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. 2025 Dec 11;26(12):e70188. doi: 10.1111/mpp.70188

Phenylacetate Hydroxylase SsPhacA Modulates p‐Coumaric Acid Accumulation to Regulate the Mating/Filamentation of Sporisorium scitamineum

Bo Xiong 1,2, Nannan Zhang 1, Yirong Guo 1, Yawen Lei 1, Jiayun Wu 1, Changqing Chang 2,✉, Enping Cai 1,2,✉
PMCID: PMC12698384  PMID: 41382358

ABSTRACT

Sugarcane smut, caused by Sporisorium scitamineum, is one of the most severe sugarcane diseases. A key pathogenic step is dikaryotic mycelium formation via sexual reproduction, but its regulatory mechanism is unclear. In this study, we identified a phenylacetate hydroxylase gene, SsPHACA, that is highly expressed during the sexual mating/filamentation of S. scitamineum and enriched within the phenylalanine metabolic pathway. SsPHACA deletion mutants presented significantly reduced sexual mating/filamentation and pathogenicity. Additionally, deletion of SsPHACA resulted in marked downregulation of the pheromone‐responsive transcription factor gene SsPRF1, a critical regulator of sexual mating/filamentation in S. scitamineum, as well as its downstream genes at the a‐ and b‐locus genes. Constitutive expression of SsPRF1 restored the sexual mating/filamentation of the SsPHACA deletion mutant. Metabolomic analyses revealed that SsPhacA mediates phenylalanine metabolism in S. scitamineum and modulates the accumulation of p‐coumaric acid, an intermediate in phenylalanine metabolism. Exogenous supplementation with p‐coumaric acid increased the transcriptional level of SsPRF1 and partially restored sexual mating/filamentation in SsPHACA deletion mutants. In summary, our results demonstrate that SsPhacA mediates the phenylalanine metabolic pathway to modulate p‐coumaric acid accumulation, which increases the transcriptional level of SsPRF1, thereby regulating sexual reproduction in S. scitamineum. These findings not only identify a new regulatory factor involved in the sexual mating/filamentation of S. scitamineum, but also provide a new theoretical foundation for the development of disease control strategies targeting metabolic pathways.

Keywords: mating/filamentation, p‐coumaric acid, phenylacetate hydroxylase, phenylalanine metabolism, Sporisorium scitamineum

SsPhacA regulates the phenylalanine metabolic pathway to modulate p‐coumaric acid accumulation, which increases the transcriptional level of SsPRF1, thereby regulating sexual reproduction in S. scitamineum.

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1. Introduction

Sugarcane (Saccharum spp.) is a globally significant source of sugar and a sustainable bioenergy source, contributing approximately 85% of the global sugar supply (Wu et al. 2024). Sugarcane smut caused by Sporisorium scitamineum is one of the most severe diseases affecting sugarcane, with an annual incidence rate of 15% to 30% in China (Rajput et al. 2021). The fungal life cycle comprises three distinct phases: haploid yeast‐like sporidia, dikaryotic hyphae, and diploid teliospores (Taniguti et al. 2015). For host infection, two haploid sporidia of opposite mating types must fuse to form a dikaryotic mycelium (Que et al. 2015; Yan, Cai, et al. 2016). Sexual mating and filamentous growth in S. scitamineum exhibit conserved regulation with Ustilago maydis , relying on two mating‐type loci (a and b) (Fedler et al. 2009; Kämper et al. 1995). The a locus encodes components of the mating factor a (MFA) and pheromone‐responsive a (PRA), mediating recognition of opposite mating‐type sporidia and conjugation tube formation (Gillissen et al. 1992; Taniguti et al. 2015). The b locus governs dikaryotic filament formation and virulence activation by encoding two heterodimeric transcription factors, bE and bW (Schlesinger et al. 1997; Yan, Zhu, et al. 2016). Studies have shown that the pheromone response transcription factor 1 gene (SsPRF1) regulates the expression of the two mating‐type loci (a and b), thereby controlling the pathogenicity of smut fungi (Zhu et al. 2019; Cai et al. 2021). However, the factors that regulate SsPRF1 have not been fully elucidated.

Studies have shown that aromatic amino acid metabolism is essential for regulating the sexual reproduction of S. scitamineum (Cui et al. 2022). Among aromatic amino acids, phenylalanine plays a crucial role in the growth and development of fungi (Li et al. 2023). Phenylalanine metabolism involves a series of enzymatic reactions such as uptake, transamination, and decarboxylation, forming a dual network of energy conversion and physiological regulation (Dai et al. 2021). This involves multilevel mechanisms including gene expression regulation, enzyme activity modulation, and feedback inhibition by metabolites (Yao et al. 2023), and its complexity reflects the plasticity of fungal amino acid metabolism. In S. scitamineum, metabolomic analysis revealed that the aromatic amino acid transaminase SsAro8 positively regulates sexual mating/filamentation and biofilm formation by modulating tryptophol synthesis (Cui et al. 2022). Studies have indicated that the cytochrome P450 monooxygenase system plays an important role in the metabolism of aromatic amino acids in fungi (Park et al. 2020).

The P450 monooxygenase system is widely involved in primary and secondary metabolism in fungi, covering physiological processes such as drug degradation, toxin synthesis, and pigment production (Urlacher and Girhard 2019), and particularly plays a key role in amino acid metabolism, lipid metabolism, and the synthesis of cell wall and membrane components (Permana et al. 2023). Phenylacetate hydroxylase PhacA, a member of the cytochrome P450 monooxygenase family, has the core function of catalysing hydroxylation reactions in fungal metabolic processes, a property that is crucial for fungi to adapt to environmental stress (Mingot et al. 1999). In Aspergillus nidulans, PhacA‐deficient strains cannot grow using phenylacetic acid as the sole carbon source but can utilise derivatives such as 2‐hydroxyphenylacetic acid (Mingot et al. 1999; Emri et al. 2003), confirming their ability to catalyse the ortho‐hydroxylation of phenylacetic acid, which is the initial step in the catabolism of phenylacetic acid. In Penicillium chrysogenum, the homologous protein PahA is induced by phenylacetic acid and catalyses the 2‐hydroxylation of phenylacetic acid, reducing the availability of precursors for penicillin G biosynthesis (Emri et al. 2003; Rodríguez‐Sáiz et al. 2001); thus, the expression level of PahA is negatively correlated with penicillin production in the strain. These findings reveal a conserved mechanism by which the PhacA family regulates secondary metabolism. However, the biological function of PhacA in smut fungi remains unclear.

In this study, a phenylacetate hydroxylase gene SsPHACA, whose transcription is significantly increased during the sexual mating/filamentation stage of S. scitamineum, was enriched in the phenylalanine metabolic pathway. SsPhacA reduces the accumulation of the metabolite p‐coumaric acid by regulating the metabolic flux of phenylalanine metabolism. Further research revealed that p‐coumaric acid regulates the sexual mating/filamentation of S. scitamineum by modulating the transcriptional level of SsPRF1. In conclusion, our results indicate that SsPHACA mediates the phenylalanine metabolic pathway to regulate p‐coumaric acid, which increases the transcriptional level of SsPRF1, thereby controlling the sexual mating/filamentation of S. scitamineum. This study not only identifies a new regulatory factor for the sexual mating/filamentation of S. scitamineum but also provides a theoretical basis for the development of targeted disease control strategies.

2. Results

2.1. SsPhacA Is Involved in Regulating the Sexual Mating of S. scitamineum

Analysis of the transcriptome data at 0 h, 24 h, and 72 h after mixing wild‐type MAT‐1 and MAT‐2 revealed a total of 498 differentially expressed genes (DEGs) across three comparison groups (WT‐24 h vs. WT‐0 h, WT‐72 h vs. WT‐0 h, and WT‐72 h vs. WT‐24 h) (Figure 1a). Further KEGG enrichment analysis was performed on the upregulated DEGs in the WT‐72 h vs. WT‐0 h comparison group, and pathways such as phenylalanine metabolism, tryptophan metabolism, and tyrosine metabolism were enriched (p adj < 0.05) (Figure 1b; Figure S1). Phenylalanine, tryptophan, and tyrosine are three key aromatic amino acids. The specific metabolites produced by aromatic amino acids metabolism play important roles in regulating the physiological behaviours of fungi, such as morphological transition and toxin production (Li et al. 2023; Chen et al. 2004; Albuquerque and Casadevall 2012; Cui et al. 2022). Therefore, we focused on the aromatic amino acid metabolic pathway, and within this pathway, we found that one of the 498 DEGs, a phenylacetate hydroxylase gene (CDR88137.1, designated as SsPHACA), was significantly upregulated in the phenylalanine metabolic pathway (Figure 1b).

FIGURE 1.

FIGURE 1

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The expression level of the phenylalanine hydroxylase gene SsPHACA increases during the sexual mating stage of Sporisorium scitamineum. (a) Overlapping relationships of differentially expressed genes (DEGs) among the comparison groups of wild‐type (WT)‐24 h vs. WT‐0 h, WT‐72 h vs. WT‐0 h, and WT‐72 h vs. WT‐24 h. WT (MAT‐1 × MAT‐2) strains cultured on YePSA medium for 0, 24, and 72 h were collected for transcriptomic analysis. Each circle represents the set of DEGs in one comparison group, the overlapping area indicates the DEGs shared by multiple groups, and the nonoverlapping area represents the DEGs unique to each group. (b) KEGG enrichment analysis of the upregulated DEGs in the WT‐72 h vs. WT‐0 h group. Bubble charts were used to visualise the enriched terms of the DEGs. A p‐value < 0.05 was set as the threshold for statistical significance. The false discovery rate (FDR) was < 0.05. (c) Transcriptional profiling analysis of SsPHACA in the WT. The MAT‐1 × MAT‐2 strain was cultured on YePSA medium for 0, 12, 24, 36, 48, and 60 h. Actin was used as the internal reference gene. The experiment included three biological replicates, each with three technical replicates (n = 3). The error bars represent the means ± standard errors of the means. ★ p < 0.05, ★★★ p < 0.001; NS indicates no statistical significance.

Phylogenetic tree analysis revealed that SsPhacA clusters with homologous proteins from basidiomycetes (such as Ustilago trichophora and Sporisorium reilianum), whereas it has a relatively distant evolutionary relationship with homologous proteins from ascomycetes (such as Penicillium and Aspergillus) (Figure S2a), indicating that it is evolutionarily conserved among basidiomycetes. Protein domain prediction of the SsPhacA protein sequence via the SMART platform (URL: http://smart.embl‐heidelberg.de/) indicated that the protein contains a transmembrane region and a highly conserved cytochrome P450 functional domain (Figure S2b), suggesting that SsPhacA may be a conserved member of the cytochrome P450 enzyme family. Reverse transcription‐quantitative PCR (RT‐qPCR) analysis of the expression profile of SsPHACA at different time points during the sexual mating of S. scitamineum showed that its expression level increased with the duration of sexual mating, peaking at 48 h, followed by a decrease (Figure 1c), indicating that SsPHACA may be involved in the regulation of sexual mating in S. scitamineum.

2.2. SsPhacA Mediates the Transcription of SsPRF1 to Regulate the Mating/Filamentation of S. scitamineum

To examine the role of SsPHACA in the mating/filamentation of S. scitamineum, we constructed SsPHACA deletion mutants (ssphacAΔ‐1 and ssphacAΔ‐2) and SsPHACA complementation strains (ssphacAΔ/PHACA‐1 and ssphacAΔ/PHACA‐2) via homologous recombination. The results of the PCR analysis of these strains revealed that specific bands of SsPHACA could be amplified by internal specific primers in the wild‐type strains (MAT‐1 and MAT‐2) and complementation strains (ssphacAΔ/PHACA‐1 and ssphacAΔ/PHACA‐2) (Figure S3a). In contrast, no such bands were detected in the deletion mutants (ssphacAΔ‐1 and ssphacAΔ‐2), while specific bands of different sizes from those of the wild‐type were amplified by specific external primers (Figure S3a). Moreover, RT‐qPCR verification revealed that the relative expression level of SsPHACA in the deletion mutants was close to 0, and there was no significant difference in the expression level between the complementation strains and the wild‐type strains (Figure S3b), confirming that the expression of SsPHACA in the complementation strains had been restored. These results indicate that the SsPHACA deletion mutants and SsPHACA complementation strains were successfully constructed.

We subsequently analysed the ability of these SsPHACA deletion mutants and SsPHACA complementation strains to form dikaryotic mycelia through sexual mating. The SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2) presented a significant defect in their ability to form white mycelia through sexual mating, whereas this ability was restored in the complementation strains (ssphacAΔ/PHACA‐1 × ssphacAΔ/PHACA‐2) (Figure 2a). These findings indicate that functional deficiency of SsPHACA severely impacts the sexual mating/filamentation of S. scitamineum. RT‐qPCR was used to analyse the relative expression levels of the key pheromone response transcription factor gene SsPRF1, as well as the downstream genes SsMFA1, SsPRA1, SsbE, and SsbW, which regulate the sexual mating/filamentation of S. scitamineum. Compared with the wild‐type MAT‐1, the transcriptional levels of SsPRF1, SsMFA1, SsPRA1, and SsbE in ssphacAΔ‐1 were significantly lower (p < 0.05) (Figure 2b). We speculated that SsPhacA regulates the sexual mating/filamentation of S. scitamineum by modulating the transcription of SsPRF1, which is known to be a central transcriptional regulator of the sexual mating/filamentation of S. scitamineum (Zhu et al. 2019).

FIGURE 2.

FIGURE 2

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SsPHACA‐mediated transcription of SsPRF1 is essential for sexual mating/filamentation in Sporisorium scitamineum. (a) Mating assay of wild‐type (MAT‐1 × MAT‐2), SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2), and SsPHACA complementation strains (ssphacAΔ/PHACA‐1 × ssphacAΔ/PHACA‐2). Haploid cells of opposite mating types were mixed in equal volumes and spotted onto YePSA medium. The plates were incubated at 28°C for 48 h, and the results were photographed. (b) Reverse transcription‐quantitative PCR analysis was performed to determine the transcriptional levels of SsPRF1, SsMFA1, SsPRA1, SsbE, and SsbW in the MAT‐1 and ssphacAΔ‐1 strains after growth on YePSA plates for 48 h. The experiment included three biological replicates, each with three technical replicates (n = 3). The error bars represent the means ± standard errors of the means (SEMs). (c) Transcriptional profiling of SsPRF1 in the wild‐type (MAT‐1 × MAT‐2), SsPHACA deletion mutant (ssphacAΔ‐1 × ssphacAΔ‐2), and SsPRF1 constitutive expression strains (ssphacAΔ/con‐PRF1‐1 × ssphacAΔ/con‐PRF1‐2). The experiment included three biological replicates, each with three technical replicates (n = 3). The error bars represent the means ± SEMs. (d) Mating assay of wild‐type (MAT‐1 × MAT‐2), SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2), and SsPRF1 constitutive expression strains (ssphacAΔ/con‐PRF1‐1 × ssphacAΔ/con‐PRF1‐2). Haploid cells of opposite mating types were mixed in equal volumes and spotted onto YePSA medium. The plates were incubated at 28°C for 48 h, and the results were photographed. For (b) and (c), Actin was used as the internal reference gene; ★ p < 0.05, ★★★ p < 0.001; NS indicates no statistical significance.

To test this hypothesis, we constructed SsPRF1 constitutive expression strains (ssphacAΔ/con‐PRF‐1 and ssphacAΔ/con‐PRF‐2) with SsPHACA deletion mutants (ssphacAΔ‐1 and ssphacAΔ‐2) via homologous recombination. RT‐qPCR analysis revealed that the expression level of SsPRF1 in these strains was restored to the wild‐type level (Figure 2c). Further sexual mating analysis revealed that the ability of the SsPRF1 constitutive expression strains (ssphacAΔ/con‐PRF‐1 × ssphacAΔ/con‐PRF‐2) to form white colonies was consistent with that of the wild‐type (Figure 2d). These findings confirm that SsPHACA regulates the sexual mating/filamentation of S. scitamineum by modulating the transcription of SsPRF1.

2.3. SsPhacA Is Essential for Phenylalanine Metabolism

As a member of the cytochrome P450 monooxygenase family, the phenylacetate hydroxylase PhacA has a core function in catalysing hydroxylation reactions during fungal metabolism, a characteristic that is crucial for fungi to adapt to environmental stress (Mingot et al. 1999). Therefore, we analysed the effect of SsPhacA on the metabolism of S. scitamineum through metabolomic analysis. The results revealed that in the comparison between the SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2) and wild‐type (MAT‐1 × MAT‐2), 77 upregulated metabolites (UPs), 166 downregulated metabolites (DWs), and 1163 downregulated metabolites were not significantly different (NoDiff) (Figure 3a). These findings indicate that the deletion of SsPhacA affects the metabolic profile of S. scitamineum.

FIGURE 3.

FIGURE 3

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The role of SsPhacA in phenylalanine metabolism in Sporisorium scitamineum. (a) Overall distribution of differentially abundant metabolites in the PhacA‐72 h vs. wild‐type (WT)‐72 h comparison group. Each dot in the volcano plot represents a metabolite, with significantly upregulated metabolites (UP) indicated by red dots, significantly downregulated metabolites (DW) indicated by blue dots, and metabolites with no significant difference (NoDiff) indicated by grey dots. (b) KEGG enrichment analysis of differentially expressed genes and differentially abundant metabolites in the PhacA‐72 h vs. WT‐72 h group. Count: The number of metabolites or genes enriched in the pathway. The colour indicates the p value; the redder the colour is the smaller the p value, and the more significant the pathway enrichment. (c) Statistical analyses were performed on differentially abundant metabolites across pathways. Blue represents downregulation, and red represents upregulation. log2 (Fold Change) refers to the fold change in the upregulation or downregulation of metabolites. For (a), (b) and (c), these experiment included three replicate samples (n = 3). The p‐value and false discovery rate (FDR) < 0.05.

The results of KEGG metabolic pathway classification revealed that the differentially abundant metabolites were enriched mainly in categories such as global and overview maps (36.8%), amino acid metabolism (29.4%), lipid metabolism (4.4%), and cofactor and vitamin metabolism (2.9%) (Figure S4). Among these pathways, the high proportion of amino acid metabolism suggested its core position (Figure S4). Through integrated analysis of metabolomic and transcriptomic data, the phenylalanine metabolism pathway was found to be significantly enriched in analyses (Figure 3b), suggesting that SsPhacA influences the accumulation of related metabolites by regulating the metabolic flux of phenylalanine metabolism. Statistical analysis was conducted on differentially abundant metabolites in pathways such as arginine and proline metabolism, phenylalanine metabolism, biosynthesis of amino acids, phenylalanine, tyrosine and tryptophan biosynthesis, and tyrosine metabolism, which were obtained from the comparison between the SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2) and wild‐type (MAT‐1 × MAT‐2). Four metabolites (phenylalanine, phenylacetaldehyde, 2‐hydroxy‐3‐phenylpropenoate, and p‐coumaric acid) in the phenylalanine metabolism pathway decreased in the SsPHACA deletion mutants (Figure 3c; Figure S5), suggesting that SsPhacA affects the concentrations of these metabolites in cells.

2.4. SsPhacA Regulates the Mating/Filamentation of S. scitamineum by Modulating the p‐Coumaric Acid Level to Promote the Transcription of SsPRF1

We used high‐performance liquid chromatography (HPLC) to quantify the difference in intracellular p‐coumaric acid levels between the wild‐type (MAT‐1 × MAT‐2) and SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2). The peaks for the p‐coumaric acid standard, wild‐type, and SsPHACA deletion mutants sample appeared between 9.1 and 9.4 min. However, the peak height of the wild‐type strain was higher than that of the SsPHACA deletion mutants (Figure 4a). After calculating the sample peak areas and using the standard curve for quantification, we found that the level of p‐coumaric acid in the SsPHACA deletion mutants was significantly lower than that in the wild‐type (Figure 4b).

FIGURE 4.

FIGURE 4

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p‐Coumaric acid regulates the mating/filamentation of Sporisorium scitamineum by promoting the transcriptional regulation of SsPRF1. (a) and (b) High‐performance liquid chromatography (HPLC) determination of p‐coumaric acid content. The wild‐type (WT; MAT‐1 × MAT‐2) and SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2) samples were collected with consistent growth status on YePSA medium. Intracellular p‐coumaric acid was extracted with ethyl acetate, followed by detection using HPLC. The intracellular content of p‐coumaric acid was calculated from the peak area using a standard curve. The bar chart shows the mean ± SE from three independent biological replicates, each with three technical replicates (n = 3). (c) Assessment of the role of exogenous supplementation with p‐coumaric acid in the mating/filamentation of wild‐type (MAT‐1 × MAT‐2) and SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2). Haploid cells of opposite mating types were mixed in equal volumes and spotted onto minimal medium with or without the addition of 10 μM p‐coumaric acid. DMSO, dimethyl sulphoxide. The plates were incubated at 28°C for 24 h, after which the results were photographed. The scale bar is 1 mm. (d) Reverse transcription‐quantitative PCR (RT‐qPCR) analysis of the transcriptional levels of SsPRF1 following exogenous supplementation with p‐coumaric acid. Haploid cells of opposite mating types were mixed (MAT‐1 × MAT‐2 and ssphacAΔ‐1 × ssphacAΔ‐2) in equal volumes and grown on minimal medium supplemented with or without 10 μM p‐coumaric acid for 36 h. RT‐qPCR analysis was conducted to quantify the transcriptional levels of SsPRF1, with Actin serving as the internal reference gene. The experiment included three biological replicates, each with three technical replicates (n = 3). The error bars represent the means ± standard errors of the means. ★ p < 0.05; NS indicates no statistical significance.

Subsequently, exogenous supplementation of the decreased metabolites was performed to analyse the sexual mating/filamentation of the SsPHACA deletion mutants. The results showed that l‐ornithine, 4‐guanidinobutanal, S‐adenosyl‐l‐methionine, l‐methionine, l‐proline, or l‐phenylalanine had no effect on the sexual mating/filamentation of the SsPHACA deletion mutants (Figure S6a). However, exogenous supplementation with a low concentration of p‐coumaric acid (0.1 and 1 μM) showed no significant effect on promoting the sexual mating of the SsPHACA deletion mutant, whereas a high concentration (10 and 100 μM) could promote the formation of dikaryotic mycelia of this mutant (Figure 4c; Figure S6b). Furthermore, supplementation with p‐coumaric acid could increase the transcription level of SsPRF1 in SsPHACA deletion mutants (Figure 4d). These results indicate that SsPhacA modulates the accumulation of p‐coumaric acid by mediating the phenylalanine metabolism pathway, thereby affecting the transcriptional level of SsPRF1 and regulating the sexual mating/filamentation of S. scitamineum.

2.5. SsPhacA Positively Modulates Cell Membrane Stability in S. scitamineum

Gene Ontology (GO) functional enrichment analysis of the transcriptome revealed that the significantly enriched (p < 0.05) cellular components in the SsPHACA deletion mutants included membrane parts, intrinsic components of membranes, and integral components of membranes, which is suggestive that the deletion of SsPHACA may affect the expression of genes related to cell membrane structure and function. In addition, molecular functions such as transition metal ion binding and oxidoreductase activity were enriched biological processes such as oxidation–reduction processes, transmembrane transport, and carbohydrate metabolism were significantly enriched (Figure 5a).

FIGURE 5.

FIGURE 5

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SsPhacA positively regulates the stability of the cell membrane of Sporisorium scitamineum. (a) GO enrichment analysis of differentially expressed genes (DEGS) in the MAT‐1 × MAT‐2 and ssphacAΔ‐1 × ssphacAΔ‐2 strains. Bubble plots were generated by selecting the 10 most significant terms in biological process, cellular component, and molecular function. The abscissa represents the ratio of the number of DEGs annotated to a GO term to the total number of DEGs. The ordinate represents the GO terms. The size of the dots indicates the number of genes annotated to the GO terms, and the colour gradient from red to purple represents the significance level of enrichment. This experiment included three replicate samples (n = 3). False discovery rate (FDR) < 0.05. (b) The tolerance of the MAT‐1, ssphacAΔ‐1, and ssphacAΔ/PHACA‐1 strains to sodium dodecyl sulphate (SDS) was evaluated. The fresh haploid sporidia were allowed to grow to an OD600 of 1.0, after which 10‐fold diluted cells were subsequently spotted onto YePSA medium with or without 0.011% SDS. Images were taken 3–4 days after cultivation.

To analyse the effect of SsPhacA on the integrity of the cell membrane structure, tolerance to sodium dodecyl sulphate (SDS), a cell membrane disruptor, was tested. The results revealed that the growth of the ssphacAΔ‐1 strain was significantly inhibited in YePSA medium supplemented with 0.011% SDS, whereas there was no significant difference in the growth of the complementation strain (ssphacAΔ/PHACA‐1) compared with that of the wild‐type MAT‐1 (Figure 5b). These findings indicate that SsPhacA regulates cell tolerance to SDS stress by maintaining the integrity of the cell membrane structure.

2.6. SsPhacA Is Indispensable for the Pathogenicity of S. scitamineum

The formation of dikaryotic mycelia through sexual mating/filamentation is a key step in the infection process of S. scitamineum. Therefore, the effects of SsPhacA on the infectivity and pathogenicity of sugarcane were tested through inoculation assays. Sugarcane seedlings inoculated with the wild‐type (MAT‐1 × MAT‐2) and complementation strains (ssphacAΔ/PHACA‐1 × ssphacAΔ/PHACA‐2) presented typical “black whip” symptoms in sugarcane, whereas those inoculated with the mutant (ssphacAΔ‐1 × ssphacAΔ‐2) barely developed typical symptoms of smut disease (Figure 6a). Statistical analysis revealed that the disease incidence rates of sugarcane seedlings inoculated with the wild‐type and complementation strains were both greater than 60%, whereas that of seedlings inoculated with the SsPHACA deletion mutants was only 2.5%, indicating a significant reduction in the pathogenicity of the SsPHACA deletion mutants (Figure 6b). These results demonstrate that SsPhacA is essential for the infection and pathogenicity of S. scitamineum.

FIGURE 6.

FIGURE 6

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SsPhacA is required for Sporisorium scitamineum pathogenicity. (a) Symptoms induced by the wild‐type (MAT‐1 × MAT‐2), SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2), and SsPHACA complementation strains (ssphacAΔ/PHACA‐1 × ssphacAΔ/PHACA‐2) in sugarcane. Haploid S. scitamineum cells of opposite mating types were mixed in equal volumes and injected into the vicinity of the stem growing points of 5‐leaf‐stage seedlings of the susceptible sugarcane variety XTT22. The seedlings were subsequently cultivated in a greenhouse under natural light cycles for 3–6 months, during which the occurrence of black whip symptoms was observed. The black whip symptom is indicated by the red arrows. The scale bar is 5 cm. (b) Incidence of sugarcane disease induced by the wild‐type (MAT‐1 × MAT‐2), SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2), and SsPHACA complementation strains (ssphacAΔ/PHACA‐1 × ssphacAΔ/PHACA‐2). The incidence rate (%) was calculated as the number of sugarcane plants with “black whip” symptoms/total number of plants ×100. Each sample was analysed with three biological replicates, with 12 sugarcane seedlings per replicate (n = 12). The error bars represent the mean ± standard error of the means. ★★ p < 0.01; NS indicates no statistical significance.

3. Discussion

PhacA, a member of the cytochrome P450 monooxygenase family, has been predominantly characterised in ascomycete fungi (e.g., A. nidulans and P. chrysogenum), where it is primarily involved in phenylacetate catabolism and secondary metabolism (Mingot et al. 1999; Rodríguez‐Sáiz et al. 2001). In this study, phylogenetic analysis of SsPhacA revealed that it clusters with PhacA homologues from basidiomycete species (e.g., U. trichophora, S. reilianum), indicating evolutionary conservation of this protein within the Basidiomycota phylum. Notably, we identified a new role for SsPhacA in regulating sexual reproduction and pathogenicity in smut fungi, a function that has not been reported for PhacA orthologues in other fungal lineages. This role of SsPhacA differs markedly from that of its ascomycetous counterparts, which mainly modulate penicillin biosynthesis or carbon source (phenylacetate) utilisation (Mingot et al. 1999; Rodríguez‐Sáiz et al. 2001; Emri et al. 2003). Instead, SsPhacA integrates metabolic signals (phenylalanine metabolism) with developmental programmes (mating/filamentation) in S. scitamineum, indicating functional divergence of the PhacA family across fungal lineages. This functional specialisation may reflect the adaptive evolution of S. scitamineum to its biotrophic lifestyle, where precise control of sexual development is critical for host colonisation.

Phenylalanine metabolism is a conserved pathway in fungi, with roles in energy production, secondary metabolite synthesis, and stress responses (Dai et al. 2021; Li et al. 2023). Our metabolomic and transcriptomic analyses collectively demonstrated that SsPhacA modulates metabolic flux through the phenylalanine pathway, specifically regulating p‐coumaric acid accumulation. This finding expands the known functions of phenylalanine metabolism in fungal pathogenesis, which has previously been linked to tryptophol synthesis and polyamine metabolism in S. scitamineum (Cui et al. 2022; Yin, Cui, et al. 2024; Yin, Hu, et al. 2024). However, the enzymatic activity of SsPhacA—whether it directly hydroxylates phenylalanine or intermediate metabolites to generate p‐coumaric acid—remains to be directly demonstrated. Notably, exogenous supplementation with p‐coumaric acid restored sexual mating/filamentation in SsPHACA deletion mutants, establishing a metabolic‐to‐transcriptional regulatory axis. This contrasts with reports in Saccharomyces cerevisiae and Candida albicans , where phenylalanine‐derived metabolites such as phenylethanol primarily function in quorum sensing and biofilm formation (Li et al. 2023; Gori et al. 2011; Wei et al. 2025). To our knowledge, this is the first demonstration that p‐coumaric acid acts as a signalling intermediate to modulate fungal sexual reproduction, highlighting the functional diversity of metabolites across fungal species.

The SsPRF1 is a well‐characterised master regulator of sexual mating/filamentation in S. scitamineum, controlling the expression of a‐ and b‐locus genes (Zhu et al. 2019; Cai et al. 2021). Our study extends this regulatory network by identifying p‐coumaric acid as a metabolic signal that modulates SsPRF1 transcription. This finding links metabolic pathways with the canonical pheromone‐responsive signalling cascade (e.g., the MAPK and cAMP/PKA pathways) (Cai et al. 2021; Deng et al. 2018), suggesting that SsPRF1 integrates both environmental cues (pheromones) and metabolic status (phenylalanine flux) to coordinate developmental transitions. However, the molecular mechanism by which p‐coumaric acid upregulates the transcription of SsPRF1 remains to be further explored. Such integration likely enhances the adaptability of S. scitamineum during host infection, where nutrient availability fluctuates. Therefore, exploring whether the level of p‐coumaric acid in sugarcane tissues affects its susceptibility to disease in the future may provide a reference for breeding strategies for disease‐resistant varieties.

In addition to its role in phenylalanine metabolism, our transcriptomic and phenotypic analyses revealed that SsPhacA contributes to cell membrane integrity, as evidenced by the hypersensitivity of SsPHACA deletion mutants to SDS. This finding is consistent with the known functions of cytochrome P450 monooxygenases in lipid metabolism and membrane component synthesis (Permana et al. 2023; Urlacher and Girhard 2019). Membrane stability is critical for fungal cell‐to‐cell communication during mating (Henderson et al. 2019; Hegemann et al. 2015). Thus, SsPhacA may regulate pathogenicity through dual mechanisms: modulating metabolic signals for sexual development and maintaining membrane integrity for environmental adaptation. Additionally, targeting SsPhacA or p‐coumaric acid synthesis with small‐molecule inhibitors may represent a novel approach for smut disease control, meriting further biochemical studies.

In summary, our findings establish a novel regulatory pathway in S. scitamineum whereby SsPhacA mediates phenylalanine metabolism to modulate p‐coumaric acid accumulation, which modulates SsPRF1 transcription and sexual mating/filamentation (Figure 7). This work advances our understanding of how metabolic networks interface with developmental programmes in pathogenic fungi and provides a theoretical basis for designing targeted strategies to manage sugarcane smut.

FIGURE 7.

FIGURE 7

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A proposed working model of SsPhacA. SsPhacA regulates the phenylalanine metabolic pathway to modulate p‐coumaric acid accumulation, which increases the transcriptional level of SsPRF1, thereby regulating sexual reproduction in Sporisorium scitamineum.

4. Materials and Methods

4.1. Culture Conditions for S. scitamineum

The wild‐type S. scitamineum strains (MAT‐1 and MAT‐2) were isolated and identified by Yan, Zhu, et al. (2016) and preserved in our laboratory. A small number of haploid sporidia of S. scitamineum were picked from the plate and transferred to 5 mL of sterile YePS medium (yeast extract‐peptone‐sucrose; pH 6.5). The culture was then incubated overnight at 28°C with shaking at 200 rpm to prepare the seed culture. Details of the strains used in this study are listed in Table S1.

4.2. Construction of Plasmids and Strains

4.2.1. Deletion of SsPHACA

The DNA sequences of SsPHACA and its 1.5 kb upstream and downstream flanking regions were retrieved from the NCBI database. Specific primers were designed on the basis of these upstream and downstream sequences. Using genomic DNA from the wild‐type strain as a template, the ~1.5 kb upstream and downstream fragments of SsPHACA were amplified with high‐fidelity DNA polymerase (Vazyme; the same enzyme was used for all subsequent PCR cloning). These fragments were then fused with a truncated hygromycin resistance gene (HYG R ) via the split‐marker method to construct deletion DNA double fragments (Chang et al. 2019). The double fragments were introduced into protoplasts of the wild‐type MAT‐1 and MAT‐2 strains via polyethylene glycol (PEG)‐mediated protoplast transformation (Chang et al. 2019). Deletion mutants (ssphacAΔ‐1 and ssphacAΔ‐2) were obtained through multiple rounds of selection on YePS medium supplemented with hygromycin B (200 μg/mL; Merck). The detailed primer sequences are listed in Table S2.

4.2.2. Complementation of SsPHACA

Each correctly constructed homologous recombination deletion mutant contains an inserted DNA fragment of approximately 3.0 kb (including the HYG R sequence). Therefore, the HYG R sequence was targeted for replacement, and the zeocin resistance gene (ZEO R ) was used as the selection marker. A DNA fragment containing the SsPHACA gene with its native promoter and terminator was amplified from wild‐type genomic DNA using the primers SsPHACA‐com‐F/SsPHACA‐com‐R. The PCR product was seamlessly integrated into the pEASY‐COM vector (Cai et al. 2021) via the ClonExpress II One Step Cloning Kit (Vazyme) to generate the plasmid pEASY‐COM‐SsPHACA. Using this plasmid as a template, two complementation DNA fragments were amplified: one fused with the left homology arm, the full‐length SsPHACA, and a portion of ZEO R (using the universal primers COM‐LB‐F/COM‐LB‐R) and the other fused with a portion of ZEO R and the right homology arm (using the universal primers COM‐RB‐F/COM‐RB‐R). These two fragments were subsequently cotransformed into protoplasts of ssphacAΔ‐1 and ssphacAΔ‐2 via PEG‐mediated protoplast transformation (Chang et al. 2019). Complementation strains (ssphacAΔ/PHACA‐1 and ssphacAΔ/PHACA‐2) were selected on YePS medium supplemented with zeocin (100 μg/mL, Invitrogen). The detailed primer sequences are listed in Table S2.

4.2.3. Constitutive Expression of SsPRF1

Similar to gene complementation, a DNA fragment fused with the GPA promoter, SsPRF1, and ZEO R was inserted into a specific nonfunctional locus in the S. scitamineum genome via double‐fragment homologous recombination (Cai et al. 2024). Specifically, the coding sequence fragment of SsPRF1 was amplified from wild‐type cDNA via PCR. The PCR product was seamlessly integrated into the pEASY‐OE vector (Cai et al. 2021) via the ClonExpress II One Step Cloning Kit to generate the plasmid pEASY‐OE‐SsPRF1. Using this plasmid as a template, two DNA fragments were amplified: one fused with the left homology arm, GPA‐SsPRF1, and a portion of ZEO R (using universal primers con‐LB‐F/con‐LB‐R), and the other fused with a portion of ZEO R and the right homology arm (using universal primers con‐RB‐F/con‐RB‐R). These two fragments were subsequently cotransformed into protoplasts of ssphacAΔ‐1 and ssphacAΔ‐2 via PEG‐mediated protoplast transformation (Cai et al. 2024). Strains with constitutive SsPRF1 expression (ssphacAΔ/con‐PRF1‐1 and ssphacAΔ/con‐PRF1‐2) were selected in YePS medium supplemented with zeocin (100 μg/mL). The detailed primer sequences are listed in Table S2.

4.3. Nucleic Acid Extraction and PCR Identification of Transformants

Wild‐type, SsPHACA deletion mutant, SsPHACA complementation, and SsPRF1 constitutive expression transformants were cultured in YePSA medium at 28°C. Mycelia were collected, quickly frozen in liquid nitrogen, and ground into a powder. Genomic DNA was extracted via an SDS‐based DNA extraction method (Aristóteles et al. 2005), and total mRNA was extracted via the RNeasy Mini Kit (Qiagen) according to the manufacturer's instructions. cDNA was synthesised from mRNA via the HiScript II 1st Strand cDNA Synthesis Kit (Vazyme).

For transformant identification, genomic DNA was used as a template for PCR amplification with the specific primers SsPHACA‐in‐F/SsPHACA‐in‐R and SsPHACA‐ex‐F/SsPHACA‐ex‐R using 2 × Rapid Taq Master Mix (Vazyme). Additionally, RT‐qPCR was performed to detect the transcription of SsPHACA and SsPRF1 using cDNA as a template, with ACTIN as the internal reference gene and specific primers (RT‐qPCR‐SsPHACA‐F/R and RT‐qPCR‐SsPRF1‐F/R). The transformants were verified via the use of the wild‐type strain as a control. All primer sequences are listed in Table S2.

4.4. RT‐qPCR Analysis

4.4.1. Transcriptional Profile of SsPHACA

Wild‐type MAT‐1 and MAT‐2 strains were cultured in YePSA medium to undergo sexual mating/filamentation for 0, 12, 24, 36, 48, and 60 h. Mycelia were collected to extract total RNA, which was then reverse‐transcribed into cDNA. qPCR was performed using ChamQ SYBR qPCR Master Mix (Vazyme) with specific primers (RT‐qPCR‐SsPHACA‐ F/R) to analyse the transcription of SsPHACA, with the wild‐type strain used as a control and ACTIN as the internal reference gene (Sun et al. 2019).

4.4.2. Transcriptional Analysis of SsPRF1 and Its Downstream Genes

Wild‐type MAT‐1 and ssphacAΔ‐1 strains were cultured in YePSA medium and allowed to undergo sexual mating/filamentation for 48 h. Mycelia were collected to extract total RNA, which was reverse‐transcribed into cDNA. qPCR was performed via the use of ChamQ SYBR qPCR Master Mix with specific primers (RT‐qPCR‐SsPRF1‐F/R, RT‐qPCR‐SsMFA1‐F/R, RT‐qPCR‐SsPRA1‐F/R, RT‐qPCR‐SsbE‐F/R, RT‐qPCR‐SsbW‐F/R) to analyse the transcription of SsPRF1 and its downstream genes, with the wild‐type strain used as a control and ACTIN as the internal reference gene.

4.4.3. RT‐qPCR Analysis of the Effect of p‐Coumaric Acid on SsPRF1 Transcription

Wild‐type MAT‐1 × MAT‐2 and ssphacAΔ‐1 × ssphacAΔ‐2 strains were cultured in minimal medium with or without p‐coumaric acid to undergo sexual mating/filamentation for 48 h. Mycelia were collected to extract total RNA, which was reverse‐transcribed into cDNA. qPCR was performed via ChamQ SYBR qPCR Master Mix with specific primers (RT‐qPCR‐SsPRF1‐F/R) to analyse the transcription of SsPRF1 in the wild‐type strain as a control, and ACTIN was used as the internal reference gene.

All the samples were analysed with three biological replicates, each with three technical replicates. The relative gene expression levels were calculated using the 2−ΔΔCt method (Livak and Schmittgen 2001).

4.5. Transcriptome Analysis

To collect samples for transcriptome sequencing related to sexual mating/filamentation, wild‐type MAT‐1 and MAT‐2 strains were separately cultured in YePS medium until they reached a consistent growth with an OD600 value of 1.0. Subsequently, equal volumes of the two strains were mixed, evenly spread onto YePSA medium, and incubated at 28°C. Fungal cells were collected after 0, 24, and 72 h of incubation for total RNA extraction. To compare the transcriptome differences between the wild‐type and the SsPHACA deletion mutant, the wild‐type (MAT‐1 and MAT‐2) and SsPHACA deletion mutants (ssphacAΔ‐1 and ssphacAΔ‐2) were each cultured in YePS medium until they reached an OD600 value of 1.0. Subsequently, MAT‐1 × MAT‐2, as well as ssphacAΔ‐1 × ssphacAΔ‐2, were mixed in equal volumes and evenly spread on YePSA medium for incubation at 28°C. Fungal cells were collected at 24 and 72 h of culture for total RNA extraction. mRNA was enriched from total RNA via oligo(dT) magnetic beads. After fragmentation, first‐strand cDNA was synthesised using random hexamer primers. Library construction was performed via the NEBNext Ultra RNA Library Prep Kit for Illumina. Transcripts were assembled de novo using StringTie software, and novel transcripts were annotated against databases such as Pfam, SUPERFAMILY, GO, and KEGG. The assembled fragments were subsequently mapped to the published S. scitamineum genome (Taniguti et al. 2015, Que et al. 2015). DEGs were analysed by alignment against databases including the NCBI NR Database and KOG Database. Each sample treatment was repeated three times.

4.6. Metabolome Analysis

The wild‐type (MAT‐1 and MAT‐2) and SsPHACA deletion mutants (ssphacAΔ‐1 and ssphacAΔ‐2) were each cultured in YePS medium to achieve an OD600 of 1.0. Thereafter, the strains (MAT‐1 × MAT‐2 and ssphacAΔ‐1 × ssphacAΔ‐2) were mixed in equal volumes, uniformly spread on YePSA medium, and incubated at 28°C. Fungal cells were harvested at 72 h post‐incubation. A 100 mg aliquot of mycelia was quickly frozen in liquid nitrogen, ground into a powder, and transferred to a microfuge tube. Five hundred microlitres of 80% methanol aqueous solution was added, and the mixture was vortexed, incubated on ice for 5 min, and centrifuged at 15,000 g at 4°C for 20 min. The supernatant was diluted with mass spectrometry‐grade water to a final methanol concentration of 53%. Metabolomic analysis was performed by Novogene Bioinformatics Technology Co. Ltd. using ultrahigh‐performance liquid chromatography combined with high‐resolution mass spectrometry (Want et al. 2010; Dunn et al. 2011). The identified metabolites were annotated against the KEGG (https://www.genome.jp/kegg/pathway.html), HMDB (https://hmdb.ca/metabolites), and LIPIDMaps database (http://www.lipidmaps.org/). The data were processed using the metabolomics data analysis software metaX, followed by principal component analysis (PCA) and partial least squares‐discriminant analysis (PLS‐DA) to determine the VIP values of each metabolite (Wen et al. 2017; Boulesteix and Strimmer 2007). Statistical significance between groups was calculated using the t test (p value), and the fold change (FC) of each metabolite between groups was computed (Kieffer et al. 2016). Differentially abundant metabolites were screened on the basis of the default criteria: VIP > 1.0, p < 0.05, and FC ≥ 2.0 or FC ≤ 0.5. Subsequently, differential metabolites were identified using the following criteria: VIP > 1.0, FC > 1.2 or FC < 0.833, and p < 0.05 (Luo et al. 2025). Volcano plots and bubble plots were generated using the R package ggplot2. Each sample treatment was repeated three times.

4.7. Determination of p‐Coumaric Acid Content

Wild‐type (MAT‐1 × MAT‐2) and SsPHACA deletion mutants (ssphacAΔ‐1 × ssphacAΔ‐2) were grown to consistent status. Approximately 0.1 g of the powdered sample was placed into a 15 mL centrifuge tube, 1 mL of ethyl acetate was added. The tube was placed on a shaker for thorough oscillation and mixing for 1 min, then water‐bath ultrasonic extraction was performed for 30 min. The supernatant was collected and dried with nitrogen gas, then the residue was reconstituted with 0.25 mL of methanol, diluted appropriately, and filtered through a 0.22 μm filter membrane. Subsequently, the sample was analysed by HPLC using the Agilent 1260 with a Hypersil GOLD C18 column (250 mm × 4.6 mm, particle size 5 μm). The parameters were column temperature, 30°C; flow rate, 1.0 mL/min; injection volume, 20 μL; 1.5% acetic acid in water; wavelength, 310 nm; elute with acetonitrile. Each sample treatment was repeated three times.

4.8. Analysis of Sexual Mating/Filamentous Growth

The wild‐type strains, SsPRF1 constitutive expression strains, SsPHACA deletion mutants, and complementation strains were cultured overnight in YePS medium. When the cultures reached a consistent growth status with an OD600 of 1.0, mycelia were collected by centrifugation, washed twice with sterile phosphate‐buffered saline (PBS), and resuspended in an equal volume of PBS. Suspensions of strains with opposite mating types were mixed in equal volumes as needed. A 1.5 μL aliquot of the mixed suspension was spotted onto YePSA/minimal medium with or without phenylalanine metabolites and incubated statically at 28°C. The results were recorded by photography. The experiment included three biological replicates, each with two technical replicates.

4.9. Analysis of Cell Membrane Stability

The MAT‐1, ssphacAΔ‐1, and ssphacAΔ/PHACA‐1 strains were cultured overnight in YePS medium. When the cultures reached a consistent growth status with an OD600 of 1.0, mycelia were collected by centrifugation at 1500 g for 3 min, washed twice with sterile PBS, and resuspended to an OD600 of 1.0. The suspensions were serially diluted 10‐fold to concentrations of 100, 10−1, 10−2, and 10−3. A 1.5 μL aliquot of each dilution was spotted onto YePSA medium with or without SDS, air‐dried, and incubated statically at 28°C for 3–4 days. The results were recorded by photography. The experiment included three biological replicates, each with two technical replicates.

4.10. Pathogenicity Analysis

The wild‐type, SsPHACA deletion mutant, and SsPHACA complementation strains were cultured overnight in YePS medium. When the cultures reached a consistent growth status with an OD600 of 1.0, mycelia were collected by centrifugation at 4000 rpm for 3 min, washed twice with sterile 1 × PBS, and resuspended to an OD600 of 1.0. Suspensions of the wild‐type, SsPHACA deletion mutant, and SsPHACA complementation strains with the corresponding opposite mating types were mixed in equal volumes as needed. A 0.2 mL aliquot of the mixed suspension was injected near the growing point of the stem in 5‐leaf‐stage seedlings of the susceptible sugarcane variety XTT22. The seedlings were then cultivated in a greenhouse under natural light cycles at 25°–35°C for 3–6 months, and the occurrence of smut whip symptoms was observed and statistically analysed. Each sample was analysed with three biological replicates, with no fewer than 12 sugarcane seedlings per replicate.

4.11. Domain Prediction and Phylogenetic Analysis

The amino acid sequence of the SsPhacA protein was retrieved from NCBI (https://www.ncbi.nlm.nih.gov/protein/; Sequence ID: CDR88137.1), and the domains of SsPhacA were predicted using the SmartBLAST tool (https://blast.ncbi.nlm.nih.gov/smartblast/). The domain distribution map of SsPhacA was drawn using IBS v. 1.0.1 software. Phylogenetic analysis of PhacA proteins from various basidiomycetous and ascomycetous species was performed using the maximum‐likelihood method in MEGA 7.

4.12. Statistical Analysis

Statistical significance was assessed using Bonferroni's multiple comparison test to compare means. The results were considered statistically significant if the p value was < 0.05. Histograms were generated via GraphPad Prism v. 5 software.

Author Contributions

Bo Xiong: investigation; methodology; formal analysis; writing – original draft. Nannan Zhang: methodology; formal analysis; data curation; writing – original draft. Yirong Guo: investigation; formal analysis. Yawen Lei: investigation; methodology. Jiayun Wu: data curation; formal analysis. Changqing Chang: conceptualization; funding acquisition; resources; supervision; writing – review and editing. Enping Cai: conceptualization; project administration; data curation; funding acquisition; supervision; writing – review and editing.

Conflicts of Interest

The authors declare no conflicts of interest.

Supporting information

FIGURE S1: KEGG enrichment analysis of the upregulated differentially expressed genes. Wild‐type (MAT‐1 × MAT‐2) strains cultured on YePSA medium for 0, 24, and 72 h were collected for transcriptomic analysis. Bubble charts were used to visualise the enriched terms of the DEGs in the WT‐24 h vs. WT‐0 h and WT‐72 h vs. WT‐24 h group. A p‐value < 0.05 was set as the threshold for statistical significance. The false discovery rate (FDR) was < 0.05.

MPP-26-e70188-s001.tif (771.8KB, tif)

FIGURE S2: Phylogenetic and domain analysis of the phenylacetate hydroxylase SsPhacA from S. scitamineum. (a) The evolutionary history of PhacA proteins from various basidiomycetous and ascomycetous species was inferred using the Maximum Parsimony method. The phylogenetic maximum parsimony tree was obtained using the subtree‐pruning‐regrafting (SPR) algorithm with a search level of 0, in which the initial trees were obtained by the random addition of sequences (10 replicates). The analysis involved 23 amino acid sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7. (b) Domain analysis of the SsPhacA protein sequence was performed via the SmartBLAST tool (https://blast.ncbi.nlm.nih.gov/smartblast/).

MPP-26-e70188-s002.tif (1.1MB, tif)

FIGURE S3: Generation and verification of the ssphacAΔ‐1, ssphacAΔ‐2, ssphacAΔ/PHACA‐1, and ssphacAΔ/PHACA‐2 mutants. (a) PCR amplification was conducted using specific internal‐F/R or external‐F/R primers to verify the replacement of the targeted gene with the HYG R selectable marker. Molecular markers (in bp) are indicated. (b) RT‐qPCR was performed to analyse the transcription of SsPHACA. The strains were grown on YePSA medium for 24 h, followed by total RNA extraction; RT‐qPCR was then conducted with ACTIN as the internal reference gene. The experiment included three biological replicates, each with three technical replicates. The error bars represent the means ± standard errors of the means (SEMs). The statistical significance of the differences was evaluated via one‐way analysis of variance (ANOVA) followed by Bonferroni's multiple‐comparison test (★ p < 0.05; ★★★ p < 0.001; NS, not significant).

MPP-26-e70188-s007.tif (1,020.5KB, tif)

FIGURE S4: KEGG classification analysis of differentially abundant metabolites. Wild‐type (MAT‐1 × MAT‐2) and SsPHACA deletion mutant (ssphacAΔ‐1 × ssphacAΔ‐2) strains cultured on YePSA medium for 72 h were collected for differentially abundant metabolites analysis. The abscissa represents the percentage of the number of metabolites annotated under a certain KEGG pathway to the total number of annotated metabolites. The right ordinate shows the first‐level classification of the KEGG pathway, and the left ordinate shows the second‐level classification of the KEGG pathway.

MPP-26-e70188-s004.tif (324.2KB, tif)

FIGURE S5: The effect of SsPhacA on the phenylalanine metabolic pathway. The circles represent metabolites, among which the green solid circles represent annotated metabolites, the red circles represent upregulated differentially abundant metabolites, and the blue circles represent downregulated differentially abundant metabolites.

MPP-26-e70188-s003.png (49KB, png)

FIGURE S6: Exogenous supplementation of the decreased metabolites was performed to assess the sexual mating/filamentation of S. scitamineum. (a) Effect of different metabolites on sexual mating of the SsPHACA deletion mutant. Haploid cells of opposite mating types were mixed (MAT‐1 × MAT‐2 and ssphacAΔ‐1 × ssphacAΔ‐2) in equal volumes and spotted onto minimal medium with or without the addition of 10.0 μM L‐Ornithine, 4‐Guanidinobutanal, S‐adenosyl‐L‐methionine, L‐Methionine, L‐Proline, and L‐Phenylalanine. (b) Effect of different concentrations of p‐coumaric acid on sexual mating of the SsPHACA deletion mutant. Haploid cells of opposite mating types were mixed (MAT‐1 × MAT‐2 and ssphacAΔ‐1 × ssphacAΔ‐2) in equal volumes and spotted onto minimal medium with or without the addition of 0.1, 1.0, 100.0 μM p‐coumaric acid. All plates were incubated at 28°C for 18 h, and the results were photographed. The scale bar is 1.0 mm.

MPP-26-e70188-s006.tif (11.4MB, tif)

TABLE S1: Details of the strains used in this study.

MPP-26-e70188-s008.doc (38.5KB, doc)

TABLE S2: The primers and sequences used in this study.

MPP-26-e70188-s005.doc (59.5KB, doc)

Acknowledgements

This research was funded by the GDAS’ Project of Science and Technology Development (2022GDASZH‐2022010102), the National Natural Science Foundation of China (32200167), the Natural Science Foundation of Guangdong Province (2023A1515012440; 2024A1515010901). The funders had no role in study design, data collection, and interpretation, or the decision to submit the work for publication. In addition, we are very grateful to Professor Phillip Jcakson (Commonwealth Scientific and Industrial Research Organisation, Australia) for the helpful suggestions and the English revisions of the manuscript.

Xiong, B. , Zhang N., Guo Y., et al. 2025. “Phenylacetate Hydroxylase SsPhacA Modulates p‐Coumaric Acid Accumulation to Regulate the Mating/Filamentation of Sporisorium scitamineum .” Molecular Plant Pathology 26, no. 12: e70188. 10.1111/mpp.70188.

Bo Xiong and Nannan Zhang contributed equally to this work.

Contributor Information

Changqing Chang, Email: changcq@scau.edu.cn.

Enping Cai, Email: dlcep@foxmail.com.

Data Availability Statement

The authors confirm that data supporting this study's findings are available in the article and/or its Supporting Information. The data that support the findings of this study are available from the corresponding author upon reasonable request.

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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: KEGG enrichment analysis of the upregulated differentially expressed genes. Wild‐type (MAT‐1 × MAT‐2) strains cultured on YePSA medium for 0, 24, and 72 h were collected for transcriptomic analysis. Bubble charts were used to visualise the enriched terms of the DEGs in the WT‐24 h vs. WT‐0 h and WT‐72 h vs. WT‐24 h group. A p‐value < 0.05 was set as the threshold for statistical significance. The false discovery rate (FDR) was < 0.05.

MPP-26-e70188-s001.tif (771.8KB, tif)

FIGURE S2: Phylogenetic and domain analysis of the phenylacetate hydroxylase SsPhacA from S. scitamineum. (a) The evolutionary history of PhacA proteins from various basidiomycetous and ascomycetous species was inferred using the Maximum Parsimony method. The phylogenetic maximum parsimony tree was obtained using the subtree‐pruning‐regrafting (SPR) algorithm with a search level of 0, in which the initial trees were obtained by the random addition of sequences (10 replicates). The analysis involved 23 amino acid sequences. All positions containing gaps and missing data were eliminated. Evolutionary analyses were conducted in MEGA7. (b) Domain analysis of the SsPhacA protein sequence was performed via the SmartBLAST tool (https://blast.ncbi.nlm.nih.gov/smartblast/).

MPP-26-e70188-s002.tif (1.1MB, tif)

FIGURE S3: Generation and verification of the ssphacAΔ‐1, ssphacAΔ‐2, ssphacAΔ/PHACA‐1, and ssphacAΔ/PHACA‐2 mutants. (a) PCR amplification was conducted using specific internal‐F/R or external‐F/R primers to verify the replacement of the targeted gene with the HYG R selectable marker. Molecular markers (in bp) are indicated. (b) RT‐qPCR was performed to analyse the transcription of SsPHACA. The strains were grown on YePSA medium for 24 h, followed by total RNA extraction; RT‐qPCR was then conducted with ACTIN as the internal reference gene. The experiment included three biological replicates, each with three technical replicates. The error bars represent the means ± standard errors of the means (SEMs). The statistical significance of the differences was evaluated via one‐way analysis of variance (ANOVA) followed by Bonferroni's multiple‐comparison test (★ p < 0.05; ★★★ p < 0.001; NS, not significant).

MPP-26-e70188-s007.tif (1,020.5KB, tif)

FIGURE S4: KEGG classification analysis of differentially abundant metabolites. Wild‐type (MAT‐1 × MAT‐2) and SsPHACA deletion mutant (ssphacAΔ‐1 × ssphacAΔ‐2) strains cultured on YePSA medium for 72 h were collected for differentially abundant metabolites analysis. The abscissa represents the percentage of the number of metabolites annotated under a certain KEGG pathway to the total number of annotated metabolites. The right ordinate shows the first‐level classification of the KEGG pathway, and the left ordinate shows the second‐level classification of the KEGG pathway.

MPP-26-e70188-s004.tif (324.2KB, tif)

FIGURE S5: The effect of SsPhacA on the phenylalanine metabolic pathway. The circles represent metabolites, among which the green solid circles represent annotated metabolites, the red circles represent upregulated differentially abundant metabolites, and the blue circles represent downregulated differentially abundant metabolites.

MPP-26-e70188-s003.png (49KB, png)

FIGURE S6: Exogenous supplementation of the decreased metabolites was performed to assess the sexual mating/filamentation of S. scitamineum. (a) Effect of different metabolites on sexual mating of the SsPHACA deletion mutant. Haploid cells of opposite mating types were mixed (MAT‐1 × MAT‐2 and ssphacAΔ‐1 × ssphacAΔ‐2) in equal volumes and spotted onto minimal medium with or without the addition of 10.0 μM L‐Ornithine, 4‐Guanidinobutanal, S‐adenosyl‐L‐methionine, L‐Methionine, L‐Proline, and L‐Phenylalanine. (b) Effect of different concentrations of p‐coumaric acid on sexual mating of the SsPHACA deletion mutant. Haploid cells of opposite mating types were mixed (MAT‐1 × MAT‐2 and ssphacAΔ‐1 × ssphacAΔ‐2) in equal volumes and spotted onto minimal medium with or without the addition of 0.1, 1.0, 100.0 μM p‐coumaric acid. All plates were incubated at 28°C for 18 h, and the results were photographed. The scale bar is 1.0 mm.

MPP-26-e70188-s006.tif (11.4MB, tif)

TABLE S1: Details of the strains used in this study.

MPP-26-e70188-s008.doc (38.5KB, doc)

TABLE S2: The primers and sequences used in this study.

MPP-26-e70188-s005.doc (59.5KB, doc)

Data Availability Statement

The authors confirm that data supporting this study's findings are available in the article and/or its Supporting Information. The data that support the findings of this study are available from the corresponding author upon reasonable request.

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