homology modelling and molecular docking with luvangetin.

FIGURE 6.

FIGURE 6

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Bioinformatic analysis of cell wall‐degrading proteins in Fusarium verticillioides. (A) Phylogenetic analysis of differentially expressed genes of F. verticillioides associated with cell wall degradation using MEGA11 software based on gene sequences. (B) Phylogenetic analysis of differentially expressed proteins of F. verticillioides associated with cell wall degradation using MEGA11 software based on amino acid sequences. (C) Homology modelling of F. verticillioides β‐glucosidase (FvBgls3) using SWISS‐MODEL server and its molecular docking with luvangetin using CB‐Dock2 server. Red regions: barrel domain; yellow regions: sandwich domain; orange regions: PA14 domain and green regions: fibronectin type III domain. Numbers 1, 2 and 3 represent three binding sites of luvangetin with FvBgls3.

The 3D structure of F. verticillioides GH3 family β‐glucosidase (FvBgls3) featured classical β‐glucosidase structural domains: barrel domain (red regions in Figure 6C), sandwich domain (yellow regions), PA14 domain (orange regions) and fibronectin type III domain (green regions). The catalytic active sites are predominantly located on the barrel domain and sandwich domain (Gudmundsson et al. 2016). The fibronectin type III domain contributes to protein stability, whereas the PA14 domain facilitates substrate binding to β‐glucosidases (Yoshida et al. 2010). In FvBgls3, the most conserved nucleophilic catalytic residue (MSDW) (Frankel 1993) of β‐glucosidases was situated within the barrel domain (Figure 6C). Molecular docking results indicated strong binding affinity between FvBgls3 and luvangetin, with binding sites including R157, Y193, D225, S349, E452, Y457, D571 and E575. Among these, residues Y193, D571 and E575 exhibit the strongest binding interactions, primarily mediated through hydrogen bonding. Similarly, homology modelling and molecular docking were performed on β‐glucosidases of three additional representative Fusarium species (Fusarium oxysporum, Fusarium graminum and Fusarium equiseti, Figure S5). The results demonstrated that residues Y193, D571 and E575 exhibited the strongest binding affinity with luvangetin in the three Fusarium species β‐glucosidases. Furthermore, multiple sequence alignment of β‐glucosidases from four Fusarium species (F. verticillioides, F. oxysporum, F. graminum and F. equiseti), Aspergillus niger (Dan et al. 2000), Talaromyces emersonii and Aspergillus fumigatus confirmed that Y193, D571 and E575 were highly conserved in diverse species (Figure S6, red framed). We expressed and purified FvBgls3138‐580 , which contains the barrel domain (catalytic active centre), nucleophilic catalytic residue (MSDW), A14 domain and three binding sites (Y193, D571 and E575), using the pET28a (His‐tagged) vector and Escherichia coli BL21 (DE3) prokaryotic expression system. By introducing point mutations (Y193A, D571A and E575A) into FvBgls3138‐580, mutant proteins (FvBgls3138‐580‐M) were generated (Figure 7A). The purified His‐tagged FvBgls3138‐580 and FvBgls3138‐580‐M are shown in Figure 7B using Coomassie briliant blue staining. β‐glucosidase hydrolyses pNPG to generate p‐nitrophenol (pNP), which develops a yellow colour under alkaline conditions (Zhang et al. 2017). Using this method, we measured the enzymatic activities of FvBgls3138‐580 and FvBgls3138‐580‐M, and assessed the effect of different concentrations of luvangetin. As shown in Figure 7C, in the absence of luvangetin treatment, FvBgls3138‐580 produced a deep yellow colour after reacting with pNPG, confirming its β‐glucosidase activity. However, the yellow intensity progressively diminished with increasing luvangetin concentrations. At 300 mg/L luvangetin treatment, FvBgls3138‐580 generated negligible pNP, similar to the negative control. In contrast, pNP production in FvBgls3138‐580‐M remained largely unaffected by luvangetin addition, consistently exhibiting bright yellow colouration. After 300 mg/L luvangetin treatment, the β‐glucosidase activity of FvBgls3138‐580 decreased from 20.3 to 5.3 U/mg, corresponding to 73.8% inhibition. In contrast, FvBgls3138‐580‐M activity declined only from 17.7 to 13.6 U/mg (22.9% inhibition), demonstrating significantly weaker suppression compared to the inhibition rate observed in FvBgls3138‐580 (Figure 7D). Statistically significant differences in inhibition rates by luvangetin were consistently observed between the FvBgls3138‐580 and its mutant protein at all three concentrations tested (Figure 7E). Isothermal titration calorimetry (ITC) data revealed that luvangetin bound spontaneously to FvBgls3138‐580, as indicated by the negative Gibbs free energy change (ΔG = −27.67 kJ/mol). The high affinity constant (Ka) and low dissociation constant (Kd) further confirmed the strong binding capacity of luvangetin. Furthermore, the negative values of enthalpy change (ΔH = −96.68 kJ/mol) and entropy change (ΔS = −231.6 KJ/mol) suggest that hydrogen bonding is the dominant interaction force between luvangetin and FvBgls3138‐580 (Figure 7F). In contrast, the binding affinity of luvangetin to FvBgls3138‐580‐M was significantly reduced (Figure 7G). The above results indicated that luvangetin directly binds to FvBgls3138‐580, thereby inhibiting its β‐glucosidase activity. When three critical binding sites were mutated to alanine (Y193A, D571A and E575A), the binding capacity of luvangetin to the FvBgls3138‐580‐M was significantly weakened, and its inhibitory effect on β‐glucosidase activity was also markedly reduced. We also performed sequence alignment on other β‐glucosidases of F. verticillioides GH family, revealing that multiple β‐glucosidases contain the three conserved residues (Y193A, D571A and E575A), which suggests that luvangetin may exert inhibitory effects on multiple β‐glucosidases (Table S2).

FIGURE 7.

FIGURE 7

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Effect of luvangetin on the activity of FvBgls3 and its mutant protein. (A) Illustration of the protein structures and mutation sites (Y193, D571 E575) of FvBgls3. Red regions: barrel domain; orange regions: PA14 domain. (B) The purified His‐tagged FvBgls3138‐580 and its mutant FvBgls3138‐580‐M visualised by Coomassie brilliant blue staining. (C) Study on the effect of luvangetin on the FvBgls3138‐580 and FvBgls3138‐580‐M activity using a pNPG assay. Luvangetin solutions in 10% ethanol (Et). (D) Statistical analysis of FvBgls3138‐580 and FvBgls3138‐580‐M activity after different concentrations (100, 200 and 300 mg/L) of luvangetin treatments. The symbols represent the following levels of significance: ****p < 0.0001, **p < 0.01, *p < 0.05, ns indicates no significant difference between the compared groups. Error bars indicate the standard deviations (SD) of three samples. Black arrows indicate the stepwise increased concentration of luvangetin. (E) Inhibition rate of different concentrations of luvangetin on FvBgls3138‐580 and FvBgls3138‐580‐M activity. (F) Isothermal titration calorimetry of luvangetin into FvBgls3138‐580. Fifty microlitres of luvangetin (20 μM) was titrated into 300 μL of FvBgls3138‐580 (0.5 mM). (G) Isothermal titration calorimetry of luvangetin into FvBgls3138‐580‐M. Fifty microlitres of luvangetin (20 μM) was titrated into 300 μL of FvBgls3138‐580‐M (0.5 mM).

2.6. The Functional Study of FvBgls3 and the Effects of Luvangetin Treatment

To further investigate the function of FvBgls3, gene knockout (FvBgls3‐KO 1#, FvBgls3‐KO 2#) and overexpression mutants (FvBgls3‐OE 1#, FvBgls3‐OE 2#) were generated using homologous recombination and the PCT74 overexpression vector, respectively. Compared with the wild‐type (WT) strain, no significant differences in growth rate were observed in the knockout mutants (Figure S8). However, the hyphae of the knockout mutant were noticeably sparser (Figure 8A). PCR using the primers H‐F/H‐R was used to detect the gene knockout mutants. In the WT strain, the full‐length sequence of FvBgls3 (2499 bp) was successfully amplified. In contrast, in the FvBgls3‐KO 1# and FvBgls3‐KO 2# mutants, the HygR gene (1026 bp) was amplified, confirming the successful knockout of FvBgls3 (Figure 8B). Overexpression mutants were verified by western blot analysis using an anti‐FLAG as the primary antibody. Consistent bands at the expected molecular weight were detected in both FvBgls3‐OE 1# and FvBgls3‐OE 2# strains (Figure 8C). Subsequent F. verticillioides inoculation assays (Figure 8D) showed that the FvBgls3 knockout mutants (FvBgls3‐KO 1#, FvBgls3‐KO 2#) exhibited significantly reduced lesion areas and relative biomass on maize leaves compared with the WT strain, whereas the FvBgls3 overexpression mutants (FvBgls3‐OE 1#, FvBgls3‐OE 2#) displayed a markedly enhanced pathogenicity (Figure 8E,F). After luvangetin treatment, infection of maize leaves by the WT and mutant strains of F. verticillioides was inhibited to varying degrees (Figure 8G,H). However, the inhibitory effect of luvangetin on the FvBgls3 overexpression mutant was significantly greater than that on the WT strain, whereas its inhibitory effect on the FvBgls3 knockout mutant was significantly weaker than that on the WT. The statistical analysis of relative biomass was consistent with these observations (Figure 8I,J). These results indicate that FvBgls3 plays a critical role in the pathogenicity of F. verticillioides and that FvBgls3 is an important target of luvangetin. Luvangetin suppresses the pathogenicity of F. verticillioides by directly binding to FvBgls3.

FIGURE 8.

FIGURE 8

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The functional study of FvBgls3 and the effects of luvangetin treatment. (A) Phenotype and pathogenicity of wild‐type (WT) and FvBgls3‐edited Fusarium verticillioides. Bar, 1 cm. Each experiment was replicated three times, independently. (B) PCR verification of FvBgl3 knockout mutants. (C) Western blot detection of FvBgl3 in overexpression mutants. (D) Inoculation of maize leaves with the WT and mutant strains of F. verticillioides at 5 days post‐inoculation (dpi). CK, control without luvagentin. Lesion area (E) and relative biomass (F) on maize leaves infected by WT and FvBgls3‐edited F. verticillioides. Each experiment was replicated three times, independently. Error bars indicate the standard deviations (SD) of three samples. The symbols represent the following levels of significance: ***p < 0.001, **p < 0.01, *p < 0.05, ns indicates no significant difference between the compared groups. After luvangetin treatment, lesion area (G) and relative biomass (H) of wild‐type and FvBgls3‐edited F. verticillioides on maize leaves. Inhibition rate of luvangetin treatment on the lesion area (I) and relative biomass (J) of wild‐type and FvBgls3‐edited F. verticillioides. on maize leaves. (K) Diagram of the molecular mechanism by which luvangetin inhibits the cell wall degradation capability of F. verticillioides.

In conclusion, F. verticillioides secretes various CWDEs (cellulases, hemicellulases and pectinases) to disrupt plant cell wall structures. Among these, cellulases comprise β‐glucosidase, endo‐β‐1,4‐glucanase and exo‐β‐1,4‐glucanase. β‐glucosidase directly cleaves β‐1,4‐glycosidic bonds, progressively hydrolysing the chains of cellulose molecules into cellobiose and ultimately yielding glucose. Luvangetin binds directly to residues Y193, D571 and E575 of β‐glucosidase (FvBgls3), inhibiting its cellulose‐degrading capacity and thereby suppressing the overall activity of F. verticillioides CWDEs (Figure 8K).

3. Discussion

3.1. Luvangetin's Inhibitory Effect on CWDEs

CWDEs, comprising cellulases, pectinases and hemicellulases, synergistically decompose polysaccharide components of plant cell walls. This process disrupts leaf tissue integrity and facilitates pathogen infection and disease progression. In previous studies, plant leaves affected by CWDEs exhibit tissue maceration and chlorosis–necrosis (Wei et al. 2022). Endoglucanases (EGs), cellobiohydrolases (CBHs) and polygalacturonases (PGs) degrade cellulose microfibril structures, compromising the cell wall scaffold (Kubicek et al. 2014). In our study, the symptoms observed on maize leaves infected by F. verticillioides were consistent with those described above (Figure 1B) and were accompanied by a significant reduction in cellulose and pectin contents, along with up‐regulation of genes related to cellulose and pectin biosynthesis (Figure 1E,F). These results indicated that F. verticillioides establishes infection by secreting CWDEs to overcome the plant's physical barrier. Following luvangetin treatment, however, infection by F. verticillioides was significantly suppressed, and the contents of cellulose and pectin, as well as the relative expression levels of their biosynthesis‐related genes, gradually recovered to levels comparable to those of healthy plants (Figure 1C–F). Notably, treatment with luvangetin alone did not alter changes in the expression of these genes (Figure S3). Therefore, we propose that luvangetin reduces the pathogenicity of F. verticillioides by inhibiting the activity of CWDEs. To further investigate the effects of luvangetin on these enzymes, crude enzyme extracts of F. verticillioides were prepared. The results demonstrated that luvangetin significantly inhibited cellulose conversion, xylan conversion, arabinose conversion, reducing sugar production (Figure 2A–D), as well as the degradation of straw, wheat bran and sugarcane bagasse by the crude enzyme preparations (Figure 3A–D). In the previous studies, supplementation of commercial Trichoderma reesei cellulases with the secretome of F. verticillioides significantly enhanced the enzymatic saccharification and increased glucose, xylose and arabinose release by 24%, 88% and 68%, respectively (Ravalason et al. 2012). This indicated that the F. verticillioides secretome contains enzymes potentially useful for lignocellulosic biomass degradation. Furthermore, this degradation capacity also assists the fungus in breaking down plant cell walls, facilitating its infection of the plant. However, the enzymes produced by fungi demonstrated significant advantages in straw processing, thereby enhancing the subsequent conversion of straw (Hu et al. 2023; Wang et al. 2022). F. verticillioides has been shown to possess considerable potential in promoting straw degradation (Gu et al. 2021; Ravalason et al. 2012). Although correctly utilising F. verticillioides to improve the utilisation efficiency of biomass resources, it is imperative to seek environmentally friendly methods to mitigate the risks posed by its CWDEs.

3.2. Molecular Mechanisms of Luvangetin‐Mediated Antifungal Activity

To elucidate the specific mechanisms by which luvangetin affects CWDEs in F. verticillioides, transcriptomic (Figure 4) and proteomic analyses (Figure 5) were performed on F. verticillioides with and without luvangetin treatment. Integrated omics analyses revealed that only the GH3 family β‐glucosidases (FvBgls3, FVEG_09248) showed significant down‐regulation at both transcriptional and translational levels (Figure 6A,B). We hypothesised that FvBgls3 plays a crucial role in F. verticillioides pathogenicity, and that luvangetin inhibits the enzymatic activity of CWDEs by suppressing the transcription and expression of FvBgls3. Subsequent molecular docking (Figure 6C), in vitro enzyme activity assays (Figure 7A–E) and ITC experiments (Figure 7F,G) all indicated that luvangetin binds directly to residues Y193, D571 and E575 of FvBgls3, inhibiting its cellulose‐degrading capacity and thereby suppressing the overall activity of F. verticillioides CWDEs. In the previous studies, we have proved that luvangetin suppresses FvFUM21‐Mediated FUM cluster's transcription by directly binding to the dsDNA helix of the promoter sequence (Chen et al. 2024). The structural uniqueness of coumarin, characterised by its furanocoumarin framework, enables specific binding affinities to critical molecular targets, making it a promising candidate for drug development (Citarella et al. 2024; Tang, Wang, et al. 2024). Sepehri et al. (2020) synthesised coumarin‐1,2,3‐triazole‐acetamide hybrids, with bioassay results demonstrating significant inhibition against β‐glucosidase. Molecular docking revealed that the coumarin moiety forms a hydrogen bond with residue G306, whereas the carbonyl oxygen of the acetamide group engages N241 via hydrogen bonding. Additional interactions were observed with catalytic site residues R312, E304, S308, P309 and V305 (Sepehri et al. 2020). Another synthesised coumarin derivative similarly exhibited potent glucosidase inhibition. Its carbonyl oxygen established hydrogen bonds with I235, K200 and S119, whereas other structural components interacted with key catalytic residues H201, Y151, A307 and G308 (Xu et al. 2018). This study demonstrated that three amino acid residues (Y193, D571 and E575) are highly conserved in Fusarium species, serving as critical sites for luvangetin‐mediated inhibition of β‐glucosidase activity.

3.3. Potential Implications for Ecofriendly Fungicide Development

To further investigate the function of FvBgls3, gene knockout and overexpression mutants were generated. These results indicate that FvBgls3 plays a critical role in the pathogenicity of F. verticillioides and that FvBgls3 is an important target of luvangetin. This study is the first to report that coumarin small molecules directly bind to and inhibit the activity of GH3 family enzymes, revealing the molecular mechanism by which luvangetin directly inhibits cell wall degradation capacity, providing new insights for ecofriendly control of Fusarium pathogens. The significance of this study lies in its potential to develop novel antifungal strategies based on natural compounds such as luvangetin. As the study demonstrates, luvangetin targets specific molecular mechanisms in F. verticillioides that are crucial for its pathogenicity, without relying on broad‐spectrum chemical fungicides, which often pose environmental and health risks. This specificity and targeted action present luvangetin as a promising candidate for the development of ecofriendly fungicides that can effectively control fungal pathogens while minimising ecological damage. Additionally, the findings highlight the importance of understanding the biochemical and molecular interactions between plant cell wall components and microbial pathogens.

4. Experimental Procedures

4.1. Materials

Luvangetin was isolated from the root of Z. avicennae in the previous period in our laboratory. F. verticillioides is deposited in the Laboratory of the Department of Plant Protection, Yangzhou University, and incubated at 25°C for subsequent experiments. Straw, wheat bran and bagasse were obtained from Huinong Technology Co. p‐Nitrophenyl‐β‐D‐galactopyranoside (pNPG) was purchased from Coolaber Technology Co. Escherichia coli DH5α and BL21 (DE3), and the expression vector pET28a (+) are preserved in the Plant Pathology Laboratory of Yangzhou University. Restriction enzymes EcoRI and HindIII were purchased from TaKaRa. Organic reagents were obtained from the Sinopharm Shanghai Chemical Reagent Company.

4.2. Measurements of Relative F. verticillioides Biomass

Total DNA of maize was extracted using the CTAB method. The biomass of F. verticillioides was assessed by qPCR, targeting the enrichment of F. verticillioides Tubulin with maize EF1a serving as the internal control. All the primer pairs used for qPCR are listed (Table S1).

4.3. Determination of Cellulose Content in Maize Leaves

Fresh leaves were defatted with ethanol‐benzene (2:1) for 24 h to remove pigments and lipids. Then 10 mL of 72% H2SO4 was added, and hydrolysis was performed at 30°C for 1 h. The hydrolysate was centrifuged to collect the supernatant. Anthrone reagent was added to the supernatant, followed by a 10‐min boiling water bath. After cooling, the absorbance was measured at 620 nm, and the cellulose content was calculated based on the glucose standard curve (Figure S4A). Each experiment was replicated three times, independently.

4.4. Determination of Pectin Content in Maize Leaves

Fresh leaves were boiled in 95% ethanol for 10 min, then ground and centrifuged to discard the supernatant. Subsequently, 0.5% ammonium oxalate solution was added to the residue, followed by extraction in an 85°C water bath for 1 h. After centrifugation, the supernatant was collected. The extract was mixed with concentrated sulphuric acid and heated in a boiling water bath for 10 min. Finally, 0.1% carbazole solution was added, and the mixture was incubated in the dark for 30 min before measuring the absorbance at 530 nm. The pectin content was calculated based on a galacturonic acid standard curve (Figure S4B). Each experiment was replicated three times, independently.

4.5. RT‐qPCR

The expression levels of target genes were detected via RT‐qPCR. TRIzol (Invitrogen) was used for total RNA extraction, and the first‐strand cDNA was obtained from 1 μg total RNA according to the manufacturer's protocol of Prime‐Script II First Strand cDNA synthesis kit (TaKaRa). Gene expression analyses were performed using a CFX96 Touch Real‐Time PCR Detection System (Bio‐Rad) based on the SsoFast EvaGreen Supermix (Bio‐Rad). Each experiment was replicated three times, independently. The actin genes of F. verticillioides and Zea mays were treated as internal controls. Cycle threshold (C t) values were calculated using the Bio‐Rad CFX Manager 3.0 software. All the primer pairs used for RT‐qPCR are listed (Table S1).

4.6. Determination of F. verticillioides Crude Enzyme Extract Activity

Fusarium verticillioides was inoculated into an enzyme‐production medium (CMC‐Na, 2.1 g; peptone 0.6 g; K2HPO4, 0.6 g; MgSO4, 0.09 g; MnSO4, 0.09 g; CaCI2, 0.09 g; Tween 80, 0.45 mL; ddH2O to 300 mL) and cultured at 28°C with agitation (150 rpm) for 3 days. The resulting fermentation broth was filtered through gauze to remove residual straw particles and mycelia. The filtrate was centrifuged at 8000 rpm for 15 min to obtain the supernatant as the crude enzyme extract. 0.5 mL of crude enzyme extract was mixed with 0.5 mL of carboxymethyl cellulose sodium (CMC‐Na) and reacted in a 50°C water bath for 30 min. The reaction was terminated by adding 1 mL of 3,5‐dinitrosalicylic acid, followed by boiling for 5 min (Karuppiah et al. 2022). After cooling, the mixture was centrifuged, and the supernatant absorbance was measured at 540 nm (A540). Cellulose conversion rate was calculated based on a glucose standard curve (Figure S4). For xylanase activity assay, xylan was used as the substrate, and xylan conversion rate was determined based on a xylose standard curve (Figure S4C) (Gao and Chen 2021). For arabinase activity assay, arabinan served as the substrate, and arabinan conversion rate was calculated based on an arabinose standard curve (Figure S4D). Total reducing sugar content was defined as the sum of generated glucose, xylose and arabinose. Each experiment was replicated three times, independently.

4.7. Enzymatic Hydrolysis of Straw, Wheat Bran and Bagasse

Straw substrates were milled and sieved to obtain particles measuring 1–2 mm. Enzymatic degradation assays were conducted by incubating straw particles with the crude enzyme extract in test tubes at 28°C with agitation (150 rpm) for 10 days, using uninoculated enzyme‐production medium as the negative control. Surface area and dry weight were subsequently analysed across treatment groups (Ma, Chen, et al. 2024). The enzymatic hydrolysis of wheat bran and bagasse followed identical procedures to those described above. Each experiment was replicated three times, independently.

4.8. SEM

The samples were fixed overnight in 10% glutaraldehyde, followed by dehydration using ethanol at concentrations of 70%, 80%, 90%, 95% and 100% sequentially. After thorough drying, the samples were coated with gold using sputter coater (MC 1000) and observed using a scanning eletron microscope (Gemini 300) (Min et al. 2024; Xiao et al. 2025).

4.9. Molecular Docking

The 3D model of the protein was constructed by homology modelling using the SWISS‐MODEL server (https://swissmodel.expasy.org/), based on its specified amino acid sequence. The mol file of luvangetin was obtained from the PubChem database (http://pubchem.ncbi.nlm.nih.gov/) (Sarwar et al. 2015). The interaction between luvangetin and protein was analysed using the CB‐Dock2 server (https://cadd.labshare.cn/cb‐dock2/php/index.php) (Liu et al. 2022), and the parameters used for docking were correlation type—shape only, FFT mode—3D, grid dimension—0.6, receptor range—180, ligand range—180, twist range—360 and distance range—40. The docked poses were visualised using PyMOL software (v. 3.1) (Seeliger and de Groot 2010).

4.10. Cloning of FvBgls3 Gene From F. verticillioides

Total RNAs were extracted from the mycelium of F. verticillioides using TRIzol according to the manufacturer's instructions (Invitrogen). First‐strand cDNA was synthesised according to the manufacturer's protocol using a Prime‐Script II First Strand cDNA synthesis Kit (TaKaRa). The possible transcripts of F. verticillioides Glycoside Hydrolase 3 family β‐glucosidase (FvBgls3) were amplified using primer pairs according to the FvBgls3 gene (XM_018898229, 2499 bp) in NCBI. Subsequently, using the FvBgls3 full‐length sequence as a template, FvBgls3 138‐580 (1329 bp) was amplified via PCR with specific primers. EcoRI and HindIII restriction sites were selected, and the fragment was constructed into the vector pET28a (+) using homologous recombination enzyme (TaKaRa), yielding the recombinant plasmid pET28a‐FvBgls3138‐580. The mutant plasmid pET28a‐FvBgls3138‐580‐M was constructed using pET28a‐FvBgls3138‐580 as the template via the overlap extension PCR method. All the primer pairs used and amplified products are listed in Tables S1 and S3.

4.11. Expression and Purification of the Proteins

The plasmid pET28a‐FvBgls3138‐580 and pET28a‐FvBgls3138‐580‐M were transformed into E. coli BL21 (DE3). The BL21 cells carrying the plasmid were cultured in 4 mL Luria Bertani (LB) liquid medium containing 50 μg/mL kanamycin at 37°C for 8 h. This bacterial suspension was then added to 500 mL of LB liquid medium containing 50 μg/mL kanamycin. After 5 h culture in an 18°C, 200 rpm incubator (Shanghai Minquan Instrument Co. Ltd.), 0.1 mM IPTG (Sangon Biotech) was added for induction, and the culture was continued under this condition for 8 h. The bacterial culture was centrifuged, and the pellet was resuspended in protein purification buffer (20 mM Tris–HCl, pH 10.3, 500 mM NaCl, 10% glycerol, 1 mM PMSF). An ultrasonic homogeniser (Thermo Fisher Scientific) was used to disrupt the bacterial cells. Ultrasonic breaking time was 3 s, interval 2 s, with working times 90, voltage 300 V, repeated three times. The resulting lysate was centrifuged to obtain the clarified supernatant. The supernatants were then incubated with Ni‐NTA Sefinose resin (Sangon Biotech) in a glass column, which was subsequently eluted with a stepwise increase in imidazole concentration (‘Prokaryotic Expression of Coat Protein Gene of Grapevine Berry Inner Necrosis Virus and Preparation of Its Polyclonal Antibody’ n.d.). The eluted recombinant His‐FvBgls3138‐580 and His‐FvBgls3138‐580‐M were concentrated using an Ultra‐15 filter unit (Millipore).

4.12. Determination of FvBgls3138 ‐580 and FvBgls3138 ‐580‐M β‐Glucosidase Activity

A pNPG solution (5 mM) was prepared in sodium acetate buffer (0.1 M, pH 5.0). The purified F. verticillioides β‐glucosidases and its mutant (FvBgls3138‐580 and FvBgls3138‐580‐M) were reacted with the pNPG solution at 37°C for 10 min, followed by adding 140 μL of sodium carbonate solution (1 M) to terminate the reaction. Each experiment was replicated three times, independently. One unit (U) of β‐glucosidase activity was defined as the amount of enzyme required to hydrolyse pNPG and release 1 μmol of p‐nitrophenol (pNP) per minute. The absorbance of the reaction mixture was measured at 405 nm, and the enzyme activity was calculated based on a pNP standard curve (Figure S4E).

4.13. ITC

Nano ITC (TA Instruments) was used to analyse the binding affinity of luvangetin with protein (FvBgls3138‐580 and FvBgls3138‐580‐M). Fifty microlitres of luvangetin (20 μM) was titrated into 300 μL of protein (0.5 mM) with an injection volume of 2 μL per step, using 10% ethanol as the blank control. The experiment was conducted at 25°C, with a titration interval of 120 s and a stirring speed of 350 rpm (Jiang et al. 2024; Liu, Du, et al. 2025). The test data were fitted using the independent model, yielding the corresponding dissociation constant Kd (the reciprocal of the binding constant Ka), stoichiometric ratio n, enthalpy change ΔH, and entropy change ΔS. The ΔG was calculated using the following equation:

ΔG=ΔH−TΔS

4.14. Gene Knockout and Overexpression of FvBgls3

Using homologous recombination technology, the FvBgls3 gene was knocked out. The upstream and downstream 600 bp homologous fragments of the target gene (FVEG_09248) were amplified and ligated to both ends of the resistance selection marker gene hygromycin phosphotransferase gene (hph), as shown in the schematic diagram (Figure S7). Protoplast transformation was performed using the polyethylene glycol (PEG) method, followed by hygromycin selection to obtain positive clones. Specific primers (H‐F/H‐R) were used to verify the gene knockout mutants. For overexpression of the target gene, the PCT74‐FvBgls3 vector was constructed. The protoplast transformation and selection methods were consistent with those described above. FLAG tag was used to detect the overexpression mutants (Zhuang et al. 2025; Zhang, Zhuang, et al. 2021). All the primer pairs used are listed in Table S1.

4.15. Statistical Analysis

SPSS software v. 17.0 (SPSS) was used for statistical analyses and two statistical methods were used in this study. In Tukey's test, different letters above each column indicate significant differences (p < 0.05) between the compared categories. For Student's t‐test, the symbols represent the following levels of significance: ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05, ns indicates no significant difference between the compared groups.

ImageJ software (http://imagej.nih.gov/ij/) was used for quantitative analysis of lesion area and surface area. BioRender Templates (https://app.biorender.com/) were used to draw pattern diagram.

Author Contributions

Kun Zhang: data curation, supervision, funding acquisition, writing – original draft, writing – review and editing, validation, conceptualization. Duxuan Liu: data curation, supervision, writing – original draft, writing – review and editing. Jing Hua: data curation, supervision, writing – original draft. Haoyu Chen: writing – original draft, writing – review and editing. Mingjie Wu: writing – original draft, writing – review and editing. Zhiqing Mao: data curation, supervision. Zhen Yang: validation, conceptualization. Xiubin Xu: validation. Yanhong Hua: validation. Chenwei Feng: writing – original draft, writing – review and editing. Jiahuan Chen: data curation, supervision, funding acquisition, writing – original draft, writing – review and editing, validation, conceptualization.

Funding

This study was supported by the National Natural Science Foundation of China (81903764 and 32372486), the Excellent Youth Fund of Jiangsu Natural Science Foundation (BK20220116), the Project of Science and Technology Development Plan for Traditional Chinese Medicine of Jiangsu Province (YB201993), the Postgraduate Research and Practice Innovation Program of the Jiangsu Province (SJCX24_2279 & SJCX23_2046), the Agricultural Science and Technology Independent Innovation Fund of Jiangsu Province (CX[24]3012), the ‘National Foreign Experts Project’ of the Ministry of Human Resources and Social Security (H20240527), the Special Scientific Research Fund of Yangzhou Health Commission (2023‐4‐08), the Yangzhou Pharmaceutical Association‐Chia Tai Tianqing Hospital Pharmacy Scientific Research Fund (YQ202205), the Open Funds of the State Key Laboratory of Plant Environmental Resilience (SKLPERKF2602) and the Young and Middle‐aged Academic Leaders of the ‘Qinglan Project’ of Yangzhou University.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

Figure S1: mpp70215‐sup‐0001‐FigureS1.docx.

MPP-27-e70215-s004.docx (323.6KB, docx)

Figure S2: mpp70215‐sup‐0002‐FigureS2.docx.

MPP-27-e70215-s009.docx (37.6KB, docx)

Figure S3: mpp70215‐sup‐0003‐FigureS3.docx.

MPP-27-e70215-s001.docx (112.4KB, docx)

Figure S4: mpp70215‐sup‐0004‐FigureS4.docx.

MPP-27-e70215-s008.docx (91.9KB, docx)

Figure S5: mpp70215‐sup‐0005‐FigureS5.docx.

MPP-27-e70215-s003.docx (484.1KB, docx)

Figure S6: mpp70215‐sup‐0006‐FigureS6.docx.

MPP-27-e70215-s010.docx (1.7MB, docx)

Figure S7: mpp70215‐sup‐0007‐FigureS7.docx.

MPP-27-e70215-s006.docx (51.1KB, docx)

Figure S8: mpp70215‐sup‐0008‐FigureS8.docx.

MPP-27-e70215-s011.docx (50.4KB, docx)

Table S1: mpp70215‐sup‐0009‐TableS1.docx.

MPP-27-e70215-s007.docx (20.4KB, docx)

Table S2: mpp70215‐sup‐0010‐TableS2.docx.

MPP-27-e70215-s002.docx (16.6KB, docx)

Table S3: mpp70215‐sup‐0011‐TableS3.docx.

MPP-27-e70215-s005.docx (23.7KB, docx)

Acknowledgements

We are grateful to each member in the plant pathology laboratory of Yangzhou University for their constructive suggestions to the MS.

Liu, D. , Hua J., Chen H., et al. 2026. “A Coumarin Compound Derived From Zanthoxylum avicennae Reduces the Pathogenicity of Fusarium verticillioides by Directly Binding to and Inhibiting Glycoside Hydrolase 3 Activity.” Molecular Plant Pathology 27, no. 2: e70215. 10.1111/mpp.70215.

Contributor Information

Kun Zhang, Email: zk@yzu.edu.cn.

Jiahuan Chen, Email: 092015@yzu.edu.cn.

Data Availability Statement

Supporting Information is available from the Online Library or from the author. The raw transcriptomic sequencing data were uploaded and deposited in NCBI (Accession No. GSE247362). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD046770 (Chen et al. 2022; Ma et al. 2019).

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Associated Data

This section collects any data citations, data availability statements, or supplementary materials included in this article.

Supplementary Materials

Figure S1: mpp70215‐sup‐0001‐FigureS1.docx.

MPP-27-e70215-s004.docx (323.6KB, docx)

Figure S2: mpp70215‐sup‐0002‐FigureS2.docx.

MPP-27-e70215-s009.docx (37.6KB, docx)

Figure S3: mpp70215‐sup‐0003‐FigureS3.docx.

MPP-27-e70215-s001.docx (112.4KB, docx)

Figure S4: mpp70215‐sup‐0004‐FigureS4.docx.

MPP-27-e70215-s008.docx (91.9KB, docx)

Figure S5: mpp70215‐sup‐0005‐FigureS5.docx.

MPP-27-e70215-s003.docx (484.1KB, docx)

Figure S6: mpp70215‐sup‐0006‐FigureS6.docx.

MPP-27-e70215-s010.docx (1.7MB, docx)

Figure S7: mpp70215‐sup‐0007‐FigureS7.docx.

MPP-27-e70215-s006.docx (51.1KB, docx)

Figure S8: mpp70215‐sup‐0008‐FigureS8.docx.

MPP-27-e70215-s011.docx (50.4KB, docx)

Table S1: mpp70215‐sup‐0009‐TableS1.docx.

MPP-27-e70215-s007.docx (20.4KB, docx)

Table S2: mpp70215‐sup‐0010‐TableS2.docx.

MPP-27-e70215-s002.docx (16.6KB, docx)

Table S3: mpp70215‐sup‐0011‐TableS3.docx.

MPP-27-e70215-s005.docx (23.7KB, docx)

Data Availability Statement

Supporting Information is available from the Online Library or from the author. The raw transcriptomic sequencing data were uploaded and deposited in NCBI (Accession No. GSE247362). The mass spectrometry proteomics data have been deposited to the ProteomeXchange Consortium (http://proteomecentral.proteomexchange.org) via the iProX partner repository with the dataset identifier PXD046770 (Chen et al. 2022; Ma et al. 2019).

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