Tangeretin Suppresses Fumonisin Production by Modulating an NmrA- and HSCARG-like Protein in Fusarium verticillioides

Abstract

Fumonisins are polyketide-derived mycotoxins posing significant health threats to humans and animals. Among these, fumonisin B₁ (FB₁) is the most prevalent mycotoxin, primarily produced by Fusarium verticillioides, especially in maize and its derived products. Tangeretin, a polymethoxyflavonoid, has been identified as having potential medicinal properties, particularly as an antioxidant. To evaluate the antifungal and anti-mycotoxigenic properties of tangeretin and to elucidate the mechanisms underlying its inhibitory effects, assessments of fungal growth, FB₁ production, conidial germination, and cellulase activity, antioxidant capacity and enzyme activities, transcriptomic analysis and gene deletion experiments were conducted. Consequently, tangeretin significantly curtailed fungal growth and FB₁ production and provided protection against pathogenic infection on corn. It affected genes associated with fungal growth, conidial development, and antioxidant response. Furthermore, tangeretin interfered with the supply of biosynthetic substrate necessary for fumonisin production, particularly impacting pathways involved in alanine metabolism, pyruvate metabolism, fatty acid degradation, and sphingolipid metabolism. Notably, tangeretin downregulated four biosynthetic genes (Fum2, Fum3, Fum10 and Fum11) that are involved in the final steps of fumonisin formation. It likely disrupted the MAPK signaling pathway and targeted a putative NmrA- and HSCARG-like protein Fv_Tan1, which was identified as having positive effects on fungal growth and mycotoxin biosynthesis. This study presents a promising approach for controlling fumonisin contamination in agricultural settings.

1. Introduction

Fumonisins are linear polyketide-derived mycotoxins primarily produced by various Fusarium species [1] and certain Aspergillus species [2]. Among these, F. verticillioides is a major contributor [3] and a global pathogen responsible for Fusarium ear rot in maize [4]. Fumonisin contamination is frequently observed in agricultural products and poses serious health hazards, including neurotoxicity, immunotoxicity, and reproductive toxicity. The International Agency for Research on Cancer (IARC) classifies fumonisins as group 2B human carcinogens [5]. Consequently, maximum allowable levels have been established in many countries and regions [6]. Within this category, fumonisin B₁ (FB₁) is the most prevalent and potent, particularly in corn and corn-derived products, significantly escalating health risks [7]. Therefore, developing innovative strategies to effectively control fumonisin contamination is of critical importance.

Although authorized fungicides effectively control plant pathogens, their overuse may lead to chemical residues that threaten human health and promote resistance among phytopathogens. As a result, natural plant extracts have garnered increasing attention as potential substitutes or supplements to synthetic fungicides [8]. These extracts contain bioactive components with medicinal properties, such as anti-inflammatory, antioxidant, and even anti-tumor activities [9]. Many medicinal ingredients, notably phenolic and flavonoid compounds, exhibit antifungal activities [10]. Interestingly, some of these active compounds demonstrate fungicidal activity without anti-mycotoxigenic effects, while others offer both antifungal and anti-mycotoxigenic benefits. By identifying inherent and unique molecular differences through comparative analysis, potential target genes closely associated with mycotoxin regulation can be discovered. This approach is pivotal for screening diverse plant extract ingredients, enabling effective control of mycotoxin contamination.

Phenolic and flavonoid compounds in plant extracts typically induce intracellular changes, affecting oxidative stress balance. Previous studies have shown that various regulators involved in stress responses also play roles in fumonisin biosynthesis. In addition to the specific regulator Fum21 within the fumonisins’ biosynthetic gene cluster [11,12], various external regulators—such as transcription factors, epigenetic modifiers, Mitogen-activated protein kinase (MAPK) cascades, and peroxisomal proteins—play significant roles in responding to external materials and environmental changes while modulating fumonisin biosynthesis [11]. Notably, environmental response transcriptional factors, such as velvet complex [13] and AtfA [14], positively regulate fumonisin biosynthesis. Peroxisomal proteins, including FvPex5 [15] and FvPex8 [16], are involved in maintaining cellular oxidative homeostasis and are implicated in FB₁ production. Genes within the MAPK signaling pathway are similarly involved in regulating responses to abiotic stress and fumonisin formation. Specifically, FvBck1, a component of the cell wall integrity MAP kinase pathway, is responsible for oxidative stress response in F. verticillioides [17], while the HOG-type MAP kinase Fphog1 is involved in nitrogen starvation stress response in F. proliferatum [18]. These proteins, through their roles in stress response and fumonisin production, potentially mediate the effects of various active compounds. This mediation may ultimately lead to the downregulation or inhibition of mycotoxin biosynthesis. To our knowledge, no relevant potential targets have been identified for the control of fumonisin in F. verticillioides using plant extracts. However, pyrrocidine, derived from maize kernel endophyte Sarocladium zeae induces a genetic repressor, FvZBD1, which effectively shuts off fumonisin biosynthesis [19]. In other studies concerning mycotoxin control, perillaldehyde has been found to inhibit A. flavus growth and aflatoxin production by disrupting cAMP-PKA signaling regulation [20], although this pathway does not significantly influence FB₁ biosynthesis [21].

This study identifies tangeretin, a flavonoid renowned for its promising pharmacological effects, including anti-inflammatory, neuroprotective, and anti-cancer properties [22], as a potent dual-functional agent capable of suppressing fungal growth and fumonisin biosynthesis. Tangeretin’s further investigation on controlling fumonisin contamination was also motivated by its medical relevance to neuroprotective potential, which aligns with the urgent need to mitigate fumonisin-induced neurotoxicity in contaminated crops and food products. Further study demonstrates that its inhibition on fumonisin production underscores its role in disrupting redox regulation, restricting biosynthetic substrate supply and directly impacting fumonisin biosynthetic genes. Notably, this research is the first to characterize an NmrA- and HSCARG-like protein as a potential target for tangeretin via the MAPK signaling pathway, exploring its effects on fungal growth and fumonisin production. These inhibitory activities of tangeretin could facilitate the development of alternative agents for controlling fumonisin levels. Furthermore, understanding the mechanisms of tangeretin’s action provides insights into how active substances can counteract pathogenic fungi and suppress mycotoxin biosynthesis.

2. Materials and Methods

2.1. Screening of Medicinal Compounds for Inhibition Against F. verticillioides

Five medicinal components were tested to determine their effectiveness in suppressing fungal growth against F. verticillioides, including tangeretin, resveratrol, curcumin, nobiletin and hesperidin (Yuanye, Shanghai, China). These compounds were separately dissolved into dimethyl sulfoxide (DMSO) and the corresponding concentration was adjusted to 1 mM in potato dextrose agar (PDA) medium. For fungal spores’ production, fresh mycelial plugs were cultured into liquid carboxymethyl cellulose (CMC) medium and collected by centrifuge, which was referred by the method of Tang et al. [23]. The concentration of these spores was standardized to 1 × 10⁶ spores/mL using a hemocytometer. Subsequently, five microliters of this spore suspension were inoculated on the PDA plates and then incubated at 25 °C for 7 days to assess fungal growth and inhibition.

2.2. Antifungal and Antimycotoxigenic Properties of Tangeretin

2.2.1. In Vitro Inhibitory Effects of Tangeretin

The inhibitory effects of tangeretin and resveratrol were investigated at a concentration of 1 mM in liquid PDB medium. A spore suspension (1 × 10⁶ spores/mL, v/v = 1/100) was inoculated into liquid medium with or without chemical treatment and cultured at 25 °C for 7 days with shaking at 180 rpm. For FB₁ extraction, an equal volume of acetonitrile containing 0.1% formic acid was added to the culture, followed by overnight mixing to ensure complete extraction. After centrifugation at 12,000 rpm for 10 min, 1 mL of the supernatant was filtered through a 0.2 μm polytetrafluoroethylene (PTFE) syringe filter (Pall, Port Washington, NY, USA) for subsequent analysis. FB₁ quantification was performed using high-performance liquid chromatography with fluorescence detection (HPLC-FLD, Agilent, Santa Clara, CA, USA) after precolumn derivatization. Samples and standards underwent derivatization with o-phthalaldehyde and were injected into HPLC-FLD with an excitation wavelength of 335 nm and an emission wavelength of 440 nm [24]. Chromatographic separation was achieved using a Zorbax RRHD Plus C18 column (3.0 × 50 mm, 1.8 μm; Agilent, Santa Clara, CA, USA) by a gradient elution method, which began with 50% each of solvent A (10 mM KH₂PO₄, pH 2.3) and B (methanol:acetonitrile = 1:1, v/v), shifted to 30% A and 70% B from 3 to 6 min, and returned to the initial conditions from 6.1 to 9 min.

2.2.2. Protective Efficacy of Tangeretin on Corn

To assess the protective efficacy of tangeretin in controlling F. verticillioides and its production of fumonisin, fresh corn was utilized. Initially, the fresh corn was sterilized using sodium hypochlorite, followed by thorough drying. To mitigate any potential damage from DMSO, tangeretin was dissolved in edible oil instead. This solution was evenly applied to the surface of the corn to ensure complete absorption for 24 h. Subsequently, fungal spores were inoculated onto the corn surface. The development of pathogenic disease was monitored and documented. Fumonisin production was ultimately extracted and quantified to evaluate the potential practical value of using tangeretin.

2.2.3. Effect of Tangeretin on Conidial Germination of F. verticillioides

The impact of tangeretin on the germination rate of F. verticillioides conidia was evaluated using a plate assay. A 200 μL aliquot of spore suspension (1 × 10⁶; spores/mL) was uniformly spread on PDA plates containing 1 mM tangeretin, with untreated PDA plates serving as controls. All plates were incubated overnight at 25 °C. Germination rates were calculated from the microscopic observation. Statistical comparisons were made between tangeretin-treated and control groups to assess germination inhibition.

2.3. Biochemical Analysis

To investigate the impact of tangeretin on fungal cellular architecture and oxidative stress modulation, mycelia were harvested from cultures grown with and without tangeretin treatment. The structure-related changes were assessed by measuring the enzymatic activity of cellulase as an indicator of structural alterations using Cellulase Activity Assay Kit (Solarbio, Beijing, China). The total antioxidant capacity of the mycelia was evaluated by ABTS (2,2′-azino-bis(3-ethylbenzthiazoline-6-sulfonic acid)) method provided by Total Antioxidant Capacity Assay Kit (Beyotime, Shanghai, China). Reactive oxygen species (ROS) management within the fungal cells was further analysed by measuring hydrogen peroxide (H₂O₂) levels and the activities of catalase and total superoxide dismutase (SOD) using respective assay kits (Beyotime, Shanghai, China) following the provided manual instructions. For these assays, the mycelia were grounded and homogenized with lysis buffer in a glass homogenizer using liquid nitrogen. The homogenate was then centrifuged to obtain the supernatant. Protein content in the supernatant was determined by bicinchonininc acid (BCA) method using the Enhanced BCA Protein Assay Kit (Beyotime, Shanghai, China).

2.4. Differential Transcriptomic Analyses

To uncover the potential mechanisms underlying tangeretin’s antifungal and antifumonisin effects, mycelia treated with and without tangeretin were subjected to differential transcriptomic analyses. These analyses were carried out by Hangzhou Lianchuan Biotechnology Co., Ltd. (Hangzhou, China). To construct RNA libraries, total RNA was extracted using TRIzol reagent (Thermo Fisher Scientific, Carlsbad, CA, USA) from which high-quality RNA (RIN number > 7.0) was selected for cDNA library construction. The final cDNA libraries were achieved at an average insert size of 300 ± 50 bp. Sequencing was performed on an Illumina Novaseq™ 6000 (Illumina, San Diego, CA, USA) following the recommended protocol. Raw reads were obtained from the sequencing machines. To obtain high quality clean reads, raw reads were further filtered by Cutadapt v1.9 [25]. Subsequently, the quality of these reads was verified using FastQC v0.11.9, which assessed parameters, including Q20, Q30 and the GC-content, of the cleaned data. The sequence data have been deposited in the Genome Sequence Archive [26] in the National Genomics Data Center [27], China National Center for Bioinformation / Beijing Institute of Genomics, and the Chinese Academy of Sciences (GSA: CRA017074) that are publicly accessible at https://ngdc.cncb.ac.cn/gsa accessed on 17 June 2024.

For data analysis, reads from all samples were aligned to the F. verticillioides reference genome (https://www.ncbi.nlm.nih.gov/datasets/genome/GCF_000149555.1, accessed on 6 July 2011) using HISAT2 v2.0.4 package [28], which includes initial quality-based read trimming and mapping to the reference genome. Mapped reads were assembled using StringTie v1.3.4d with default parameters [29]. All sample transcriptomes were merged to reconstruct a comprehensive transcriptome using GffCompare v0.9.8 [30]. Expression levels were quantified using StringTie v1.3.4d [29] and Ballgown v3.2 (https://bioconductor.org/packages/release/bioc/html/ballgown.html accessed on 9 March 2025) [31], calculating FPKM (fragment per kilobase of transcript per million mapped reads) values for each transcript. Differential expression analysis was performed by DESeq2 (https://bioconductor.org/packages/release/bioc/html/DESeq2.html accessed on 30 March 2025) [32], identifying genes with a false discovery rate (FDR) below 0.05 and an absolute fold change ≥ 2 as differentially expressed genes (DEGs). These DEGs were then subjected to enrichment analysis of Gene Ontology (GO) functions and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathways to determine their biological impact.

2.5. Function Analysis of Potential Target Genes

Pertinent genes identified from the crossover analysis of the differentially expressed genes across treatment groups were considered potential targets of tangeretin. They encode an NmrA- and HSCARG-like protein (FVEG_03055; Fv_Tan1) and a multidrug resistance (MDR) transporter (FVEG_04238; Fv_Tan2). Bioinformatical analyses were separately performed on them, including similarity, conserved domain, and phylogenetic relationship with other homologous proteins from mycotoxin-producing fungi by MEGA11 [33]. The NmrA- and HSCARG-like protein carried out its interaction with intracellular proteins by STRING [34]. The transmembrane helices of the MDR transporter were predicted by TMHMM v2.0 [35].

To investigate their roles in fungal growth and fumonisin production, they were separately knockout by a homologous recombination strategy. The flanking sequences of both genes were amplified separately using the primer pairs mentioned as up-F/R or down-F/R (Table S1). In parallel, hygromycin resistance gene (HYR) was also amplified from a plasmid template. Following amplification, the flanking sequences and HYR were assembled through nested PCR using the primer pair knock-F/R. The resultant PCR products were purified and used to transform into the protoplast of F. verticillioides. Transformants were identified using PCR with two primer sets: up-F/HYR-test-R and in-F/R. The first set was designed to amplify the upstream gene sequence along with a partial HYR sequence, confirming gene replacement. The second primer set aimed to amplify part of the internal gene sequence to verify the absence of any residual gene sequence in the chromosomal DNA. Furthermore, transformants were subjected to quantitative PCR (qPCR) analysis to verify the presence of a single, correct homologous recombination event involving the target gene, as referenced in Zhang et al. [36]. Subsequently, the wild-type and mutant strains were cultured in the presence of tangeretin to assess the impact of gene deletion on both growth and mycotoxin production.

2.6. Statistical Analysis

All experiments were conducted in triplicate. Data were calculated and expressed as mean ± standard error (S.E.). Significant differences between groups were determined using one-way Analysis of Variance (ANOVA) followed by a post-hoc Tukey test (p < 0.05) by IBM SPSS Statistics 26.0 (Armonk, NY, USA). Graphical representations of the data were generated using GraphPad Prism 8.0 (La Jolla, CA, USA).

3. Results

3.1. Suppression of Fungal Growth and Fumonisin Production by Tangeretin

In an evaluation of antifungal properties, five medicinal substances were selected for their effects against F. verticillioides and F. graminearum. Among these compounds, tangeretin demonstrated inhibitory effects on both Fusarium species (Figure 1A and Figure S1). Specifically, tangeretin’s inhibition of F. graminearum was notably weak, showing a significant inhibition rate of 22.3% (Figure S1). In contrast, F. verticillioides showed greater sensitivity to tangeretin with a markedly higher inhibition rate of 46.7%. In addition, resveratrol exhibited the highest inhibition against the hyphal extension of F. verticillioides (Figure S1). Based on these observations, both tangeretin and resveratrol could be considered for controlling F. verticillioides due to their inhibitory effects. Further analysis in liquid PDB medium revealed that 1 mM tangeretin significantly suppressed 36.4% of FB₁ production. In contrast, resveratrol did not demonstrate a noticeable inhibition of mycotoxin biosynthesis under similar conditions (Figure 1B).

The efficacy of tangeretin was further evaluated on fresh corn to control the growth of F. verticillioides and its production of fumonisin. Observations from the second day showed emerging fungal growth that progressively enveloped the entire corn surface by the seventh day. Contrastingly, tangeretin application effectively inhibited the proliferation of hyphae, significantly protecting the corn from fungal colonization (Figure 1C). In the presence of tangeretin, hyphal growth was notably slower and shorter compared to control samples without tangeretin intervention. Conclusively, mycotoxin analysis revealed a substantial 69.8% reduction in FB₁ production (Figure 1D), underscoring tangeretin’s potential as a protective agent against mycotoxin output. This demonstrates its practical utility in agricultural applications for the control of fumonisin production.

3.2. Interference of Tangeretin in Hyphal Extension, Conidial Development and Redox Homeostasis

A comprehensive investigation was conducted to explore the influence of tangeretin on the biological activities of F. verticillioides, with a specific focus on spore germination and oxidative stress responses. Exposure to 1 mM tangeretin significantly reduced conidial germination rates by 19.5% compared to the control (p = 0.0008, Figure 2A). This reduction in germination rates may impede mycelial extension of F. verticillioides, thereby potentially limiting fungal proliferation. Further analyses of enzymatic activity revealed significant structural changes, notably a reduction in cellulase activity (Figure 2B). Given that mycelial elongation in fungi largely relies on the hydrolysis and reformation of the cell wall facilitated by cellulase, decreased cellulase activity could directly contribute to the observed suppression of fungal hyphal extension.

Given tangeretin’s potent antioxidant activity documented in previous studies [22,37], the regulation of reactive oxygen species (ROS) was scrutinized. Notably, the total antioxidant capacity of F. verticillioides decreased by 20.2% when exposed to tangeretin (Figure 2C). Additionally, the levels of H₂O₂ reduced by 21.7% in response to tangeretin exposure (Figure 2D). The activities of the antioxidant enzymes were also evaluated including catalase and SOD. Remarkably, catalase activity significantly increased under tangeretin stress, which was possibly related with the decrease of the observed H₂O₂ (Figure 2E), while total SOD activity was notably reduced by 56.08% (Figure 2F). These findings suggest that tangeretin influences the enzymatic activities involved in ROS detoxification within F. verticillioides.

3.3. Transcriptomic Analysis of F. verticillioides Reveals Tangeretin Impacts

To explore the influence of tangeretin on F. verticillioides and its fumonisin production, a differential transcriptomic analysis was performed under tangeretin-treated and untreated conditions (Table S2). In total, 1165 differentially expressed genes (DEGs) were identified, including 297 upregulated and 868 downregulated DEGs, upon exposure to tangeretin (Figure S2).

The statistics of GO and KEGG pathway enrichment were performed to systematically delineate the inhibitory effects of tangeretin on fungal growth and mycotoxin production. Five DEGs enriched into hyphal growth (GO:0030448) were completely downregulated (Figure 3 and Figure S3), correlating with observed reductions in hyphal extension. Of the enriched genes in conidiophore development (GO:0070787), six DEGs were lower transcribed under tangeretin-treated condition, which aligned with the inhibition of conidial germination. In the enrichment of pathogenesis analysis, the transcripts of six DEGs were influenced by tangeretin, including 1 upregulated and 5 downregulated genes. Notably, genes related to stress response (GO:0006950) and cellular response to drugs (GO:0035690) were entirely downregulated when exposed to tangeretin. Moreover, two DEGs enriched in the response to oxidative stress (GO:0006979) were also lower expressed.

The KEGG pathway enrichment analysis demonstrated significant enrichment in pathways related to multi-drug resistance, particularly those coding for ABC transporters, highlighting a stress response that potentially initiates resistance mechanisms against abiotic stress. Relevant metabolic pathways, including beta-alanine metabolism (ko00410) and alanine, aspartate and glutamate metabolism (ko00250) enriched, were crucial for fumonisin biosynthesis (Figure 4). Alanine is one of the substrates from the chemical structure, which is condensed with a linear 18-carbon-long polyketide to form the fumonisin backbone [38]. In addition, pathways such as pyruvate metabolism (ko00620) and fatty acid degradation (ko00071) were enriched, which were integral to the synthesis of key fumonisin precursors and components. The metabolism of fumonisin analogue sphingolipid (ko00600) was also enriched in the KEGG pathway, suggesting that tangeretin could possibly affect sphingolipid metabolism, ultimately reducing fumonisin production.

3.4. Effects of Tangeretin on Fumonisin Biosynthetic Gene Cluster

Exposure to tangeretin significantly alters the expressions of four genes within this cluster—Fum2, Fum3, Fum10 and Fum11—resulting in reduced activity compared to the stress-free control (Figure 5A). The relative expression levels of three chosen cluster genes were further confirmed by RT-qPCR (Figure 5B). Fum2 and Fum3 encode a cytochrome P450 mono-oxygenase and a dioxygenase catalyzing the hydroxylation at C-10 and C-5 [39,40]. An acyl-CoA synthetase/acyl-protein synthetase Fum10 is responsible for the formation of tricarboxylic CoA to supply two side chains at C-14 and C-15 [11], which requires the transport of tricarboxylic acid by a tricarboxylate transporter protein encoded by Fum11 [41]. Overall, this demonstrates that tangeretin could directly influence the biosynthetic gene cluster and crucial steps in the biosynthesis of fumonisin, particularly the formation of tricarboxylic ester and two terminal steps of fumonisin formation.

3.5. Identification of Tangeretin’s Potential Target Genes in F. verticillioides

To identify specific genes targeted by tangeretin in F. verticillioides, the differences of DEGs were compared and analyzed across various treatment groups. Moreover, the study incorporated resveratrol treatment as a comparative measure to delineate individual gene functional enrichments and to identify unique genes affected by tangeretin (Figure 6A). KEGG pathway enrichment analysis revealed that MAPK signaling pathway (fvr04011; Figure S4) was significantly enriched in the tangeretin treatment group compared to the stress-free group, while no obvious enrichment was observed in resveratrol treatment. This implies that tangeretin-induced stress, which correlates with a decrease in fumonisin production, may be linked to the MAPK signaling pathway. Furthermore, through a comprehensive analysis of the functions of the DEGs, two key genes, FVEG_03055 and FVEG_04238, were ultimately identified as playing key roles in the antifungal and anti-mycotoxigenic activity of tangeretin (Figure 6B). These genes encode an NmrA- and HSCARG-like protein (designated as Fv_Tan1) and a multidrug resistance transporter (designated as Fv_Tan2), respectively. The relative transcriptional levels of these genes were further validated using RT-qPCR (Figure 6C).

Phylogenetic analysis showed that the Fv_Tan1 protein and its homologs in other Fusarium species closely relate to proteins found in A. niger and A. flavus but not those in Penicillium expansum (Figure S5). In contrast, Fv_Tan2 displayed high similarity with PEX2_100930 from P. expansum MD-8. Fv_Tan1 was predicted with other potential interaction proteins, whose expression showed no significant variation in F. verticillioides under tangeretin stress (Figure S6A; Table S3). Despite Fv_Tan1’s conservation within the NmrA-like regulator superfamily, it does not appear to be significantly involved with nitrogen metabolism as implied by protein–protein interaction analyses involving the nitrogen metabolism regulator AreA (FVEG_02033). Accordingly, AreA is more likely to interact with another NmrA-like regulator FVEG_07953 from the analysis of AreA interaction proteins (Figure S6B). Nevertheless, the expression of AreA and FVEG_07953 exhibited steadily in F. verticillioides with or without tangeretin treatment (Table S2). Furthermore, the presence of a conserved HSCARG domain in Fv_Tan1 suggests a role as an NADPH sensor, indicating a distinct mechanism of action from typical NmrA-like regulators with AreA. Moreover, the transporter Fv_Tan2 was considered to have 12 transmembrane helices potentially exerting xenobiotic resistance (Figure S7).

To confirm their roles in fungal growth and fumonisin biosynthesis in F. verticillioides, both genes were individually disrupted via homologous recombination (Figure S8). The hyphal growth of the gene-disrupted mutants became significantly slower than the wild-type strain of F. verticillioides (Figure 7A). Correspondingly, microscopic analysis revealed that Fv_Tan1 could be responsible for the maintenance of mycelial integrity, as disruptions led to irregular growth and exacerbated structural anomalies under tangeretin stress condition. Conversely, deletion of Fv_Tan2 caused no obvious influence of mycelial structure, suggesting a distinct role from Fv_Tan1. Overall, this analysis confirms the involvement of both genes in fungal growth, with Fv_Tan1 specifically important for maintaining normal hyphal structure, highlighting the nuanced genetic impact of tangeretin on F. verticillioides.

Regarding mycotoxin production, the individual deletion profoundly affected the marked reduction of FB₁ production compared to the wild-type strain (Figure 7B). Notably, the mutant deletion of Fv_Tan2 halted fumonisin production entirely. In addition, the mutant without Fv_Tan1 changed no obvious fluctuation in FB₁ production when subjected to tangeretin stress. This possibly suggests that loss of Fv_Tan1 may lessen the impact of tangeretin on mycotoxin production.

4. Discussion

Fumonisins are sphingosine-like mycotoxins linked with a variety of toxicities, including neurotoxicity, immunotoxicity and even carcinogenicity, by acting on sphingolipid metabolism, oxidative stress and apoptosis pathways [6,42,43]. F. verticillioides is the predominant fumonisin producing phytopathogen in maize. Plant-derived compounds are being explored as sustainable alternatives to the use of fungicides for the mitigation of pathogenic disease and fumonisin contamination. In this study, tangeretin was identified and evaluated for its potential to inhibit fumonisin production by F. verticillioides. While previous research has primarily focused on tangeretin’s antimicrobial properties against pathogens, such as respiratory syncytial virus [44], Phytophthora citrophthora [45], and Magnaporthe oryzae [46], this study is the first to investigate its antifungal and antimycotoxigenic potential against F. verticillioides. Our findings confirm its efficacy in inhibiting fumonisin production, thereby enhancing food safety prospects. This research establishes a theoretical foundation and underscores the practical value of tangeretin in managing fumonisin contamination. Notably, considering the neurotoxicity associated with fumonisins and the neuroprotective properties of tangeretin, this compound might also have therapeutic potential in mitigating fumonisin-induced neurotoxicity. To effectively address fumonisin control, future research should focus on improving the bioavailability of tangeretin. Nanoencapsulation presents a promising approach for enhancing the deployment of tangeretin in food safety applications, potentially leveraging green nanotechnology for efficient and cost-effective food preservation [47].

Tangeretin has been previously revealed to mitigate rice blast and inhibit the pathogenicity of M. oryzae by influencing redox signaling and ferroptosis processes [46]. A comparable mechanistic study in F. verticillioides revealed significant alterations in antioxidant activity, as evidenced by shifts in antioxidant enzymatic activities and H₂O₂ content. GO enrichment analysis indicated a notable delay in the transcription of oxidative stress-related DEGs. Specifically, DEGs associated with peroxidase activity (GO:0004601) exhibited lower transcription levels, underscoring a substantial disruption in the redox balance, consistent with findings in M. oryzae [46]. In contrast, the transcription levels of two genes (FVEG_00444 and FVEG_11478) encoding NADPH oxidase (Nox) did not show significant variation, differing from observations in M. oryzae where Nox-mediated lipid peroxidation was targeted by overexpressed Nox genes. This suggests pathogen-specific effects of tangeretin, which may interact differently with the molecular targets in F. verticillioides. In addition, peroxisomes are essential for the cellular oxidative homeostasis, infection, virulence as well as mycotoxin biosynthesis in phytopathogenic fungi [48,49]. PEX genes are involved in peroxisome biogenesis [50,51]. Components of the peroxisomal docking/translocation module (DTM) crucial for peroxisome biogenesis—such as FvPex5 (FVEG_01301) [15], FvPex8 (FVEG_05423), FvPex13 (FVEG_00529), FvPex14 (FVEG_03847), and FvPex33 (FVEG_11334) [16]—play roles in redox homeostasis and fumonisin production in F. verticillioides. Despite this, the transcription of these genes did not significantly change under tangeretin stress. Similarly, the Atf1 orthologous bZIP-type transcription factor FvAtfA (FVEG_02866) [14], which is involved in the modulation of oxidative balance, virulence, and mycotoxin biosynthesis, also showed no differential expression in response to tangeretin. This suggests that tangeretin may target other genes to influence redox homeostasis and fumonisin biosynthesis that warrants further investigation.

In contrast to resveratrol, tangeretin not only suppresses hyphal growth but also effectively inhibits fumonisin production. Comparative transcriptome analysis between tangeretin- and resveratrol-treated groups identified unique tangeretin-responsive DEGs implicated in mycotoxin regulation in F. verticillioides. The differences in enriched KEGG pathway demonstrated that tangeretin significantly interfered the MAPK signaling pathway, while resveratrol does not. The correlation between mycotoxin production and the MAPK pathway suggests that tangeretin may hinder mycotoxin accumulation through MAPK cascade. Furthermore, an NmrA- and HSCARG-like protein (Fv_Tan1) and an MDR transporter (Fv_Tan2) emerged as promising molecular targets responsive to tangeretin. The functions of these genes positively influence both fungal growth and fumonisin production. Notably, a mutant with Fv_Tan1 deletion showed no significant effect in mycotoxin production in response to tangeretin. Bioinformatic analysis of Fv_Tan1 indicates that it does not directly interact with nitrogen metabolism regulator AreA, as previously speculated. Instead, AreA is believed to interact directly with nitrogen metabolism repressor FVEG_07953, due to its high similarity with NmrA (XP_041146541.1) from A. flavus, which plays crucial roles in radial growth, aflatoxin production, and stress responses [52]. Additionally, NmrA-like proteins have been identified to play a significant role in oxidative stress responses [53,54,55]. Further exploration of gene targets revealed that the conserved domain of Fv_Tan1 aligned with that of an HSCARG-like protein, which act as an NADPH sensor, suggesting a potential relationship with redox modulation. Protein interaction analysis indicates Fv_Tan1 likely interacts with many NADH-ubiquinone oxidoreductase involved in the mitochondrial respiratory chain. During the gradual reduction process of oxygen (O₂) to water by this chain, electron leakage can lead to the formation of ROS [56]. This suggests that Fv_Tan1’s role in mycotoxin biosynthesis may be linked to its regulation on redox homeostasis. In conclusion, tangeretin inhibits fumonisin biosynthesis by disrupting normal MAPK signaling pathways and targeting the NmrA- and HSCARG-like protein Fv_Tan1. By elucidating the modes of action, this study enhances our understanding of how plant-derived compounds, like tangeretin, can inhibit mycotoxin production and suggests new avenues for developing effective mycotoxin management strategies.

5. Conclusions

Tangeretin, known for its antioxidant, anti-inflammatory and anti-tumor properties, has also demonstrated notable antifungal and anti-fumonisin effects. It significantly downregulates the expression of DEGs associated with hyphal growth, conidial development, and pathogenesis, thereby disrupting redox regulation. Additionally, tangeretin restricts substrate availability for fumonisin biosynthesis by interfering with alanine metabolism, pyruvate metabolism, fatty acid degradation and sphingolipid metabolism. Furthermore, it may directly act on the biosynthetic gene cluster responsible for fumonisin production. The compound also disrupts the MAPK signaling pathway and targets a downstream NmrA- and HSCARG-like protein (Fv_Tan1). Gene deletion of Fv_Tan1 causes a significant reduction in hyphal growth and fumonisin level. Notably, tangeretin exerts minimal influence on mycotoxin production in the mutant lacking this gene, underscoring the specificity of its target interaction. This study provides a theoretical foundation and emphasizes the potential application of tangeretin in controlling fumonisin contamination.