N⁶-methyladenosine RNA methyltransferase CpMTA1 mediates CpAphA mRNA stability through a YTHDF1-dependent m⁶A modification in the chestnut blight fungus

Abstract

In eukaryotic cells, N⁶-methyladenosine (m⁶A) is the most prevalent RNA epigenetic modification that plays crucial roles in multiple biological processes. Nevertheless, the functions and regulatory mechanisms of m⁶A in phytopathogenic fungi are poorly understood. Here, we showed that CpMTA1, an m⁶A methyltransferase in Cryphonectria parasitica, plays a crucial role in fungal phenotypic traits, virulence, and stress tolerance. Furthermore, the acid phosphatase gene CpAphA was implicated to be a target of CpMTA1 by integrated analysis of m⁶A-seq and RNA-seq, as in vivo RIP assay data confirmed that CpMTA1 directly interacts with CpAphA mRNA. Deletion of CpMTA1 drastically lowered the m⁶A level of CpAphA and reduced its mRNA expression. Moreover, we found that an m⁶A reader protein CpYTHDF1 recognizes CpAphA mRNA and increases its stability. Typically, the levels of CpAphA mRNA and protein exhibited a positive correlation with CpMTA1 and CpYTHDF1. Importantly, site-specific mutagenesis demonstrated that the m⁶A sites, A¹³⁰⁶ and A¹³⁴¹, of CpAphA mRNA are important for fungal phenotypic traits and virulence in C. parasitica. Together, our findings demonstrate the essential role of the m⁶A methyltransferase CpMTA1 in C. parasitica, thereby advancing our understanding of fungal gene regulation through m⁶A modification.

Author summary

Numerous modifications have been found in eukaryotic mRNA, with m⁶A appearing to be the predominant modification. However, the biological roles and regulatory mechanisms of these m⁶A-modified enzymes are still largely unknown in filamentous fungi. Here, we characterized an m⁶A methyltransferase, CPMTA1, in C. parasitica using a combined analysis of multiple phenotypes and multi-omics. Based on our results, CpMTA1 plays a crucial role in fungal phenotypic traits, virulence, and stress tolerance. Furthermore, m⁶A-seq and RNA-seq identified the acid phosphatase gene CpAphA as a target of CpMTA1.The m⁶A methylation of CpAphA was verified by MeRIP-qPCR and MazF analysis. We also found that an m⁶A reader protein CpYTHDF1 could recognize CpAphA mRNA and increase its stability. Importantly, the m⁶A sites A¹³⁰⁶ and A¹³⁴¹ of CpAphA mRNA are involved in the regulation of fungal phenotypic traits and virulence in C. parasitica. These results provide a comprehensive insight into the function and mechanism of CpMTA1-mediated m⁶A modification in fungi, as well as improve our understanding of m⁶A-related studies.

Introduction

RNA modification is a post-transcriptional regulatory mechanism that influences RNA processing or metabolism [1]. N⁶-methyladenine (m⁶A) is the most common RNA methylation modification observed in diverse eukaryotic cells [2]. Three types of proteins dynamically and reversibly regulate m⁶A modification: m⁶A methyltransferases (“writers”), m⁶A demethylases (“erasers”), and m⁶A-binding proteins (“readers”) [3,4].

The m⁶A writer has been identified as a methyltransferase complex in many organisms, including mice, fruit flies, zebrafish, and humans. Typically, this complex consists of METTL3, METTL14, and additional protein subunits such as Zc3h13 or WTAP [5,6]. METTL3 is the primary m⁶A writer that assumes a pivotal catalytic function in methyl group transfer from the S-adenosylmethionine (SAM) to adenine [7]. Meanwhile, METTL14 facilitates substrate binding, while WTAP is necessary for RNA methylation and METTL3/14 localization [8]. Additionally, several other m⁶A methyltransferases have been identified, including METTL16, METTL5, ZCCHC4, and METTL4 [9]. METTL16 has been reported to methylate 3′ UTR of MAT2A mRNA thus regulating its mRNA expression [10]. METTL5 is an enzyme responsible for 18S rRNA m⁶A modification, while ZCCHC4 is involved in 28S rRNA modification [11]. The METTL4 protein acts as a methyltransferase in the regulation of pre-mRNA splicing by facilitating the m⁶A methylation of U2 snRNA [12]. Moreover, m⁶A erasers, such as AlkB homolog 5 (ALKBH5) and fat mass and obesity-associated protein (FTO), selectively remove the m⁶A modification from target mRNAs [13,14]. In addition, m⁶A recognizing proteins, also reported as readers, including the YTH homologous domain protein family, mediate mRNA degradation, splicing, and translation [15].

As a highly dynamic post-transcriptional regulation, m⁶A modification plays a significant role in various RNA metabolism processes, including mRNA structure, alternative splicing, processing, nuclear export, degradation, and translation [6]. For example, the exonic splicing of the adipogenic regulatory factor RUNX1T1 was regulated by FTO through modulating m⁶A levels of splice sites, thus influencing the process of differentiation [16]. YTHDC1, an m⁶A-binding protein, facilitates methylated mRNA transport from the nucleus to cytoplasm in HeLa cells [17]. YTHDF1 promotes RNA translation in the cytoplasm by interacting with initiation factors [18]. The mRNA degradation process is mediated by YTHDF2 through directly binding to m⁶A modification sites on LHPP and NKX3-1 [19].

Recent evidence suggests that m⁶A modification has been linked to multiple biological processes in various organism, including human circadian rhythms, stem cell differentiation, sex determination, viral replication, spermatogenesis, stress response, meiosis, obesity, cancer development and other diseases [6]. Moreover, an increasing number of studies have revealed essential roles for mRNA m⁶A modification in plant development, encompassing floral transition, trichome morphology, embryo development, shoot apical meristem proliferation, and fruit ripening [20]. The biological functions of m⁶A modification in fungi, however, have only been explored in a few species. In Saccharomyces cerevisiae, Ime4 (a homolog of mammalian methyltransferase METTL3) was identified as an essential component for m⁶A modification on yeast mRNA and regulates spore formation and meiosis, triglyceride metabolism, vacuolar morphology, mitochondrial morphological abnormalities, and dysfunction [21,22]. In the rice blast fungus Magnaporthe oryzae, the m⁶A writer MTA1, an orthologue of human METTL4, mediates m⁶A modification and regulates autophagy for fungal infection [5]. ALKB1 of M. oryzae was identified as an orthologue of m⁶A demethylase Alkbh1. YTH1 and YTH2 of M. oryzae were reported as orthologues of a human m⁶A reader protein. Knockout of these m⁶A-related genes in M. oryzae resulted in reduced virulence [23]. Two putative RNA m⁶A MTases (METTL3, METTL14) have been reported in Rhizophagus irregularis [24]. Recently, it was found that RNA modifications are associated with secondary metabolite biosynthesis of Aspergillus flavus [25]. Although these reports suggest that m⁶A modifications are important for fungi, the underlying mechanisms are largely unknown.

Cryphonectria parasitica, the causal agent of chestnut blight disease, almost destroyed American chestnut forests in the early 20th century [26]. Notably, some strains of the fungus contain a single-stranded, positive-sense RNA virus termed Cryphonectria hypovirus 1 (CHV1) that can serve as a biological control agent, as well as an important model to investigate virus-host interactions. After infection with CHV1, colonies of C. parasitica change from orange to white in colour, accompanied by a reduction in virulence, growth rate, sporulation, and female fertility [27,28]. The mechanisms of hypovirulence have been studied extensively through systemic analysis of transcriptome, proteome, metabolome, and DNA methylation, resulting in the identification of genes and pathways contributing to the phenotypes of interest [29–33]. Nevertheless, the regulatory mechanisms of reversible RNA methylation modification in C. parasitica have not yet been explored.

Our previous study found that CHV1 infection caused a significant decrease in the transcription level of CpMTA1, which encodes an m⁶A methyltransferase [33]. To understand the biological role of CpMTA1 in the chestnut blight fungus, we conducted a comprehensive analysis through the creation of CpMTA1 deletion mutants. Furthermore, the target genes mediated by CpMTA1 were screened by the combined analysis of m⁶A-seq and RNA-seq results. Mechanistically, the m⁶A methylation of C. parasitica acid phosphatases (CpAphA) was further verified by MeRIP-qPCR and MazF analysis, and the interaction between CpAphA mRNA and CpYTHDF (the m⁶A reader, a homolog of human YTHDF1) was proven using RNA immunoprecipitation. Importantly, the functional significance of the m⁶A modification sites on CpAphA was examined by constructing a corresponding deletion mutant and site-specific mutagenesis. Overall, our findings provide novel insights into the roles and mechanisms of CpMTA1-mediated m⁶A modification in fungi.

Results

Characterization of m⁶A methyltransferase from C. parasitica

Among the observed decreased changes in transcription level caused by CHV1 infection, a gene tentatively identified as an m⁶A methyltransferase was chosen for further analysis [33]. The CpMTA1 gene was characterized by exploration of the C. parasitica genome [28]. The coding region of the CpMTA1 gene is composed of two exons with 336 amino acid residues and one intron. The CpMTA1 protein was found to contain a conserved MT-A70 domain (152–323 aa) through domain analysis, which was initially recognized as the SAM-binding subunit of the human N⁶-adenosine-methyltransferase (MTase) and exhibits specific methylation activity towards adenines in pre-mRNAs [5]. A phylogenetic analysis was performed to further investigate the relationship between the CpMTA1 protein and its homologous sequences. The result showed that CpMTA1 is evolutionarily conserved in filamentous fungi, suggesting similar importance for other fungi (S1 Fig).

To investigate the biological function of the CpMTA1 in C. parasitica, we constructed the CpMTA1 knockout mutant using replacement with a hygromycin resistance gene (hph). The single-spored ΔCpMTA1 mutants were screened by PCR, then confirmed by Southern blot and RT-PCR (Fig 1A–1C). To confirm the observed phenotypes of the mutants were caused by the deletion of CpMTA1, the ΔCpMTA1 mutants were complemented by re-introducing a copy of the wild-type CpMTA1 gene (S2 Fig).

Fig 1. Deletion of CpMTA1 affects the m⁶A RNA methylation of Cryphonectria parasitica.

(A): Schematic diagram of the CpMTA1 gene deletion strategy. Fragment on the hph gene (probe A) and fragment on the right arm (probe B) were used to distinguish the fragment size of the wild-type strain and ΔCpMTA1 mutants in Southern blot. Scale bar = 1 kb. (B): The Southern blot analysis was conducted in ΔCpMTA1 mutants using probe A (left) and probe B (right). Fungal total DNAs were digested with Bgl II and separated in the agarose gel by electrophoresis, then blotted using probe A and probe B, respectively. (C): The CpMTA1 gene deletion was confirmed at the RNA level by RT-PCR. As an internal reference gene, 18S rRNA was used. (D): The relative amount of m⁶A/A in CpMTA1 mutant strains was calculated with RNA methylation quantitative kit. There are significant differences between samples indicated by different letters on the bars. (ANOVA followed by Tukey’s test, p < 0.05). (E): m⁶A dot blot analysis of m⁶A levels using a specific m⁶A antibody. RNA from different samples was extracted, spotted onto membranes, and incubated with an m⁶A antibody before being detected using the ECL system. Methylene blue staining served as a loading control.

It has been reported that METTL3 possesses a catalytic DPPW motif that enables its selective recognition of m⁶A [34]. According to active site prediction and amino acid sequence alignment, a conserved DPPW (D158-W161) motif was also identified on CpMTA1, suggesting that this may be necessary for the methyltransferase activity (S3 Fig). Consequently, we introduced mutations to convert the m⁶A recognition sites from DPPW to APPA. To further illustrate the structural basis for the mutation of CpMTA1, we employed a structure prediction software, AlphaFold2, to construct a 3D model structure of CpMTA1 and APPA mutant proteins using their respective amino acid sequences. We reanalyzed the structure of active sites in CpMTA1 and observed that substitutions of Asp158 and Trp161 to Ala would likely affect the formation of hydrogen bonds with surrounding residues and possibly influence the substrate recognition of the enzyme (S4 and S5 Figs). The genes with confirmed sequencing of CpMTA1 mutations were incorporated into the CpMTA1 deletion mutant to construct the catalytic inactive mutant ΔCpMTA1-com (D158A/W161A).

To determine whether CpMTA1 is responsible for RNA m⁶A methylation in C. parasitica, we compared the total m⁶A RNA methylation levels of the parent strain KU80, ΔCpMTA1, ΔCpMTA-com and ΔCpMTA1-com (D158A/W161A) (Fig 1D). The amount of m⁶A RNA in the ΔCpMTA1 mutant was 0.034 ± 0.009% of the total RNA, which was 26.98% of that in the parent strain KU80 (0.126 ± 0.011% of the total RNA), showing a significant reduction of m⁶A RNA modification. Compared with KU80, the m⁶A modification level was fully restored in the complemented strain ΔCpMTA-com, but not the catalytically inactive mutant (D158A/W161A). Similar results were obtained when an m⁶A dot blot assay was used. As shown in Fig 1E, the m⁶A RNA methylation level of ΔCpMTA1 was lower than that of KU80 strain at all detected RNA amounts. These results demonstrate that CpMTA1 appears to be involved in mRNA m⁶A methylation of C. parasitica.

CpMTA1 is necessary for fungal phenotypic traits, virulence, and stress tolerance

To study the roles of CpMTA1 in the mycelial growth and sporulation of C. parasitica, the mutants and WT strains were compared on PDA plates. The ΔCpMTA1 mutant exhibited a growth rate defect and displayed an irregular colony margin compared to the WT strain EP155 and parent strain KU80 (Fig 2A and 2B). Moreover, the deletion of CpMTA1 led to a significant decrease in sporulation (Fig 2C). These abnormal phenotypes could be successfully rescued by the complementation of the WT-CpMTA1, but not the catalytically inactive mutant (D158A/W161A). Additionally, we found that CpMTA1 is located in the nucleus, which is consistent with a previous report (Fig 2D) [35].

Fig 2. Phenotype analysis of CpMTA1 mutant strains.

(A): Colony morphology of the mutants on PDA medium. Photographs were taken at 7 and 14 days after inoculation. The wild-type EP155, parental strain KU80, CpMTA1 deletion strain ΔCpMTA1, complementary strain ΔCpMTA1-com, and mutant complementation strain ΔCpMTA1-com (D158A/W161A) were shown. (B): Mutant colony area were measured at day 7 and 14 post-inoculation. (C): Sporulation levels of the tested strains. Spores were calculated on day 14. There are significant differences between samples indicated by different letters on the bars (ANOVA followed by Tukey’s test, p < 0.05). (D): Subcellular localization of CpMTA1-GFP fusion protein. Images were taken using light microscopy and fluorescence microscopy, respectively. DAPI staining was performed to visualize the nuclei. Scale bar, 20 μm.

To further determine whether CpMTA1 is involved in fungal virulence, the virulence test was conducted on chestnut stems and apples. As shown in Fig 3, the WT strain EP155 and KU80 were highly virulent, while the hypovirus-infected strain EP155/CHV1-EP713 incited very small cankers. The result also showed a significant virulence reduction of the ΔCpMTA1 mutants. The ΔCpMTA1-com strain restored the virulence to resemble that of wild-type strains, but the site-specific mutant exhibited similar virulence to ΔCpMTA1.

Fig 3. Virulence assay of CpMTA1 mutant strains.

(A): Cankers were induced using the indicated strains on dormant stems of Chinese chestnut. The stems were inoculated and then maintained at 26°C, and the cankers were subsequently measured and photographed at 25 days post-inoculation. (B): Cankers were induced using the tested strains on Red Fuji apples. The apples were inoculated and kept at 26°C, and cankers were measured and photographed at 10 days post-inoculation. There are significant differences between samples indicated by different letters on the bars (ANOVA followed by Tukey’s test, p<0.05).

Furthermore, using oxidative stress (H₂O₂), osmotic stress (NaCl), or cell wall integrity stress (Congo red or SDS), the ΔCpMTA1 mutants were examined for stress tolerance. The ΔCpMTA1 strains displayed more sensitivity to both H₂O₂, SDS, and NaCl compared to EP155 and KU80 strains (S6 Fig), whereas no significant difference was found on Congo red. Besides, the hypovirus CHV1-EP713 was introduced into ΔCpMTA1 strains through anastomosis with EP155/CHV1-EP713 to investigate the effect of CpMTA1 deletion on hypovirus infection. Hypovirus-infected ΔCpMTA1 strains exhibited a decreased growth rate and reduced pigmentation. Analysis of viral dsRNA levels revealed comparable accumulation of CHV1-EP713 in both ΔCpMTA1/CHV1-EP713 and EP155/CHV1-EP713 (S7 Fig). Collectively, these results support that CpMTA1 plays a crucial role in fungal phenotypic traits, virulence, and stress tolerance. Moreover, the conserved active sites (D158/W161) are important in regulating CpMTA1 function in vivo.

Identification of m⁶A modification in C. parasitica through transcriptome-wide m⁶A-seq and RNA-seq assays

To reveal the mechanism of CpMTA1 in m⁶A modification of C. parasitica, we conducted transcriptome-wide m⁶A sequencing (m⁶A-seq) and RNA sequencing (RNA-seq) analysis using the parent strain KU80 and ΔCpMTA1 mutant. We compared the m⁶A peaks detected between m⁶A immunoprecipitated (IP) samples and the corresponding input samples as control. Firstly, we analyzed the m⁶A peaks identified in KU80 to determine the distribution of peaks across the genome in C. parasitica. m⁶A methylation shows a preference for genes with 50–60% CG content (Fig 4A) and longer transcript lengths (Fig 4B), compared to the overall genome of C. parasitica. Furthermore, motif analysis of enriched m⁶A peaks revealed that DRACH and RRACH (D = A, G, U; R = A, G; H = A, U, C) were the most frequently conserved consensus motifs (Fig 4C).

Fig 4. Characterization of m⁶A modification in the KU80 and ΔCpMTA1 mutant.

(A): Line plots displaying the percentage of CG content of all genes harboring m⁶A modifications identified through m⁶A-seq. (B): Line plots displaying the relative length of transcripts in all genes harboring m⁶A modifications. (C): Predominant consensus motif found using HOMER with m⁶A-seq peaks in KU80. Number of m⁶A peaks (D) and m⁶A-modified genes (E) identified by m⁶A-seq in KU80 and ΔCpMTA1. (F): Distribution of m⁶A peaks along the whole mRNA transcripts divided into the 5’UTR, CDS, and 3’UTR. (G): The proportions of m⁶A peaks distributed in the indicated regions in KU80 and ΔCpMTA1. (H): In KU80 and the ΔCpMTA1 mutant, genes are divided into two categories according to the number of m⁶A sites in each gene. m⁶A- gene (m⁶A site = 0), m⁶A+ gene (m⁶A site > = 1). (I): Cumulative distribution of mRNA expression changes between m⁶A-modified genes (m⁶A+) and non-modified genes (m⁶A-). **, p<0.01.

In all, m⁶A-seq found 4510 and 2997 m⁶A peaks from 3150 and 2257 m⁶A-modified genes in KU80 and ΔCpMTA1 samples, respectively (Fig 4D and 4E and S1 Table). Compared to KU80, 3285 peaks disappeared in ΔCpMTA1, and 1772 peaks appeared. The other 1225 peaks were found in both KU80 and ΔCpMTA1 strains. The CpMTA1 deletion mutant exhibits a noticeable decrease in the number of m⁶A peaks. Since CpMTA1 is an m⁶A methyltransferase, the 3285 KU80-unique peaks are expected to contain potential targets of CPMTA1. We then performed gene set enrichment analysis (GSEA) to investigate the potential downstream pathways regulated by CpMTA1. As shown in S8 Fig and S2 Table, CpMTA1 was positively correlated with metabolic pathways, amino acid biosynthesis, and oxidative phosphorylation. In contrast, a negative correlation was found between CpMTA1 and MAPK signalling pathway, meiosis, and cell cycle.

Furthermore, we investigated the m⁶A distribution patterns within KU80 and ΔCpMTA1. After segment normalization by the total length of each gene portion, the results showed that m⁶A peaks were mainly distributed in the coding sequences (CDS) and the 3′ terminate (near the stop codon) (Figs 4F, 4G and S9A and S3 Table). There was no notable difference in m⁶A peak distribution between the two strains. To further examine whether gene expression is related to m⁶A modification, the expression abundance with or without m⁶A modification was compared. We found that the expression levels of m⁶A-modified genes were significantly higher than those of non-m⁶A-modified genes in two strains (Fig 4H and 4I).

Combined analysis of m⁶A-seq and RNA-seq data of KU80 and ΔCpMTA1 samples

To investigate the impact of m⁶A on gene expression at the transcriptional level, we compared the abundance between KU80 and ΔCpMTA1 samples by performing differentially expressed gene (DEG) analysis. A total of 727 and 854 genes showed a significant increase and decrease, respectively (Fig 5A and S4 Table). To verify the RNA-seq results, a total of 21 DEGs were selected for qRT-PCR analysis. The Pearson correlation analysis between qRT-PCR and RNA-seq revealed that the values were consistent (correlation coefficient R² = 0.8607, p < 0.0001), confirming the reliability of the RNA-seq (Fig 5B). Furthermore, these DEGs were used to perform Gene Ontology (GO) enrichment analysis and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis (Fig 5C and 5D). In detail, the DEGs downregulated in ΔCpMTA1 compared to those in KU80 were significantly enriched in carbohydrate metabolic process, polysaccharide binding (GO), metabolic pathways, starch and sucrose metabolism, and inositol phosphate metabolism (KEGG). Compared to those in KU80, the upregulated genes were mostly involved in oxidation-reduction process, oxidoreductase activity (GO), glutathione metabolism, cysteine and methionine metabolism (KEGG).

Fig 5. Combined analysis of m⁶A-seq and RNA-seq data of the KU80 and ΔCpMTA1 samples.

(A): RNA-seq data from KU80 and ΔCpMTA1. The cutoff criteria for differentially expressed genes was log₂|FC|≥1 and FDR≤0.05 (red color indicates an increase and blue color represents a decrease in mRNA expression.) (B): Confirmation of the expression of 21 randomly selected DEGs by qRT-PCR analysis. Log₂FC was calculated from three samples. The coefficient of determination (R²) was displayed. (C): GO-based enrichment analysis of DEGs in terms of biological process (BP), cell component (CC) and molecular function (MF). (D): KEGG pathway enrichment analysis of DEGs. (E): A dot plot showing the log₂ (FC) of mRNA expression in relation to the log₂ (FC) of differential m⁶A level. (F): A nine-image map to display the distribution of genes with a significant change in both m⁶A level and mRNA expression level in ΔCpMTA1 compared to the control KU80. Yellow represents decreased m⁶A level and increased mRNA (hypo-up). Red represents increased m⁶A level and mRNA (hyper-up). Purple represents decreased m⁶A level and mRNA (hypo-down). Green represents increased m⁶A level and decreased mRNA (hyper-down).

Subsequently, the correlation between differentially methylated genes and their corresponding mRNA expression levels was assessed by combining m⁶A-seq and RNA-seq data. It was found that the m⁶A methylation level exhibited a significantly positive association with gene expression levels (Fig 5E). A total of 272 differentially methylated and differentially expressed genes (log₂|FC|≥1 and FDR≤0.05) were shown in the nine-quadrant diagram. Most of these genes were hypomethylated and downregulated (hypo-down, 150/272), and hypermethylated and upregulated (hyper-up, 98/272). In contrast, 18 genes were hypomethylated and upregulated (hypo-up). Only six genes showed hypermethylated and down-expression (hyper-down) (Fig 5F and S5 Table).

CpAphA is a direct target gene of CpMTA1

According to the result shown in Fig 4D, we identified 3285 KU80-unique m⁶A peaks covering 2516 genes. Overlap analysis of 2516 genes and RNA-seq data revealed 198 downregulated genes with lost m⁶A modification in ΔCpMTA1 mutant are expected to contain genuine targets of CpMTA1 (Fig 6A and S6 Table). KEGG pathway enrichment analysis of 198 downregulated genes suggested that many metabolic pathways were associated with m⁶A methylation, which involves starch and sucrose metabolism, pentose and glucuronate interconversions, ether lipid metabolism, and inositol phosphate metabolism (Fig 6B and S7 Table).

Fig 6. CpAphA is a direct target gene of CpMTA1.

(A): Venn diagram showing the numbers of overlapped genes between 2516 genes corresponding to KU80-unique peaks and RNA-seq data. (B): Enrichment of KEGG metabolic pathways in 198 genes with KU80-unique m⁶A peak and downregulated expression. (C): Verification of EP155/3×Flag-CpMTA1 strain by western blotting with an anti-flag antibody or anti-β-actin antibody (control). (D): RIP assay confirmed the association between CpAphA and CpMTA1. RIP assay was performed using anti-flag antibody in EP155/3×flag-CpMTA1 strain. β-actin was used as a negative control. The fold enrichment values were normalized to that of Input. (E): qRT-PCR verification of CpAphA expression in KU80 and ΔCpMTA1. There are significant differences between samples indicated by different letters on the bars (ANOVA followed by Tukey’s test, p<0.05). (F): Integrative genomics viewer (IGV) plots displaying m⁶A peaks in CpAphA transcript. Y-axis indicates normalized numbers of reads count. The black rectangles indicate that m⁶A peaks with significantly decreased m⁶A enrichment in ΔCpMTA1 compared to KU80. (G): Fold enrichment of IP/input for peaks in the window. (H): MeRIP-qPCR analysis of m⁶A levels of CpAphA. The mRNA of the KU80 and ΔCpMTA1 mutant were used for m⁶A-IP assay with an anti-m⁶A antibody. Subsequent MeRIP-qPCR was conducted using IP assay products as the template. The asterisk represents a statistically significant difference from KU80 (p<0.01). (I): Schematic diagram of MazF enzyme assay. Scissors represent endonuclease MazF which preferentially cleaves RNA at ACA sites but not at m⁶ACA sites. The MazF ACA site within the methylation site was highlighted in red. (J): PCR amplification of cDNA prepared from MazF-digested mRNA from KU80, ΔCpMTA1 and ΔCpMTA1-com. P1/P2, P3/P4, P5/P6 and P7/P8 primer locations relative to the probed methylation site were shown in (I). The control primers P9/P10, which do not flank an ACA site, were used as negative control.

Among these 198 genes, 80 have multiple m⁶A peaks (S9B Fig and S8 Table). Ries et al. [36] has demonstrated that m⁶A regulates the fate of cytosolic mRNA by interacting with m⁶A-binding protein. This interaction is particularly effective for polymethylated mRNAs, which can bind multiple m⁶A-binding proteins with high affinity. In contrast, singly methylated mRNA has low binding affinity and cannot mediate such functions effectively. Based on this information, the top six polymethylated mRNAs from S8 Table were selected for further RNA Immunoprecipitation (RIP) assay to investigate their potential targeting by CpMTA1. The results showed a substantial enrichment of CpAphA transcripts, but not the other five genes, indicating a direct binding of CpMTA1 to CpAphA mRNA (Figs 6C, 6D and S10). CpAphA was annotated to acid phosphatase (XP_040780407.1) in NCBI (S11 Fig). Therefore, it is suggested that CpAphA may be a target gene of CpMTA1. Subsequent qRT-PCR assays showed a significant decrease in the expression level of CpAphA mRNA upon deletion of CpMTA1 (Fig 6E), consistent with the RNA-seq results. Moreover, as shown in Fig 6F and 6G, CpAphA mRNA derived from KU80, but not ΔCpMTA1, harbors four m⁶A peaks in its CDS and 3′ UTR. We then performed methylated RNA Immunoprecipitation (MeRIP) followed by CpAphA-specific qPCR to verify the change of CpAphA m⁶A modification. As expected, the four CpAphA m⁶A peaks exhibited strongly decreased methylated levels in ΔCpMTA1, confirming that CpMTA1 is the responsible methyltransferase that methylates the CDS and 3′ UTR of CpAphA (Fig 6H).

To further identify exact methylation sites of four m⁶A peaks in CpAphA transcripts, a methylation-sensitive RNA restriction enzyme MazF was used, which cleaves RNA at ACA sites but not m⁶ACA sites [37]. The ACA sequence is part of the DRACA motif observed in the CpAphA m⁶A peak 1, 3, and 4 (Fig 6I). Through motif screening analysis, we speculated that A396, A1306, A1341, and A1666 may be key methylation modification sites, which were located in CpAphA m⁶A peak 1, 3, and 4. The purified mRNA from KU80, ΔCpMTA1, and ΔCpMTA1-com were digested with MazF and then reverse transcribed to synthesize cDNA, respectively. PCR amplification of the KU80 and ΔCpMTA1-com cDNA using primers P3/P4, P5/P6 and P7/P8 yielded a corresponding product, while no product was obtained from ΔCpMTA1 cDNA. Meanwhile, no product was obtained from both tested strains using P1/P2 primers for amplification. PCR with P9/P10 primers was used as negative control (Fig 6J). Thus, the A1306, A1341, A1666 are protected from MazF cleavage in mRNA prepared from KU80 and ΔCpMTA1-com. This result supports that CpAphA is methylated at the A1306, A1341, and A1666 positions, which are regulated by the methyltransferase CpMTA1.

CpMTA1 mediates the CpAphA mRNA stability through a CpYTHDF1-dependent m⁶A modification

Previous reports suggest that RNA m⁶A modifications affect mRNA stability [38,39], which may explain why CpAphA mRNA expression is reduced in ΔCpMTA1. Therefore, we assessed the CpAphA mRNA decay rate in KU80 and ΔCpMTA1 using the transcription inhibitor actinomycin D. As presented in Fig 7A, knockout of CpMTA1 significantly shortened the half-life of CpAphA mRNA, suggesting that CpMTA1-induced repression of CpAphA mRNA expression is at least in part due to the decreased stability of CpAphA mRNA. Furthermore, western blot assay revealed that the protein level of CpAphA was significantly decreased in ΔCpMTA1 (Fig 7B).

Fig 7. The stability of CpAphA mRNA is regulated by CpMTA1 via a CpYTHDF1-dependent m⁶A modification.

(A): The expression levels of CpAphA mRNA in KU80 and ΔCpMTA1 were analyzed by qRT-PCR at indicated time points after actinomycin D treatment. The mRNA half-life (t_1/2) of CpAphA was evaluated with a nonlinear regression model. (B): The protein expression of CpAphA in KU80 and ΔCpMTA1 were determined by western blotting with an anti-flag antibody or anti-β-actin antibody (control). (C): Verification of EP155/3×Flag-CpYTHDF1 strain by western blotting with an anti-flag antibody or anti-β-actin antibody (control). (D): RIP assay confirmed the association between CpAphA and m⁶A reader protein CpYTHDF1. RIP assay was performed using the anti-flag antibody in EP155/3×flag-CpYTHDF1 strain. The negative control was β-actin. The fold enrichment values were normalized to that of Input. (E): qRT-PCR analysis of CpAphA mRNA expression in KU80 and ΔCpYTHDF1. (F): The expression levels of CpAphA mRNA in KU80 and ΔCpYTHDF1 were determined by qRT-PCR at indicated time points after actinomycin D treatment and the decay rate of CpAphA was evaluated with a nonlinear regression model. (G): The protein expression of CpAphA in KU80 and ΔCpYTHDF1 was determined by western blotting with an anti-flag antibody or anti-β-actin antibody (control). There are significant differences between samples indicated by different letters on the bars (ANOVA followed by Tukey’s test, p < 0.05).

Meanwhile, m⁶A “readers” play an essential role in controlling the fate of the m⁶A marked mRNA [40]. To identify the specific m⁶A reader of CpAphA, we searched for the homologous protein in the C. parasitica genome database using human m⁶A reader YTHDF1/2/3 as a query, respectively. As a result, we identified a homologous protein of YTHDF1, but not for YTHDF2/3. We named the C. parasitica m⁶A reader protein (XP_040773278) as CpYTHDF1, which contains a YTH domain. Furthermore, RIP assays confirmed the direct interaction between the CpYTHDF1 and CpAphA mRNA in C. parasitica (Fig 7C and 7D). Additionally, CpAphA mRNA level was significantly decreased upon the deletion of CpYTHDF1, indicating that CpYTHDF1 is necessary for the stable expression of CpAphA (Fig 7E). Consistently, the half-life time of CpAphA mRNA was markedly shortened in ΔCpYTHDF1 compared with the KU80 (Fig 7F). Moreover, the mutant ΔCpYTHDF1 exhibited a notable decrease in CpAphA protein expression, in accordance with findings on mRNA stability (Fig 7G). Together, these findings suggest that CpMTA1 regulates the CpAphA mRNA stability through a CpYTHDF1-dependent m⁶A modification.

The m⁶A sites A¹³⁰⁶ and A¹³⁴¹ of CpAphA mRNA are important for fungal phenotypic traits and virulence in C. parasitica

To further explore the effect of the m⁶A methylation site of CpAphA on the phenotypic traits and virulence in C. parasitica, the CpAphA deletion mutant was constructed firstly. The single-spored transformants were screened by PCR and confirmed by Southern blot. Further, we successfully complemented a ΔCpAphA mutant by re-introducing a copy of the WT CpAphA gene (Fig 8A and 8B). Moreover, we mutated the m⁶A methylation sites of CpAphA by changing A to C and transformed the mutated genes into the ΔCpAphA mutant, respectively. Transformants ΔCpAphA-com (A¹³⁰⁶C), ΔCpAphA-com (A¹³⁴¹C) and ΔCpAphA-com (A¹⁶⁶⁶C) were confirmed and subsequently used for further analysis. As shown in Fig 8C and 8D, the growth rate and sporulation of the ΔCpAphA mutant were decreased compared to the EP155 and KU80 strains. As expected, the abnormal phenotypes were restored in the complemented strain ΔCpAphA-com. The ΔCpAphA-com (A¹³⁰⁶C) and ΔCpAphA-com (A¹³⁴¹C) mutants grew much more slowly on PDA plates and displayed reduced pigment and conidial spores, while the ΔCpAphA-com (A¹⁶⁶⁶C) mutant showed similar phenotypes as the WT and ΔCpAphA-com strains. Additionally, the mutation of A¹³⁰⁶C and A¹³⁴¹C resulted in a substantial decrease in mRNA expression and stability of CpAphA, while the mutation of A¹⁶⁶⁶C did not (Fig 8E and 8F). Similar changes were observed in the protein level of CpAphA with western blot analysis (S12 Fig).

Fig 8. Construction and phenotype analysis of CpAphA mutant strains.

(A): Schematic diagram of the CpAphA gene deletion strategy. Southern blot was performed to distinguish the wild-type strain and CpAphA knockout mutants using fragment on the hph gene (probe A) and fragment on the right arm (probe B). Scale bar, 1 kb. (B): Southern blot results of ΔCpAphA mutants with probe A (left) and probe B (right). Fungal total DNAs were digested with Hind III and Xho I then separated by electrophoresis, and then blotted using probe A and probe B, respectively. (C): Colony morphology of the mutants on PDA plates. Photographs were taken at 7 and 14 days post-inoculation. The wild-type EP155, parental strain KU80, CpAphA deletion strain ΔCpAphA, complementary strain ΔCpAphA-com, mutant complementation strains ΔCpAphA-com(A¹³⁰⁶C), ΔCpAphA-com(A¹³⁴¹C) and ΔCpAphA-com (A¹⁶⁶⁶C) were shown. (D): Sporulation levels were counted in the indicated strains. Spores were analyzed at 14 days post-inoculation. (E): qRT-PCR analysis of CpAphA mRNA expression in ΔCpAphA-com, ΔCpAphA-com(A¹³⁰⁶C), ΔCpAphA-com(A¹³⁴¹C) and ΔCpAphA-com (A¹⁶⁶⁶C). (F): The expression levels of CpAphA in strains were quantified by qRT-PCR at indicated time points after actinomycin D treatment and the decay rate of CpAphA was evaluated with a nonlinear regression model. There are significant differences between samples indicated by different letters on the bars (ANOVA followed by Tukey’s test, p < 0.05).

Furthermore, the virulence assay showed the ΔCpAphA, ΔCpAphA-com (A¹³⁰⁶C) and ΔCpAphA-com (A¹³⁴¹C) mutants incited significantly smaller cankers. The virulence of the ΔCpAphA-com strain showed similar virulence with the WT strain. Moreover, mutation of A¹⁶⁶⁶C had no obvious impact on virulence (Fig 9). These results indicate that CpAphA is responsible for fungal phenotypic traits and virulence of C. parasitica, and the methylation sites of A¹³⁰⁶ and A¹³⁴¹ play a crucial role in regulating the expression level and stability of CpAphA in vivo.

Fig 9. Virulence assay of CpAphA mutant strains.

(A): Cankers were induced by the indicated strains on dormant stems of Chinese chestnut. The stems were inoculated and then maintained at 26°C, and the cankers were subsequently measured and photographed at 25 days post-inoculation. (B): Cankers were induced by the tested strains on Red Fuji apples. The inoculated apples were kept at 26°C and cankers were measured and photographed at 10 days post-inoculation. There are significant differences between samples indicated by different letters on the bars (ANOVA followed by Tukey’s test, p < 0.05).

Transcriptomic insight into the regulatory role of CpAphA

To further investigate the regulatory role of CpAphA in C. parasitica, RNA-seq analysis was conducted for KU80 and ΔCpAphA strains. Hierarchical clustering of the RNA-seq data revealed significant differences in the heat map of mRNA expression patterns between KU80 and ΔCpAphA (S13A Fig). A total of 965 upregulated genes and 4018 downregulated genes were identified in ΔCpAphA compared with KU80 (S13B Fig and S9 Table), suggesting that CpAphA is a global regulator gene. Additionally, KEGG enrichment analysis showed that the upregulated genes were enriched in biosynthesis of amino acids, ABC transporters, and metabolic pathways, while downregulated genes were enriched in DNA replication, cell cycle, meiosis, and mismatch repair (S13C Fig). Furthermore, GO pathway enrichment analysis indicated that upregulated genes were mostly involved in oxidation-reduction process, carboxylic acid metabolic process, integral component of plasma membrane, plasma membrane part, and oxidoreductase activity; whereas downregulated genes were significantly involved in cell cycle DNA replication, nuclear DNA replication, DNA strand elongation, nuclear replication fork, symporter activity, and sugar transmembrane transporter activity (S13D Fig).

Discussion

Numerous modifications have been found in eukaryotic mRNA, with m⁶A appearing to be the predominant modification [41–43]. However, the biological roles and regulatory mechanisms of these m⁶A-modified enzymes are still largely unknown in filamentous fungi. In this study, we characterized an m⁶A methyltransferase, CPMTA1, in C. parasitica using a combined analysis of multiple phenotypes and multi-omics. Based on our results, CpMTA1 plays a crucial role in fungal phenotypic traits, virulence, and stress tolerance. Furthermore, integrated analysis of m⁶A-seq and RNA-seq identified CpAphA as a target of CpMTA1. We found that an m⁶A reader protein CpYTHDF1 could recognize CpAphA mRNA and increase its stability. Importantly, the m⁶A sites A¹³⁰⁶ and A¹³⁴¹ of CpAphA mRNA are involved in the regulation of fungal phenotypic traits and virulence in C. parasitica. The schematic model shown in Fig 10 summarizes our findings.

Fig 10. CpMTA1 mediates the CpAphA mRNA stability via a CpYTHDF1-mediated m⁶A modification.

The MT-A70 domain-containing family is considered to be the most prevalent m⁶A methyltransferases. These include Ime4 and Kar4 in yeast, as well as METTL3 and METTL14 in humans [21,44]. However, there was not found homologous proteins of human METTL3 and METTL14 in C. parasitica. Consistently, M. oryzae MTA1, exhibits orthology with human METTL4, not METTL3 [5]. Recently, Fusarium graminearum was discovered to contain MTA1, a homolog of METTL4, as a sole MT-A70-containing protein. Furthermore, species within Pezizomycotina, which is comprised entirely of filamentous fungi, may have experienced the loss of the m⁶A writers Ime4 and Kar4. In contrast, budding yeasts (Saccharomycotina) may have undergone the loss of the m⁶A writer MTA1 [45]. Here, we showed that the CpMTA1 protein has the conserved MT-A70 domain. Phylogenetic analysis also found that CpMTA1 is much more closely related to M. oryzae MoMTA1 and human METTL4 than to METTL3 (S1 Fig). As a subclade of the MT-A70 family, METTL4 is separated from the METTL3 and METTL14 subclades. Despite METTL4 being reported to be a U2 snRNA N⁶-adenosine methyltransferase in Drosophila and human [12], it is not clear whether METTL4 homologs can regulate m⁶A methylation of mRNA. Here, we found that the deletion of the CpMTA1 led to a notable reduction in overall m⁶A levels (Fig 1), and the m⁶A-seq analysis revealed that 3285 KU80-unique m⁶A peaks were lost in ΔCpMTA1 mutant (Fig 4), suggesting an important role for CpMTA1 in the m⁶A modification of mRNA in C. parasitica.

Our results found that the knockout strain ΔCpMTA1 still retains some level of m⁶A methylation (Fig 1), consistent with the result reported in M. oryzae [5]. Similarly, depletion of METTL3 and METTL14 is known to cause a ~60% reduction in m⁶A modification in some cell types [46]. These observations suggest the presence of additional m⁶A methyltransferases. More recently, METTL16 has also been identified as an m⁶A methyltransferase. Originally thought to target only a few transcripts, including 3 noncoding RNAs (U6 snRNA, MALAT1 and XIST) and one mRNA (MAT2A), METTL16 has now been shown to function as an m⁶A writer to deposit m⁶A into hundreds of specific mRNA targets [47]. It is a conserved protein with homologs found from vertebrates to yeast and bacteria [48]. Using human METTL16 as a query, we also identified a homologous protein XP_040778198 in C. parasitica, named CpMETTL16. Our preliminary results showed that knockout of CpMETTL16 gene resulted in a significant decrease in m⁶A abundance. It would also be interesting to investigate if CpMETTL16 could compensate for activity in ΔCpMTA1 in further studies.

Previous studies have reported that loss of the m⁶A methyltransferase can affect stem cell differentiation, animal and plant development, viral infection, spermatogenesis, sex determination, stress response, and cancer development [49]. The disruption of the MTA1 gene has a significant impact on vegetative growth, conidial formation, appressorium and pathogenicity in M. oryzae [5]. However, extensive efforts to generate homozygous MTA1 knockout were unsuccessful, indicating its essential function in F. graminearum [45]. In this study, the homozygous ΔCpMTA1 mutant was viable and showed a significant decrease in growth rate, sporulation, virulence, and stress tolerance (Figs 2 and 3). Because severe growth retardation of the CpMTA1 knockout strains was found, decreases in the virulence of ΔCpMTA1 may simply result from the growth defect. Therefore, if virulence determinants are defined as factors that exclusively impact virulence, CpMTA1 may not be considered a specific virulence factor but rather a vital gene for normal fungal growth and development. Consistently, the gene expression profile showed significant changes in response to the deletion of CpMTA1. Specifically, the loss of CpMTA1 resulted in a decrease in 854 gene expressions and an increase in 727 gene expressions (Fig 5). Among these genes, CpAphA was identified as a crucial target of CpMTA1. While our focus was on the regulatory role of CpAphA, it is possible that other target genes also contribute to fungal development and virulence of ΔCpMTA1. One of the downregulated genes was Glycoside hydrolase family 12 (xgeA), which has been linked to reduced virulence in Fusarium oxysporum when deleted or its enzyme activity lost [50]. Two other noteworthy differentially expressed genes are subtilisin-like protein (aorO) and citrate synthase (gltA). Previous reports have demonstrated that Prb1, a subtilisin-like protease, is essential for virulence and phenotypical traits in C. parasitica [51]. Additionally, deletion of citrate synthase led to a significant reduction in growth rate, sporulation, and virulence of C. parasitica [32], resembling the phenotype observed with ΔCpMTA1.

By m⁶A-seq analysis, we revealed that m⁶A peaks of C. parasitica were significantly enriched in RRACH and DRACH, which are the conserved m⁶A motif among mammals [52,53]. In comparison, m⁶A modifications in plants involve complicated sequence preferences, such as RRACH, URUAH, AAACCV (V means U, A, or G), and WKUAH (W means U or A; K means G or U) motifs [54]. The transcript-specific m⁶A localization has also been investigated at the transcriptome-wide level in C. parasitica. Our data showed that m⁶A peaks in C. parasitica transcriptome were abundant in the CDS region and near stop codons (Fig 4). Though m⁶A distribution around the stop codon or in the 3′ UTR are conserved among various plants and mammals, m⁶A deposition in the CDS region can also be found in some plants [54]. The m⁶A modification in strawberry are highly enriched in the CDS region adjacent to the start codon, besides the occurrence in the stop codon and 3′ UTR region [55]. Moreover, m⁶A modifications in pak-choi leaves and apple are most abundant in the CDS region, followed by the 3′ UTR region [56,57]. Therefore, the m⁶A distribution appears to differ among different species, suggesting that m⁶A modifications can mediate all stages of the mRNA life-cycle.

Correlation analysis of m⁶A-seq and RNA-seq data suggested that the m⁶A methylation level was significantly positively correlated with the gene expression level in C. parasitica (Fig 5). This observation was consistent with some previous studies, while it was different from others [58]. The difference may be attributed to the species types and collection of samples. For instance, m⁶A within the 3’ UTR or near the stop codon exhibits the capacity to decrease mRNA stability in normally growing maize seedling, tomato fruit, and strawberry fruit, while m⁶A enriching in the CDS region tends to increase mRNA stability in ripe strawberry fruit [59]. Coincidentally, the mRNA expression and stability of CpAphA were decreased in the ΔCpMTA1 mutant, proving that the m⁶A methylation positively mediates mRNA abundance.

Importantly, m⁶A modification can also influence RNA fate by m⁶A readers, which preferentially recognize and bind modified nucleotides. Currently, various m⁶A reader proteins have been reported in mammals, including the YTHDF family, YTH domain containing proteins (YTHDC1/2), IGF2 mRNA binding proteins (IGF2BP1/2/3), eukaryotic initiation factor 3 (eIF3), and heterogeneous nuclear ribonucleoproteins (HNRNP) family [60]. Among them, the YTHDF family proteins are the most studied m⁶A readers, located in the cytoplasm including YTHDF1, YTHDF2, and YTHDF3. By influencing translation and stability of target mRNAs, they affect the expression of downstream molecules, as well as various biological processes. However, the role of each protein is different, YTHDF1 improve RNA translation, and further promote its RNA stability, YTHDF2 facilitates RNA decay and YTHDF3 has a bidirectional regulation effect [61]. To identify the YTHDF homologous proteins in C. parasitica, the amino acid sequence of human YTHDF1/2/3 were used as a query for the BLASTp similarity search against the C. parasitica genome, respectively. Only one m⁶A reading protein was identified, which is homologous to human YTHDF1. Our data showed that the m⁶A reader protein CpYTHDF1 could recognize CpAphA mRNA, and further promote its RNA stability, which is consistent with the function of YTHDF1 in mammals. In contrast, YTH1 and YTH2 proteins that contain a YTH domain have been characterized in M. oryzae [23]. Protein sequence alignment confirmed that C. parasitica CpYTHDF1 is indeed closer to M. oryzae YTH1, but far from YTH2. Because m⁶A reading proteins in fungi are rarely reported, further studies are needed to demonstrate the function and regulatory mechanism of these proteins.

It was observed that the deletion of CpAphA gene resulted in a decrease in growth rate, sporulation, and pathogenicity, which was consistent with the ΔCpMTA1 strain (Fig 8). Therefore, CpMTA1 may regulate C. parasitica growth and virulence by downregulating CpAphA expression. CpAphA was annotated to a metallo-dependent phosphatase (XP_040780407.1) by NCBI. RNA-seq analysis of ΔCpAphA suggested that CpAphA is a global regulator through influencing multiple genes (S13 Fig). Previous evidence also indicates important functions of individual metallo-dependent protein phosphatases (PPM) isoform in signalling and cellular processes, including senescence, proliferation, apoptosis, and metabolism in animals [62] and plants [63]. Nevertheless, there is a lack of report regarding the regulatory effects of m⁶A modification on phosphatase protein. Further analysis of the detailed regulatory mechanisms of CpAphA will facilitate our understanding of the role of protein phosphatases in fungi.

Moreover, the methylation sites of A¹³⁰⁶ and A¹³⁴¹ of CpAphA, which are located in CDS region, were found to be essential for regulating the expression level and stability of CpAphA in vivo, but not in the 3’ UTR region (A¹⁶⁶⁶) (Fig 8). After analyzing the amino acid sequence, we found that the mutation at position A¹³⁰⁶C did not change its corresponding amino acid, while the mutation at position A¹³⁴¹C resulted in the substitution of Asn⁴¹⁵ with Thr. Through the functional domain and active site analysis of CpAphA protein (S11 Fig), we found that Asn⁴¹⁵ was located in the MPP-PAP domain, but not in the active site. Together, these data suggest that the mutation of A¹³⁰⁶ affects mRNA deposition rather than protein function, while the mutation of A¹³⁴¹ may affect mRNA deposition and protein function. Meanwhile, previous studies have reported that m⁶A “readers” are required in m⁶A-regulated diverse downstream signaling pathways [61]. Although our data showed that the m⁶A reader protein CpYTHDF1 could recognize CpAphA mRNA, the exact binding site has not been determined currently. We hypothesized that CpYTHDF1 does not recognize the m⁶A-modified A¹⁶⁶⁶ site, so mutating this site does not change the fate of CpAphA mRNA. It remains to be determined which specific regions of CpAphA mRNA are recognized by m⁶A reader in C. parasitica.

To our knowledge, this is the first study to demonstrate that CpAphA is a direct downstream target of CpMTA1-mediated m⁶A modification, revealing the mechanisms by which CpMTA1 can manipulate CpAphA and regulate fungal phenotypic traits and virulence. Together, the present findings provide a comprehensive insight into the function and mechanism of CpMTA1-mediated m⁶A modification in filamentous fungi, and expand the understanding of related studies.

Materials and methods

Fungal strains and culture conditions

Wild-type (WT) C. parasitica strain EP155 (ATCC 38755), its isogenic hypovirulent strain EP155/CHV1-EP713 (hypovirus CHV1-EP713 infected EP155, ATCC 52571)

[64], the highly efficient gene deletion strain KU80 (Δcpku80 of EP155) [65], and mutant strains were cultured on potato glucose agar (PDA, Difco) medium at 26°C with a 12 h light/dark cycle [66] for phenotypic analyses, DNA and RNA extraction. For protein extraction, liquid EP complete medium was used as described previously [67].

Construction of fungal mutants

The knockout of CpMTA1 gene was performed through homologous recombination method as described previously [65]. The upstream (983 bp) and downstream (927 bp) sequences of the CpMTA1 gene and the hygromycin B resistance marker (2145 bp) were amplified from C. parasitica genome DNA and plasmid pCPXHY2, respectively. These three fragments were joined by overlap extension PCR and the resulting fused cassette was transformed into KU80 strain using PEG-mediated protoplast transformation. The positive transformants were identified by PCR, RT-PCR and Southern blot analysis, followed by single-spore isolation to achieve nuclear homogeneity [68]. To complement the CpMTA1 knockout mutant strain, the entire open reading frame (ORF) of CpMTA1 gene, along with its promoter, was amplified using PCR and subsequently inserted into the pCPXG418 vector, which harboured the geneticin resistance gene (G418) [69]. The resulting vector pCPXG418-CpMTA1 was transformed into the knockout strain ΔCpMTA1. Complemented strains were then confirmed by PCR, qRT-PCR and Southern blot analysis. The Mut Express II fast mutagenesis kit V2 (Vazyme Biotech) was used to construct point mutation of CpMTA1 with pCPXG418-CpMTA1 as the template. Each mutant vector was verified by DNA sequencing and transformed into ΔCpMTA1.

To study the subcellular localization of CpMTA1, the CpMTA1 ORF was inserted into pCPXG418-GFP vector, and the recombinant plasmid pCPXG418-CpMTA1-GFP was transformed into ΔCpMTA1. A positive GFP-tagged strain was chosen for subcellular localization analysis on an Olympus BX51 fluorescence microscope. The construction of gene deletion, complementation, and point mutation of the CpAphA gene were performed using similar methods described above.

To construct strains expressing N-terminally 3×flag-tagged CpMTA1 and CpYTHDF1 fusion protein, CpMTA1 ORF and CpYTHDF1 ORF were cloned into a modified pCPXG418-3×flag vector, respectively. The correct pCPXG418-3×flag-CpMTA1 and pCPXG418-3×flag-CpYTHDF1 plasmid were transformed into the WT EP155 strain separately, and the expression of CpMTA1 and CpYTHDF1 was validated by PCR and western blotting. The construction of strains expressing C-terminally 3×flag-tagged CpAphA fusion protein were performed with a similar method. S10 Table lists the primer sequences used in this study.

Detection of methylation level of m⁶A RNA

Total RNA was extracted from C. parasitica by the MiniBEST Plant RNA Extraction Kit (Takara). The methylation level of m⁶A RNA was detected with the colorimetric EpiQuik m⁶A RNA methylation quantification kit (Epigentek, USA) according to the manufacturer’s protocol. After coating 200 ng RNA and the m⁶A standard onto the assay wells, capture and detection antibody solutions were sequentially added. Using a wavelength of 450 nm (OD₄₅₀), the absorbance of each well was measured to estimate m⁶A levels. The relative m⁶A RNA methylation levels were then calculated.

m⁶A dot blot assay

Total RNA was extracted from the KU80, ΔCpMTA1, ΔCpMTA1-com and ΔCpMTA1 (D158A/W161A), and spotted onto a Hybond-N+ membrane. The sample volume used was 1.5 μL, followed by UV crosslinking for 3 minutes at a strength of 1500 mJ/cm² after drying the membrane. Subsequently, the membrane was blocked in 5% milk phosphate buffered saline Tween (PBST) for 1 h before being incubated with an m⁶A antibody (EpiGentek) overnight at 4°C. After washing with PBST, the membrane was incubated with the secondary antibody for 1 h at room temperature. Finally, visualization was carried out using the ECL detection system (AI600 images).

Fungal phenotype and virulence analysis

The WT strain EP155, KU80, and EP155/CHV1-EP713 were compared to the constructed mutant strains in terms of phenotypic and virulent properties. Phenotypic changes in growth rate, conidiation and pigmentation were analyzed as previously described [31]. After the fungi were cultured on PDA for 14 days, the conidia from each sample were eluted and counted with a hemacytometer. To test the stress tolerance, PDA was used as the base medium supplemented with different chemical reagents (H₂O₂, SDS, NaCl and Cogo Red). To detect fungal virulence, the dormant Chinese chestnut (Castanea mollissima) stems or Red Fuji apple were inoculated with strain samples, respectively. Stems or apples were incubated at 26°C for 25 days or 10 days to allow lesion development. Finally, canker size was observed and analyzed [68].

m⁶A-seq analysis

The C. parasitica strain KU80 and ΔCpMTA1 were used for m⁶A-seq analysis, and three biological replicates were used for each strain. RNA was prepared from mycelial mats grown on PDA at 7 d post-inoculation. A total of 10 μg RNA was extracted for each sample and fragmented into short fragments (about 100 nt) using fragmentation buffer. A portion of untreated fragmented RNA was used for RNA-seq as input control. Another portion of fragmented RNA and anti-m⁶A antibody were incubated at 4°C for 2 h in IP buffer. Next, cDNA libraries were constructed from m⁶A-containing fragments (IP) and untreated input control fragments. The cDNA fragments were ligated to Illumina sequencing adapters by paired-end strategy. The m⁶A-seq and input RNA-seq were performed using Illumina HiSeq 4000 of Gene Denovo Biotechnology Co (Guangzhou, China).

The IP RNA-seq and input RNA-seq raw data were filtered by Fastp software (version 0.18.0), removing reads containing adapters and low-quality bases (Qvalue≤20) [56]. With HISAT2. 2.4 software [70], clean data from the IP and input samples were mapped to the C. parasitica reference genome (https://genome.jgi.doe.gov/portal/Crypa2/Crypa2.download.ftp.html). According to the background information (input RNA-seq data), exomePeak2 software [71] was used to identify m⁶A peaks with default settings. PeakAnnotator (version 2.0) was performed to annotate m⁶A peaks to the C. parasitica annotation file [72]. The exomePeak2 software was used to identify the differentially methylated peaks between the KU80 and ΔCpMTA1 with a criterion of log₂|FC|≥1 and FDR≤0.05.

RNA-seq analysis

The input sequencing reads from m⁶A-seq were used for RNA-seq analysis as described previously [73]. Using StringTie v1.3.1, mapped reads were assembled in a reference-based approach for each sample [74]. The FPKM (fragment per kilobase of transcript per million mapped reads) value was calculated to quantify gene expression level and variations by RSEM software [75]. Significant DEGs analyse were determined using DESeq2 [76], and defined by FDR≤0.05 and log₂|FC|≥1 in the two groups.

Quantitative real-time PCR

Total RNA was extracted from the strain samples using MiniBEST Plant RNA Extraction Kit (Takara), and then cDNA was produced using HiScript III RT SuperMix (Vazyme Biotech, R323). With the SYBR Green PCR Master Mix (Takara), quantitative real-time PCR (qRT-PCR) was performed on the LightCycler480II real-time PCR system (Roche).

Methylated RNA immunoprecipitation coupled with qRT-PCR

For methylated RNA Immunoprecipitation (MeRIP)-qPCR, the m⁶A RNA Enrichment Kit (Epigentek, USA) was used according to the manufacturer’s instructions. A total of 10 μg RNA was extracted for each sample, and reduced into fragments of 300 or fewer nucleotides. Magnetic beads containing 10 μg anti-m⁶A antibody or IgG were used to immunoprecipitate RNA samples. The IP or input assay products were then analyzed with qRT-PCR using primers (S10 Table).

RNA restriction enzyme MazF assay

RNase MazF (TaKaRa 2415A) was used to confirm the methylation site on CpAphA mRNA as described previously [77]. Here, 12.5 μg of total RNA was subjected to isolate poly(A) mRNA using VAHTS mRNA Capture Beads (Vazyme) and mixed with 4 μL MazF buffer, 0.5 μL RNase inhibitor and 1 μL MazF (TaKaRa). Then the 20 μL mixture was incubated at 37°C for 2 h. Next, RNA was resuspended in 12 μL RNase-free water, and cDNA was synthesized by HiScript III RT SuperMix (Vazyme Biotech, R323) kit. The resulting cDNA was employed in subsequent PCR reactions with special primers (S10 Table), and the products were analyzed by a TBE gel.

Western blot

Total fungal proteins were extracted with NP40 lysis buffer (Solarbio) as described previously [78]. After boiled with SDS loading buffer, the protein was separated using 12% SDS-PAGE and transferred to a PVDF membrane (Millipore, USA). Next, the membrane was blocked and incubated with anti-flag antibody (Abmart) or anti-β-actin antibody (ABclonal) at 4°C overnight. Finally, the membrane was detected using an ECL detection reagent (Coolaber, China).

RNA immunoprecipitation

The RIP assay was performed using the RNA Immunoprecipitation (RIP) Kit (BerSinBio, Bes5101) based on the provided directions. Briefly, the collected fungal cells were lysed with RIP lysis buffer on ice, and DNase was used to remove DNA. Then the cell lysate was immunoprecipitated with anti-flag antibody (1:100) at 4°C for 16 h, and incubated with protein A/G beads for 1 h. Finally, co-precipitated RNAs were detected using qRT-PCR. The primers used in this assay are shown in S10 Table.

mRNA stability detection

To determine the CpAphA mRNA stability in KU80 and ΔCpMTA1 strains, actinomycin D (GlpBio Technology, CA, USA) was added to the EP liquid medium to the final concentration of 20 μM. The mock control used was DMSO. At the indicated time point of 0 h, 8 h, and 24 h, fungal samples were collected for RNA extraction. CpAphA mRNA expression was detected by qRT-PCR [79].

Statistical analysis and reproducibility

All statistical differences among the samples were carried out by Tukey’s test and analysis of variance using IBM SPSS Statistics 22 software. All data are shown as mean ± SD with error bars. A p-value < 0.05 was considered statistically significant. Unless otherwise noted, all experiments were repeated three times.

Supporting information

S1 Data. Excel spreadsheet containing, in separate sheets, the underlying numerical data and statistical analysis for Figs 1D, 2B, 2C, 3A, 3B, 5B, 6D, 6E, 6H, 7A, 7D, 7E, 7F, 8D, 8E, 8F, 9A, 9B, S6 and S10.

(XLSX)

S1 Fig. Phylogenetic analysis, conserved domain, and sequence similarity comparison of CpMTA1 homologues from different organisms.

(A): Phylogenetic tree of MT-A70 domains orthologs from diverse species using MEGAX software analysis. (B): Conserved domain of CpMTA1 homologous proteins. The structural domains of these sequences were analyzed using the NCBI website (https://www.ncbi.nlm.nih.gov/Structure/bwrpsb/bwrpsb.cgi) and TBtools software. (C): Comparison of sequence similarity between CpMTA1 and other homologous proteins. The sequence similarity between CpMTA1 and other homologous proteins was determined using DNAMAN software.

(TIF)

S2 Fig. Construction and verification of ΔCpMTA1-com.

(A): Schematic diagram of the construction of CpMTA1 gene complement plasmid pCPXG418-com-CpMTA1. (B): Verification of the plasmid pCPXG418-com-CpMTA1 using EcoR I/Not I digestion. 1: The CpMTA1 gene. 2: The pCPXG418 plasmid after EcoR I/Not I digestion. 3: The pCPXG418-com-CpMTA1 plasmid. 4: The pCPXG418-com-CpMTA1 plasmid after EcoR V/ Not I digestion.

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S3 Fig. Analysis of conserved sites of CpMTA1.

(A): Prediction of conserved active sites by InterPro. (B): The sequence alignment of CpMTA1 and its orthologs was performed using CLC Genomics Workbench. The red boxes represent the conserved sites of CpMTA1.

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S4 Fig. The structure of CpMTA1 and its mutant protein was predicted using AlphaFold2.

Asp-158, Pro-159, Pro-160, and Trp-161 are highlighted in red (left). To analyze the amino acid residues at positions 158 and 161 before and after mutation, PyMOL v2.5.4 was used to create a hydrogen bond interaction map. Amino acids 158 and 161 are shown in blue, and hydrogen bonds are represented by yellow dashed lines (right).

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S5 Fig. The quality of predicted structure of CpMTA1 protein.

(A): Ramachandran plot of the predicted structure of wild-type CpMTA1 protein. (B): Ramachandran plot of the predicted structure of mutated CpMTA1 protein (APPA). (C): The predicted CpMTA1 structure by AlphaFold2 was compared with Arabidopsis METTL4 structure (PDB DOI: https://doi.org/10.2210/pdb7CVA/pdb) using PyMOL software. The root mean square deviation (RMSD) is 1.255, indicating that the similarity between the two proteins (Arabidopsis METTL4 is shown in red, CpMTA1 is shown in green).

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S6 Fig. Fungal strains were grown on PDA medium containing H₂O₂, SDS, NaCl, or Congo red.

(A): Colonies of the wild type, deletion mutant, complementation mutant and overexpression mutant were shown after 7 days of cultivation at 26°C. (B): Growth inhibition rate of strains by stressors, and colony diameter on PDA was set to 100%. All measurements were performed after 7 days of growth at 26°C and were performed in triplicate. Error bars represent the standard deviation. Different letters on the bars indicate significant differences (p < 0.05).

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S7 Fig. Colony morphology and viral RNA accumulation were examined in hypovirus-containing ΔCpMTA1 mutants.

(A): The colony morphology of hypovirus-free and hypovirus containing ΔCpMTA1 mutants was observed on PDA at day 7 post-inoculation. (B): Viral dsRNA accumulation was analyzed using agarose gel electrophoresis in hypovirus-containing ΔCpMTA1 mutants.

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S8 Fig. Gene set enrichment analysis (GSEA) of the potential downstream target genes of CpMTA1.

The top of the graph represents the enrichment score (ES): The ES value reflects the degree to which the members of the gene set are enriched at both ends of a sorted list. A positive ES indicates that the gene set is enriched at the top of the list, i.e., the pathway is activated or upregulated; while a negative ES indicates that the gene set is enriched at the bottom, i.e., the pathway is repressed or downregulated. The color of the curve is indicative of the color associated with the KEGG pathway listed on the right. The bottom of the graph shows the distribution of the rank values of all genes after sorting.

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S9 Fig. Analysis of m⁶A modification in the KU80 and ΔCpMTA1 mutant strains.

(A): Distribution of m⁶A peaks along the whole mRNA transcripts of C. parasitica detected in the KU80 strain and the ΔCpMTA1 mutant. (B): Number of the m⁶A-modified transcripts containing different m⁶A peak numbers in the KU80 strain and the ΔCpMTA1 mutant.

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S10 Fig. RIP assay detected the association between CpMTA1 and potential target genes.

RIP assay was performed using an anti-flag antibody in EP155/3×flag-CpMTA1 strain. β-actin was used as a negative control. The fold enrichment values were normalized to that of Input. The same letters on the bars mean no significant difference between samples (ANOVA followed by Tukey’s test).

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S11 Fig. Analysis of conserved domains of CpAphA protein in NCBI.

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S12 Fig. Expression of CpAphA protein was verified by western blotting using an anti-flag antibody. Anti-β-actin antibody was used for the control.

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S13 Fig. RNA-seq analysis identified transcripts affected by CpAphA deletion.

(A): Heatmap showing relative expression levels of genes in ΔCpAphA relative to KU80 strain based on the RNA-seq data. Cutoff criteria for differentially expressed genes included log₂|FC|≥1 and FDR≤0.05 (red color indicates an increase and blue color represents a decrease in mRNA expression.) (B): Volcano plot showing the DEGs between KU80 and ΔCpAphA. (C): KEGG pathway enrichment analysis of DEGs. (D): GO-based enrichment analysis of DEGs in terms of biological process (BP), cell component (CC) and molecular function (MF).

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S1 Table. All m⁶A peaks of transcripts identified by m⁶A-seq.

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S2 Table. GSEA analysis of genes with unique m⁶A peak in KU80.

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S3 Table. Distribution of m⁶A peaks along the whole mRNA transcripts.

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S4 Table. Differentially expressed genes in KU80 and ΔCpMTA1.

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S5 Table. Genes with a significant change in both m⁶A level and mRNA expression in CpMTA1 deletion strain compared to the control KU80.

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S6 Table. Analysis of the genes with unique m⁶A peak in KU80 and down-regulated expression in ΔCpMTA1, or up-regulated expression in ΔCpMTA1.

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S7 Table. KEGG pathway enrichment analysis of the genes with unique m⁶A peak in KU80 and down-regulated expression in ΔCpMTA1.

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S8 Table. The number of m⁶A peaks on genes with unique m⁶A peak in KU80 and down-regulated expression in ΔCpMTA1.

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S9 Table. Differentially expressed genes in KU80 and ΔCpAphA.

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S10 Table. Primers used in this study.

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Data Availability

All relevant data supporting the findings of this study are available within the article or supplementary information. Raw data of the m6A-seq and RNA-seq were submitted to the sequence read archive (SRA) database with a combined accession no. (SRP475797). The direct link is https://www.ncbi.nlm.nih.gov/sra/?term=SRP475797.

Funding Statement

This work was supported by the National Natural Science Foundation of China (32160623 to R.L., https://www.nsfc.gov.cn), Innovation Project of Guangxi Graduate Education (YCBZ2022032 to L.Z., https://keyanyun.myclub2.com/) and Guangxi Natural Science Foundation (2021GXNSFAA196036 to R.L., https://gkg.kjt.gxzf.gov.cn/).The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.