m⁶A demethylase CpALKBH regulates CpZap1 mRNA stability to modulate the development and virulence of chestnut blight fungus

ABSTRACT

As the most abundant eukaryotic mRNA modification, N⁶-methyladenosine (m⁶A) plays a crucial role in regulating multiple biological processes. This methylation is regulated by methyltransferases and demethylases. However, the regulatory role and mode of action of m⁶A demethylases in fungi remain poorly understood. In this study, we demonstrate that CpALKBH is a demethylase in Cryphonectria parasitica that removes m⁶A modification from single-stranded RNA in vitro. The deletion of CpALKBH resulted in a significant increase in the m⁶A methylation levels, along with decreases in the growth rate, sporulation, and virulence in C. parasitica. Additionally, CpZap1—a transcription factor—was identified as a downstream target of CpALKBH demethylase based on RNA sequencing analysis. We confirmed that CpALKBH demethylase regulates CpZap1 mRNA stability in an m⁶A-dependent manner. Furthermore, through MazF assay, we found that methylation of CpZap1 at position 1935A is regulated by both CpALKBH demethylase and CpMTA1 methyltransferase. CpZap1 significantly influences the fungal phenotype and virulence, thereby restoring the abnormal phenotype observed in ∆CpALKBH mutants. Collectively, our findings highlight the essential role of CpALKBH as an m⁶A demethylase in the development and virulence of C. parasitica, while also elucidating the molecular mechanisms through which m⁶A modification impacts CpZap1 mRNA stability.

IMPORTANCE

N⁶-methyladenosine (m⁶A) is the most abundant eukaryotic mRNA modification and is involved in various biological processes. Methyltransferases and demethylases regulate the m⁶A modification, but the regulatory role of m⁶A demethylases in fungi remains poorly understood. Here, we demonstrated that CpALKBH functions as a demethylase in Cryphonectria parasitica. The deletion of CpALKBH leads to a significant increase in m⁶A levels and a reduction in fungal growth, sporulation, and virulence. We identified CpZap1 as a downstream target of CpALKBH, with CpALKBH regulating CpZap1 mRNA stability in an m⁶A-dependent manner. Additionally, our findings indicate that methylation at position 1935A of CpZap1 is regulated by both the CpALKBH demethylase and the CpMTA1 methyltransferase. Given its critical role in fungal development and virulence, overexpression of CpZap1 can rescue abnormal phenotypes of ∆CpALKBH mutant. Overall, these findings contribute to improving our understanding of the role of m⁶A demethylase in fungi.

INTRODUCTION

RNA modification, a key mechanism of epigenetic regulation, is widely observed at the transcriptome level. Among these modification, N⁶-methyladenosine (m⁶A) is the most abundant and functionally important internal modification in mRNAs, initially identified in eukaryotic cells in 1974. Other prominent modifications include N6, 2′-O-dimethyladenosine (m⁶Am), N1-methyladenosine (m¹A), 2′-O-methylation (2′-OMe), and 5-methylcytosine (m⁵C) (1). Specifically, m⁶A refers to the methylation occurring at the N6 position of adenosine. In contrast, m⁶Am is a terminal modification found at the second base next to the 5′ cap in many mRNAs, where it is 2′-O-methylated and further methylated at the N6 position (2). Similar to m⁶A, adding a methyl group to the N1 position of adenosine creates m¹A modification (3). Among them, m⁶A is the predominant internal modification in eukaryotic mRNA, representing 80% of RNA methylation (4–6). This modification regulates gene expression by various methods, including alternative splicing, localization, transportation, translation, degradation, and stability (7–10). m⁶A modification modulates a wide range of biological processes, such as stem cell differentiation, development in both animals and plants, responses to stress, viral infections, sex determination, cancer progression, and immune responses. Additionally, its significance has been demonstrated in various aspects of plant development, including flowering, trichome development, embryo growth, and fruit ripening (11). Therefore, m⁶A modification holds significant importance in eukaryotes (12, 13).

The dynamic and reversible process of m⁶A modification, involving RNA methyltransferases (writers), demethylases (erasers), and m⁶A-binding proteins (readers) (14), is crucial for post-transcriptional regulation in various kingdoms. The m⁶A demethylases, including fat mass and obesity-associated protein (FTO) demethylase and alpha-ketoglutarate-dependent dioxygenase alkB homolog 5 (ALKBH5) demethylase, catalyze the removal of m⁶A methylation. Both these proteins—members of α-ketoglutarate (α-KG)-dependent dioxygenase family—facilitate m⁶A demethylation through Fe²⁺ and α-KG-dependent mechanisms (6). FTO demethylase, identified as the first confirmed m⁶A demethylase, catalyzes the demethylation of various substrates. These include m⁶A and cap m⁶Am in mRNA, m¹A in tRNA, m⁶A and m⁶Am in snRNA (15). However, it has been observed that internal m⁶A modifications in mRNA serve as major substrates for FTO demethylase in various cell types (16). Distinct from FTO demethylase, ALKBH5 is an m⁶A-specific demethylase as it displays no activity toward m⁶Am. Recent reports have found that m⁶A demethylases play critical roles in various developmental processes and human diseases (17). For instance, FTO demethylase contributes to oncogenesis in acute myeloid leukemia, breast cancer, and melanoma via posttranscriptional regulation (18). This role is facilitated by targeting critical transcripts, including ASB2 and RARA (19). In mammals, m⁶A modification can also be affected by the silencing or overexpression of ALKBH5 demethylase. The ALKBH5 was found to be required for mouse fertility, immunity inflammation, and the progression of cancer (17, 20). Additionally, a knockout of Arabidopsis m⁶A demethylase ALKBH9B has been found to negatively regulate virus accumulation and systemic invasion, linked to higher m⁶A levels in viral RNA (21).

Contrary to mammals and plants, few studies have explored the role of m⁶A modification in fungi. METTL3 is the primary m⁶A writer that transfers the methyl group from S-Adenosylmethionine (SAM) to N⁶ adenine (22). IME4, a METTL3 homolog in Saccharomyces cerevisiae is crucial for triglyceride metabolism, meiosis, mitochondrial impairment, and vacuolar morphology (23). In addition, MTA1—the m⁶A writer—in Pyricularia oryzae has been found to mediate m⁶A modification and regulate autophagy during fungal infection. PoALKB1 in P. oryzae was also reported as an ortholog of human demethylase ALKBH1 (24, 25). Moreover, the functional significance of m⁶A methylation in the biosynthesis of aflatoxin in Aspergillus flavus has been investigated. Notably, m⁶A site A332 of the aflatoxin biosynthetic pathway gene aflQ has been found to significantly influence aflatoxin production both on culture media and on crop kernels (26). However, recent studies have not sufficiently explored the biological significance of m⁶A demethylase and m⁶A modification in fungal development and virulence.

Here, to advance our knowledge of the role of RNA methylation in phytopathogenic fungi, we focused on Cryphonectria parasitica—an important plant pathogen that is destructive to chestnut forests. C. parasitica has been widely used as a model filamentous fungus to explore host-virus interactions and fungal pathogenicity due to its ability to support the replication of diverse mycoviruses (27). Various transcriptomic, proteomic, metabolomic, and DNA methylomic studies have unveiled a series of growth- or virulence-related genes (28–32). However, the impact of mRNA methylation/demethylation in the modulation of fungal traits has not been reported in C. parasitica.

This study identified a fungal protein exhibiting m⁶A demethylase activity (CpALKBH) in C. parasitica and investigated its biological functions by constructing CpALKBH deletion mutants. Additionally, we screened putative genes targeted by CpALKBH using RNA sequencing (RNA-seq). Furthermore, the association between the transcription factor CpZap1 mRNA and CpALKBH demethylase was confirmed by RNA immunoprecipitation (RIP). The m⁶A methylation of CpZap1 was also verified through methylated RNA immunoprecipitation (MeRIP) RT-qPCR and MazF analyses. Moreover, CpMTA1 was found to be the methyltransferase that catalyzes CpZap1 m⁶A modification. The functional significance of CpZap1 in C. parasitica was also elucidated by constructing gene deletion mutant and RNA-seq analysis. In summary, our results offer new insights into the functions and mechanisms of CpALKBH-mediated m⁶A demethylation in filamentous fungi.

RESULTS

Characterization of m⁶A demethylase CpALKBH and its role in the development and virulence of C. parasitica

To identify the homologous protein of m⁶A demethylase ALKBH5 in the C. parasitica genome (33), the amino acid sequence of Homo sapiens ALKBH5 (NP_060228.3) was used to perform a BLASTp search against the C. parasitica genome (taxid: 660469) available on the NCBI database. This led to the identification of an α-KG-dependent dioxygenase homologous protein (NCBI accession: XP_040772748) in C. parasitica—CpALKBH. CpALKBH comprises three exons and two introns, encoding a 350-amino acid long protein. Evolutionary phylogenetic relationship analysis showed that the CpALKBH protein is more closely related to homologous proteins of fungi but separated from those of mammals, and it is classified within the ALKBH subfamily of the Fe (II)/2-oxoglutarate (2OG) dioxygenase superfamily (Fig. 1A). The predicted three-dimensional structure of CpALKBH encompasses a highly conserved double-stranded beta-helix fold, a characteristic feature of the α-KG-dependent dioxygenase superfamily and essential for its catalytic core function (34) (Fig. 1B). Furthermore, we found that the purified CpALKBH protein could remove m⁶A modification of the synthesized RNA probe (containing a GAACA motif) (Fig. 1C and D). This indicates that CpALKBH is a protein identified in C. parasitica as RNA m⁶A demethylase activity.

Fig 1.

Characterization of the m⁶A demethylase CpALKBH in C. parasitica. (A) Phylogenetic tree of CpALKBH orthologs from diverse species using MEGAX software analysis. Conserved domain of CpALKBH 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. The sequence similarity between CpALKBH and its homologous proteins was analyzed using DNAMAN software for comparison. (B) CpALKBH protein structure prediction using AlphaFold2. The predicted DSBH domain of CpALKBH (222–345 aa) is labeled in red. (C) The purified CpALKBH protein was analyzed by SDS-PAGE. M shows molecular mass marker; lane 1 shows uninduced total proteins, lane 2 shows IPTG-induced total proteins, lane 3 shows supernatant proteins; lane 4 shows precipitate proteins; lane 5 shows purified CpALKBH proteins. (D) Demethylation of m⁶A in ssRNA by CpALKBH was demonstrated in vitro using RNA dot blot assays. The red-marked part represents the m⁶A motif. Different quantities of ssRNA were used for dot blot assay (200 ng, 400 ng, and 800 ng). The heat-inactivated CpALKBH protein did not have enzymatic activity and was used as a control. The loading control was performed by staining with Methylene blue (MB).

To explore the biological role of CpALKBH in C. parasitica, the CpALKBH deletion mutant was generated by replacing the CpALKBH gene with a hygromycin-resistant gene. The single-spored ΔCpALKBH mutants were verified through PCR, qRT-PCR, and Southern blotting analysis. To ensure that the phenotypic changes were actually caused by the deletion of CpALKBH, we complemented ΔCpALKBH mutants in trans with a wild-type allele of CpALKBH (Fig. 2A through C; Fig. S1).

Fig 2.

The deletion of CpALKBH alters the m⁶A RNA methylation level in C. parasitica. (A) CpALKBH gene deletion strategy in schematic form. Southern blotting used a fragment from the probe A (hph gene) and the probe B (a fragment from the right arm) to compare the fragment sizes between the KU80 strain and CpALKBH deletion mutants. The scale bar represents 1 kb. (B) Using probe A and B, Southern blotting analysis was conducted on ΔCpALKBH mutants and KU80. Genomic DNA from fungi was digested with Bsp1407I, electrophoresed on agarose gels, then probed with probe A and probe B. The strains used were EP155 (wild-type), KU80 (parental strain), ∆CpALKBH (CpALKBH deletion strain), and ∆CpALKBH-com (complementary strain). EP155 and KU80 were used as control. (C) The deletion of the CpALKBH gene was verified using RT-PCR, with the 18S rRNA gene serving as an internal control. (D) Methylation level of m⁶A RNA was determined with an RNA methylation detection kit. A standard deviation from three independent experiments is shown with the error bars. Treatment differences are indicated by different letters above the bars. (ANOVA followed by Tukey’s test, P < 0.05).

To examine whether CpALKBH is responsible for RNA m⁶A demethylation in C. parasitica, global m⁶A methylation levels were quantified by ELISA in all the RNA samples extracted from the control strain KU80, ΔCpALKBH, and ΔCpALKBH-com. In the ΔCpALKBH mutant, the m⁶A RNA content reached 0.256% ± 0.001% of the total RNA, representing a 1.95-fold increase compared to the KU80 strain (0.087% ± 0.006% of the total RNA), highlighting a marked rise in m⁶A modification. The m⁶A modification level returned to normal in the complemented strain ΔCpALKBH-com (Fig. 2D). These results demonstrate that CpALKBH is involved in the m⁶A demethylation of C. parasitica.

To investigate the function of CpALKBH, the growth patterns of mutants were compared with the wild-type strains. The growth rate of ΔCpALKBH was slower than that of EP155 and KU80 strains, and its colony margin was irregular (Fig. 3A). Additionally, the CpALKBH deletion significantly reduced sporulation in the mutant compared to the wild-type strain (Fig. 3B). Furthermore, the abnormal phenotype of the knockout mutant was fully restored in the complementation strain.

Fig 3.

Analysis of phenotype, sporulation, and virulence of ΔCpALKBH mutant strains. (A) Photographs of mutant colonies were taken at 7 and 14 days post-inoculation to assess colony morphology. The strains displayed included EP155, KU80, ΔCpALKBH, and ∆CpALKBH-com. (B) The sporulation levels of the indicated strains were measured on day 14. (C) The dormant Chinese chestnut stems were inoculated with the tested strains and kept at 26°C for 25 days. (D) The Red Fuji apples were inoculated with the tested strains at 26°C for 10  days. A standard deviation from three independent experiments is shown with the error bars. Treatment differences are indicated by different letters above the bars. (ANOVA followed by Tukey’s test, P < 0.05).

To further investigate whether CpALKBH is essential for the virulence of C. parasitica, chestnut stems and red Fuji apples were used. The hypovirulent strain EP155/CHV1-EP713 was used as the control. Compared to that of EP155 and KU80 strains, the virulence of ∆CpALKBH was significantly decreased. The complemented strain ∆CpALKBH-com restored the mutant virulence to a level comparable to that of the parent strain (Fig. 3C and D). The findings collectively substantiate the critical role of CpALKBH in fungal development and virulence.

Transcriptomic analysis reveals the regulatory function of CpALKBH

To further elucidate the regulatory role of CpALKBH, the mycelial samples of KU80 and ΔCpALKBH strains were collected 7 days post-inoculation for transcriptome sequencing (RNA-seq). A total of 37.5–43.8 million and 43.3–48.9 million reads were obtained for KU80 and ΔCpALKBH strains, respectively. After removing adapter sequences and low-quality reads, 37.3–43.5 million and 43.1–48.7 million reads were used for further analysis (Table S1). Hierarchical clustering of RNA-seq data revealed notable differences in the mRNA expression heat maps between the KU80 and ΔCpALKBH groups (Fig. 4A). RNA-seq database identified 954 upregulated genes and 1,883 downregulated genes in ΔCpALKBH compared to KU80 strain [log₂(fold change) >1, P < 0.05] (Fig. 4B; Table S2). The reliability of the RNA-seq data was verified using qRT-PCR analysis of 10 randomly selected DEGs. A strong correlation (correlation coefficient R² = 0.9791, P < 0.001) between RNA-seq and qRT-PCR results further confirmed their consistency (Fig. 4C; Table S2). As shown in Fig. 4D and E, these DEGs were subjected to gene ontology (GO) enrichment and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway analysis. Specifically, the DEGs downregulated in the ΔCpALKBH group were significantly associated with KEGG pathways such as pentose and glucuronate interconversions, starch and sucrose metabolism, methane metabolism, amino sugar and nucleotide sugar metabolism, and GO terms like polysaccharide catabolic process and extracellular region. In comparison to the KU80 strain, the upregulated genes in the ΔCpALKBH group were primarily associated with amino acid biosynthesis, 2-oxocarboxylic acid metabolism, and ABC transporters (KEGG), as well as alpha-amino acid metabolism, small-molecule biosynthesis, and the sulfite reductase complex (NADPH) (GO).

Fig 4.

RNA-seq analysis identified transcripts affected by CpALKBH. (A) RNA-seq heatmap showing relative gene expression in the ΔCpALKBH compared to the KU80. (B) The DEGs between the KU80 and ΔCpALKBH strains were shown with a Volcano plot. mRNAs log₂FC > 1 in ΔCpALKBH relative to KU80 (P value < 0.01) were highlighted in red. mRNAs log₂FC < −1 in ΔCpALKBH relative to KU80 (P value < 0.01) were highlighted in blue. (C) The expression of 10 randomly chosen DEGs was validated through RNA-seq and qRT-PCR analysis. From three biological samples, Log₂FC values and the coefficient of determination (R²) were calculated. (D) DEG enrichment analysis based on biological process (BP), cellular component (CC), and molecular function (MF). (E) Analyses of DEGs enriched for KEGG pathways. DEG, differentially expressed gene.

CpALKBH regulates the stability of CpZap1 mRNA through an m⁶A-dependent manner

To identify the downstream targets of CpALKBH effectively, we overlapped the above RNA-seq data with previously obtained m⁶A-seq results of the KU80 strain (accession no. SRP475797). The m⁶A-seq identified 4510 m⁶A peaks from 3150 m⁶A-modified genes in the KU80 strain. By overlapping m⁶A-modified genes and DEGs, we identified 184 upregulated genes and 423 downregulated genes with m⁶A modification, which are potential CpALKBH targets (Fig. 5A). Considering that transcription factors are regulated by RNA demethylases in an m⁶A-dependent manner (35), we conducted a screening of 423 downregulated genes and identified a single transcription factor, CpZap1, for further investigation (Table S3). CpZap1 was annotated with a classical C2H2 zinc finger domain (XP_040772497.1) through the NCBI database, which was well conserved among different organisms (Fig. S2). C2H2 transcription factors are a major family of fungal zinc finger regulators found in all eukaryotes. They primarily regulate fungal development, stress tolerance, and metabolism in plant-pathogenic fungi (36). To assess whether CpZap1 has transactivation activity, the entire CpZap1 cDNA was cloned into a pGBKT7 vector and introduced into Y2HGold yeast cells. Yeast cells transformed with the fusion plasmid (pGBKT7-CpZap1) or positive control grew on the SD/-Trp/-Leu/-His/-Ade medium, whereas yeast cells transformed with the negative control did not grow (Fig. S3A). Additionally, subcellular localization showed that CpZap1 was located in the nucleus (Fig. S3B). These findings indicate that CpZap1 acts as a transcription factor.

Fig 5.

CpALKBH regulates CpZap1 mRNA stability in an m⁶A-dependent manner. (A) Venn diagram illustrating the number of overlapping genes between the m⁶A-seq and RNA-seq data. (B) The EP155/3 × Flag-CpALKBH strain was verified using Western blotting with an anti-Flag antibody and anti-β-actin antibody. (C) RIP assay demonstrated the interaction between CpZap1 mRNA and CpALKBH protein. It was performed with an anti-Flag antibody in the EP155/3 × Flag-CpALKBH strain. Negative controls were made with β-actin. Normalizing fold enrichment values to input was done. (D) qRT-PCR analysis confirmed CpZap1 expression levels in KU80 and ΔCpALKBH. (E) Integrative genomics viewer (IGV) plots show m⁶A peaks on the CpZap1 transcript in KU80, with the Y-axis representing normalized read counts. A schematic of the MazF enzyme assay was also included, with scissors indicating the MazF; the MazF ACA site within the methylation site is highlighted in red (bottom). (F) MeRIP-qRT-PCR analysis with anti-m⁶A antibody was conducted to detect m⁶A levels of CpZap1 in KU80 and ΔCpALKBH mutant. (G) CpZap1 expression levels were measured by qRT-PCR in KU80 and CpALKBH after actinomycin D treatment, and the mRNA half-life (t_1/2) of CpZap1 was assessed using a nonlinear regression model. (H) PCR amplification was performed on cDNA from MazF-digested mRNA. The positions of primers P1 and P2 relative to the targeted methylation site are illustrated in E. Control primers P3 and P4, which do not flank an ACA site, were also utilized. (I) The result of (H) was analyzed using ImageJ. The relative intensity of the (P1 + P2)/(P3 + P4) was calculated. The product level of strains with primers P1 + P2 in KU80, ΔCpALKBH, and ΔCpALKBH-com was normalized with the control P3 + P4, respectively. A standard deviation from three independent experiments is shown with the error bars. Treatment differences are indicated by different letters above the bars (ANOVA followed by Tukey’s test, P < 0.05).

To examine the interaction between CpALKBH and CpZap1 transcripts, RIP of the EP155/3 × Flag-CpALKBH strain was performed. Fig. 5B and C showed that CpZap1 mRNA was significantly enriched with anti‐Flag antibody. This revealed that CpZap1 mRNA was a direct target of CpALKBH. Furthermore, the significantly downregulated expression of CpZap1 in ΔCpALKBH was confirmed by qRT-PCR (Fig. 5D). As CpALKBH is an m⁶A demethylase, it was presumed to regulate the expression of CpZap1 by removing its m⁶A modification. The KU80 m⁶A-seq findings revealed a significant m⁶A peak (chr8:1334739–1334863) distributed in the 3'UTR region of CpZap1 mRNA (Fig. 5E). MeRIP was then performed in combination with CpZap1-specific qRT-PCR to detect the changes in CpZap1 m⁶A modification following CpALKBH deletion. The findings verified that m⁶A methylation was readily detectable on CpZap1 mRNA in the KU80 strain. CpALKBH deletion further increased the m⁶A level in CpZap1 mRNA compared with that in the control (Fig. 5F), thereby confirming the role of CpALKBH as the m⁶A demethylase responsible for CpZap1 demethylation. To further evaluate the stability of CpZap1 mRNA after CpALKBH deletion, an RNA decay assay was performed by the transcription inhibitor actinomycin D in KU80 and ΔCpALKBH strains. As illustrated in Fig. 5G, the deletion of CpALKBH markedly reduced the half-life of CpZap1 mRNA, thereby indicating its impact on CpZap1 mRNA stability.

To determine the specific methylation sites on CpZap1 transcripts, we employed the MazF RNA restriction enzyme. It cleaves RNA at ACA sites but not m⁶ACA sites in a methylation-sensitive manner (37). The ACA sequence is a part of the GAm⁶ACA sequence of CpZap1 m⁶A peaks shown in Fig. 5E. Through motif screening analysis, 1935A at 3′UTR was speculated to be a key methylation modification site. The purified mRNA isolated from the KU80, ΔCpALKBH, and ΔCpALKBH-com strains was subjected to digestion with MazF, followed by reverse transcription to generate cDNA. To exclude the possibility of genomic DNA (gDNA) contamination, primers targeting the CpZap1 intron were also designed for the assay (Fig. S4A). The cDNA, thus, obtained was amplified using primers P1/P2, resulting in the generation of corresponding products. The cDNA yields from ΔCpALKBH were significantly higher than those from the KU80 and ΔCpMTA1-com strains, as demonstrated in Fig. 5H and I. Meanwhile, PCR with primers P3/P4 was used as a negative control, as this region did not contain a potential MazF cleavage site. This result suggests that CpZap1 undergoes methylation at the 1935A position—a process regulated by the demethylase CpALKBH. Taken together, these observations collectively support that CpALKBH mediates CpZap1 mRNA stability in an m⁶A‐dependent manner.

CpMTA1 catalyzes m⁶A modification of CpZap1

The dynamic and reversible characteristics of m⁶A modification in eukaryotic cells has already been established (38). In a previous study, we identified that CpMTA1 is an m⁶A methyltransferase in C. parasitica (39). Therefore, we investigated the potential role of CpMTA1 in catalyzing the m⁶A modification of CpZap1. Initially, we assessed the impact of CpMTA1 deletion on the expression level of CpZap1 and observed 6.5-fold increase in the expression level in ΔCpMTA1 compared to that in the KU80 strain (Fig. 6A). Furthermore, RIP assay revealed a direct interaction between CpZap1 mRNA and CpMTA1 in the EP155/3 × Flag-CpMTA1 strain (Fig. 6B). These findings indicate that CpZap1 may be a substrate for CpMTA1 methyltransferase. To verify this, MeRIP combined with qRT-PCR was performed to analyze the levels of CpZap1 m⁶A methylation following CpMTA1 deletion. The findings revealed a small CpZap1 m⁶A peak indicating significantly decreased methylation levels in ΔCpMTA1 mutant compared to that in the KU80 strain, confirming that CpMTA1 functions as the methyltransferase responsible for catalyzing the m⁶A modification of CpZap1 (Fig. 6C).

Fig 6.

CpMTA1 catalyzes the m⁶A modification of CpZap1. (A) qRT-PCR analysis confirming CpZap1 expression levels in KU80 and ΔCpMTA1. (B) RIP assay demonstrated the interaction between CpZap1 mRNA and CpMTA1 protein. It was performed with an anti-Flag antibody in the EP155/3 × Flag-CpMTA1 strain. Negative controls were made with β-actin. Normalizing fold enrichment values to input was done. (C) MeRIP-qPCR analysis was conducted to assess m⁶A levels of CpZap1. mRNA from the KU80 and ΔCpMTA1 mutant strains was used for m⁶A-IP, employing an anti-m⁶A antibody. The resulting IP products served as templates for the subsequent MeRIP-qPCR. (D) PCR amplification was performed on cDNA synthesized from MazF-digested mRNA. A standard deviation from three independent experiments is shown with the error bars. Treatment differences are indicated by different letters above the bars (ANOVA followed by Tukey’s test, P < 0.05).

Furthermore, we verified that the deletion of CpMTA1 resulted in a decreased m⁶A level at the 1935A site of CpZap1 mRNA. The purified mRNA from the KU80, ΔCpMTA1, and ΔCpMTA1-com strains was digested by MazF, by reverse transcription to synthesize cDNA. No contamination of gDNA was confirmed (Fig. S4B). PCR amplification of the resulting cDNA with primers P1/P2 yielded a corresponding product with cDNA derived from the KU80 and ΔCpMTA1-com strains but not from the ΔCpMTA1 strain (Fig. 6D). The findings showed that the 1935A site of CpZap1 was protected from MazF cleavage in mRNA from strains containing a functional methyltransferase, indicating that CpMTA1 could catalyze CpZap1 mRNA methylation at the 1935A site in vivo.

The regulatory impact of CpZap1 on C. parasitica development, virulence, and transcriptional activity

To investigate the function of CpZap1 in the development and virulence of C. parasitica, a CpZap1 null mutant was generated by replacing CpZap1 with the hph gene. PCR and Southern blotting were used to screen and confirm the single-spored transformants. Additionally, wild-type CpZap1 was introduced into the ∆CpZap1 to complement the mutant (Fig. 7A and B). In addition, we introduced CpZap1 into the ΔCpALKBH mutant, which was named ∆CpALKBH/CpZap1-OE. qRT-PCR analysis revealed that the expression level of CpZap1 was significantly increased in this mutant compared with ∆CpALKBH (Fig. S5). The growth rate and sporulation of ∆CpZap1 significantly reduced compared to those of the wild-type strains (Fig. 7C and D). The observed abnormal phenotypes were successfully restored in the complemented strain ∆CpZap1-com. The ∆CpALKBH/CpZap1-OE strain showed a phenotype similar with that of the wild-type strain. Moreover, the virulence tests on chestnut stems and red Fuji apples were performed. The findings showed that the pathogenicity of ∆CpZap1 mutants was completely lost, and the virulence of ∆CpZap1-com strains could be restored to the wild-type level. Moreover, ∆CpALKBH/CpZap1-OE strains incited cankers similar with those of the EP155 and KU80 strains (Fig. 8A and B). This indicates that CpZap1 plays an important role in C. parasitica development and virulence.

Fig 7.

Construction and phenotype analysis of CpZap1 mutant strains. (A) CpZap1 gene deletion strategy in schematic form. Southern blotting used a fragment from the probe A (hph gene) and the probe B (a fragment from the right arm) to compare the fragment sizes between the KU80 strain and CpZap1 deletion mutants. The scale bar represents 1 kb. (B) Using probe A and B, Southern blotting analysis was conducted on ΔCpZap1 mutants and KU80. Genomic DNA from fungi was digested with Bsp1407I and Xho I, electrophoresed on agarose gels, and then probed with probe A and probe B. The strains used were EP155 (wild-type), KU80 (parental strain), ∆CpZap1 (CpZap1 deletion strain), and ∆CpZap1-com (complementary strain). EP155 and KU80 were used as control. (C) Photographs of mutant colonies were taken at 7 and 14 days post-inoculation to assess colony morphology. The strains displayed included EP155, KU80, ∆CpZap1, ∆CpZap1-com, and ∆CpALKBH/CpZap1-OE. (D) Sporulation levels of the tested strains were assessed on day 14. A standard deviation from three independent experiments is shown with the error bars. Treatment differences are indicated by different letters above the bars (ANOVA followed by Tukey’s test, P < 0.05).

Fig 8.

Virulence assay of CpZap1 mutant strains. (A) The dormant Chinese chestnut stems were inoculated with the tested strains and kept at 26°C for 25 days. (B) The Red Fuji apples were inoculated with the tested strains at 26°C for 10  days. A standard deviation from three independent experiments is shown with the error bars. Treatment differences are indicated by different letters above the bars (ANOVA followed by Tukey’s test, P < 0.05).

To elucidate the crucial role of the m⁶A methylation site of CpZap1 in C. parasitica, we introduced the extended CpZap1 (CDS + 3′UTR) with key residue 1935A into the ΔCpALKBH mutant, named ΔCpALKBH/CpZap1 + 3′UTR. Furthermore, we mutated the m⁶A methylation site of CpZap1 by changing A to C, resulting in the transformant ΔCpALKBH/CpZap1 + 3′UTR (A1935C) (Fig. S6A). Compared with the KU80 and ΔCpALKBH strains, the ΔCpALKBH/CpZap1 + 3′UTR strain grew much more slowly and showed significantly decreased conidial spores and virulence. However, the phenotype and virulence of the site-specific mutant ΔCpALKBH/CpZap1 + 3′UTR (A1935C) were similar to the KU80 strain (Fig. S6B and C). Furthermore, the m⁶A level of CpZap1 in the ΔCpALKBH/CpZap1 + 3′UTR was significantly elevated compared to that in KU80 and ΔCpALKBH. In contrast, theA1935C mutation resulted in a substantial decrease in the m⁶A level of CpZap1 (Fig. S6D). Additionally, the ΔCpALKBH/CpZap1 + 3′UTR strain demonstrated a marked decrease in the stability of CpZap1 mRNA, while the mutation of A1935C did not (Fig. S6E). This finding suggests that the methylation of 1935A plays an important role in regulating the CpZap1 mRNA stability and influencing the observed phenotypic switch in C. parasitica.

To further explore the regulatory mechanism of CpZap1, RNA-seq was conducted on the KU80 and ∆CpZap1. This analysis identified 1,056 upregulated genes and 1,693 downregulated genes in the ∆CpZap1 strain compared to the KU80 strain (Fig. S7A; Table S4). The reliability of the RNA-seq data was verified by performing qRT-PCR analysis of eight randomly selected DEGs. A strong correlation (correlation coefficient R² = 0.8424, P < 0.001) between qRT-PCR and RNA-seq results further validated their consistency (Fig. S7B; Table S4). To elucidate the primary functional categories represented by the DEGs, GO enrichment analysis was performed (Fig. S8A). The upregulated genes were mostly involved in ion transport, metal ion transport, intrinsic component of membrane, ion transmembrane transporter activity, and active transmembrane transporter activity. The downregulated DEGs were significantly enriched in mitotic DNA replication, nuclear DNA replication, nuclear replication fork progression, and catalytic activity. Moreover, we performed an enrichment analysis based on the KEGG pathways (Fig. S8B). The most enriched pathways for the upregulated genes were amino sugar and nucleotide sugar metabolism, MAPK signaling pathway, and tryptophan metabolism. However, the downregulated genes were enriched in DNA replication, mismatch repair, nucleotide excision repair, and cell cycle.

Overlapping genes between ΔCpALKBH and ΔCpZap1

As both ΔCpALKBH and ΔCpZap1 strains exhibited similar virulence deficiency, we reasoned that CpALKBH and CpZap1 might function through a similar molecular mechanism. To address this, DEGs induced by CpALKBH and CpZap1 deletion were chosen for further analysis. The overlapping analysis showed that 585 genes were downregulated in both ΔCpALKBH and ΔCpZap1 strains. Similarly, 240 genes were upregulated in both ΔCpALKBH and ΔCpZap1 strains (Fig. 9A and B; Table S5). The high number of overlapping DEGs between ΔCpALKBH and ΔCpZap1 indicates that they function similarly. To demonstrate the biological function of these overlapping DEGs, we performed GO and KEGG analyses. GO analysis revealed significantly enriched functional terms for 585 overlapping downregulated genes, including oxidation-reduction process, plasma membrane, and oxidoreductase activity (Fig. 9C). In KEGG analysis, significantly enriched pathways, including metabolic pathways, cell cycle, meiosis, and DNA replication, were found (Fig. 9D). Additionally, GO analysis showed significant enrichment in drug transmembrane transport, ATP-binding cassette (ABC) transporter complex, coenzyme binding, and ATPase activity for these 240 overlapping upregulated genes (Fig. 9E). In KEGG analysis, significantly enriched pathways, including ABC transporters and metabolic pathways, were found (Fig. 9F).

Fig 9.

Analysis of overlapping genes between ΔCpALKBH and ΔCpZap1 based on RNA-seq results. (A) Venn diagrams displaying the overlapped DEGs from RNA-seq (downregulated in ΔCpALKBH and ΔCpZap1, upregulated in ΔCpALKBH and ΔCpZap1). (B) The heat map was generated based on the expression levels shown in (A). (C) GO enrichment analysis of overlapping genes that were downregulated in both ΔCpALKBH and ΔCpZap1. (D) KEGG pathway enrichment analysis of overlapping genes that were downregulated in both ΔCpALKBH and ΔCpZap1. (E) GO enrichment analysis of overlapping genes that were upregulated in both ΔCpALKBH and ΔCpZap1. (F) KEGG pathway enrichment analysis of overlapping genes that were upregulated in both ΔCpALKBH and ΔCpZap1.

DISCUSSION

Recently, m⁶A modification has been identified as a pivotal mechanism in the regulation of mRNA biology (40). As a dynamic regulation process, methyltransferases and demethylases work together to regulate the m⁶A levels (41). Several studies have demonstrated that m⁶A modification is a conserved feature of mRNA and plays a critical regulatory role in fungi (23, 25, 26, 42). However, the biological effects of these m⁶A-modifying enzymes in phytopathogenic fungi remain largely unexplored. In this study, we report that CpALKBH can remove m⁶A modification from ssRNA in vitro, indicating that CpALKBH possesses m⁶A demethylase activity. Furthermore, we found that CpALKBH is essential for phenotypic characteristics and virulence of C. parasitica. In addition, CpALKBH regulates CpZap1 mRNA stability in an m⁶A-dependent manner. CpZap1 undergoes methylation at the 1935A position—a process regulated by the demethylase CpALKBH and methyltransferase CpMTA1. Moreover, CpZap1 is involved in regulating the fungal phenotype and virulence. Figure 10 presents a schematic model that summarizes our findings.

Fig 10.

A proposed model for CpALKBH regulates the mRNA stability of transcription factor CpZap1 via an m⁶A-dependent manner. In wild-type strains, CpMTA1 catalyzes the m⁶A modification of CpZap1, while CpALKBH reverses this methylation. The m⁶A modification in CpZap1 mRNA is balanced by both CpMTA1 and CpALKBH. However, in CpALKBH deletion mutants, the m⁶A level of CpZap1 mRNA increased significantly compared with the control, which resulted in a reduction in the mRNA stability of CpZap1. As an important transcription factor, the decreased mRNA of CpZap1 hinders the fungal growth, conidial spores, and pathogenicity of C. parasitica.

The AlkB (ALKBH1-8, FTO) enzyme is a member of Fe²⁺- and α-KG-dependent dioxygenases, which catalyzes the demethylation of various substrates, including DNA, RNA, and histones. In mammals, ALKBH5 has been widely recognized as an m⁶A-specific demethylase (43). However, m⁶A demethylases have still not been identified in fungi. In a recent study reported by Shi et al. (25), a putative oxidoreductase, PoAlkb1, containing an ALKB domain was identified in P. oryzae. Although PoAlkb1 was shown to be involved in virulence, it was not reported to be a demethylase. In this study, after comparing the C. parasitica genome with the human ALKBH5 sequence, an uncharacterized protein, XP_040772748 (CpALKBH)—a sole protein containing the Fe2OG dioxygenase domain in C. parasitica—was found. Subsequently, the CpALKBH protein was expressed in E. coli and its m⁶A demethylase activity was confirmed by RNA dot assay (Fig. 1). Similarly, ALKBH9B has been observed in Arabidopsis (21). Furthermore, we confirmed that CpALKBH deletion resulted in a notable increase of m⁶A levels, suggesting an important role of CpALKBH in the m⁶A modification in C. parasitica (Fig. 2). Consistent with our results, male mice deficient in ALKBH5 also exhibited elevated levels of m⁶A in their mRNA (9).

As reported in previous studies, m⁶A methyltransferase deletion can impact various biological processes, including mouse fertility, antiviral response, animal and plant development, and tumor development (44–46). In this study, the ΔCpALKBH mutant exhibited a marked reduction in growth rate, sporulation, and virulence (Fig. 3), suggesting an essential role of CpALKBH in the regulation of C. parasitica development and virulence. Furthermore, we found that the impaired phenotype of CpALKBH deletion mutant might be related to the expression of metabolism-related genes (Fig. 4). The correlation analysis of this transcriptome with m⁶A distribution is an effective method to identify the downstream targets of m⁶A demethylase (35). Through overlapping analysis of the RNA‐seq data and previously obtained m⁶A-seq findings of the KU80 strain, a total of 184 upregulated genes and 423 downregulated genes with m⁶A modification have been identified as potential targets of CpALKBH. Among these genes, CpZap1 was found to be crucial for CpALKBH functioning in C. parasitica. Although our focus was on the m⁶A modification of CpZap1, other target genes of CpALKBH may also affect CpALKBH functioning.

It has been extensively studied how m⁶A demethylase affects mRNA stability. For example, FTO specifically removes m⁶A methylation of mRNAs of DNA repair genes, leading to increased mRNA stability (47). In contrast, overexpression of ALKBH5 has been reported to significantly decrease PKMYT1 mRNA stability (48). The difference may lie in the position of m⁶A modification on mRNA. For instance, m⁶A within the 3′UTR or near the stop codon has been shown to reduce the stability of mRNA in a regular maize seedling, strawberry fruit, and tomato fruit. Conversely, m⁶A enrichment in the CDS region has been found to positively regulate the mRNA stability in the ripe strawberry fruit (49). The present study shows that the 1935A position of CpZap1 is a key methylation modification site, which is located in the 3′UTR region (Fig. 5). Coincidently, the mRNA stability of CpZap1 decreased in the ΔCpALKBH mutant, suggesting that the m⁶A methylation of CpZap1 negatively mediates mRNA abundance. Nevertheless, further research is required to understand how m⁶A modification alters mRNA stability in C. parasitica.

As a transcription factor, CpZap1 belongs to the zinc finger protein family. In light of the highly conserved sequence of CpZap1 in eukaryotes, including fungi, plants, and animals, it is reasonable to speculate that the function of CpZap1 is crucial. In S. cerevisiae, Zap1 controls zinc metabolism by regulating the expression of zinc metabolism-related genes (50). The zinc sensors bZIP19 and bZIP23 in Arabidopsis maintain the zinc levels in plants (51). This study also revealed that CpZap1 is necessary for C. parasitica development and virulence. Furthermore, the RNA-seq analysis confirmed that CpZap1 influences the expression of a large number of genes, which are mostly enriched in amino sugar and nucleotide sugar metabolism, MAPK signaling pathway, DNA replication, mismatch repair, and nucleotide excision repair. This study represents the first report on the interaction between m⁶A modification and transcription factors in fungi, highlighting their significant roles in regulating gene expression and RNA metabolism. Such regulation is crucial for various cellular biological functions and developmental processes. Identifying whether m⁶A modification regulates the mRNA stability of zinc finger protein in other species would be of interest.

Until now, this study is the first to demonstrate that CpZap1 is a direct downstream target of CpALKBH-regulated m⁶A demethylation in C. parasitica, revealing how CpALKBH influences CpZap1 and modulates fungal phenotype and virulence. Collectively, these findings offer valuable insights into the role and mechanism of CpALKBH-mediated m⁶A demethylation in filamentous fungi, then enhancing the understanding of related studies.

MATERIALS AND METHODS

The Materials and Methods within this manuscript have been previously described in our publications (39). They have been reproduced here for the convenience of the reader.

Fungal strains and culture conditions

The wild-type strain EP155 of C. parasitica (ATCC 38755), its hypovirulent isogenic strain EP155/CHV1-EP713 (EP155 infected with hypovirus CHV1-EP713, ATCC 52571), the highly efficient gene deletion strain KU80 (∆cpku80 of EP155) (52), and all the constructed mutants were maintained on potato dextrose agar (PDA) plates under a 12 h light/dark cycle at 26°C. These cultures were used for phenotypic analyses as well as for DNA and RNA preparation. The liquid complete medium was used to extract fungal proteins as described earlier (53).

Purification of CpALKBH protein

The coding sequence of CpALKBH was amplified through polymerase chain reaction (PCR) from EP155 and then inserted into the pET32a expression vector. The resulting plasmid was named as pET32a-CpALKBH, and plasmid DNA was then transformed into Escherichia coli BL21. The expression of CpALKBH protein was induced with 0.5 mM isopropyl-beta-d-thiogalactopyranoside for 12 h at 16°C. The CpALKBH protein was purified from the supernatant through nickel affinity chromatography. Purified proteins were quantified using BCA (Pierce) assays and then analyzed by SDS-PAGE. The excess protein was stored at −80°C. Primer sequences are shown in Table S6.

Three-dimensional structure prediction of CpALKBH protein

The three-dimensional structure of the CpALKBH protein was predicted utilizing AlphaFold2. Initially, the amino acid sequence of the CpALKBH protein was obtained from the NCBI database and stored in FASTA format. Subsequently, the AlphaFold2 (https://github.com/deepmind/alphafoldRoseTTAFold) prediction script was executed via the command line, with the input FASTA file and output directory specified. Upon completion, the predicted model was saved in PDB format and subjected to visualization and analysis using PyMOL software (https://pymol.org/2/). To identify the DSBH (Double-Strand Beta-Helix) domain, the structural comparison was conducted using PyMOL between the CpALKBH protein and the human ALKBH5 protein (PDB DOI: https://doi.org/10.2210/pdb4NJ4/pdb).

m⁶A demethylase activity assay

RNA dot blot assay was performed to detect the m⁶A demethylase activity of CpALKBH as described earlier (21). Briefly, 2.5 µg of CpALKBH protein was incubated with m⁶A monomethylated ssRNA (Sangon Biotech) oligonucleotide at 25°C for 3 h in a reaction mixture including 50 mM phosphate buffer saline (pH 7.3), 10 µM α-ketoglutarate, 100 µM l-ascorbic acid ascorbate, and 20 µM (NH₄)₂Fe(SO₄)₂·6H₂O. The heat-inactivated CpALKBH protein lacking any enzymatic activity was used as a control. The reaction was quenched by heating to 95°C for 10 min. RNA was extracted and spotted onto the Hybond-N+membrane. After drying, the membrane was UV crosslinked for 3 min at 1500 mJ/ cm². The membrane was blocked with 5% non-fat milk in tris buffered saline tween (TBST) for 1 h before incubating with the m⁶A antibody (EpiGentek) overnight at 4°C. Ethylene blue staining was conducted using a 0.02% methylene blue solution in sodium acetate, serving as a loading control. Following TBST washing, a secondary antibody was incubated at room temperature for 1 h. Visualization was performed with the ECL detection system (AI600 images).

Mutant construction and verification

The CpALKBH deletion mutant was constructed through homologous recombination (52). The upstream and downstream sequences of CpALKBH and the hygromycin B resistance gene were amplified using EP155 genomic DNA and pCPXHY2 plasmid (31), respectively. Through overlapping PCR, these three fragments were connected, and the fused cassette was then transformed into KU80 protoplast. The PDA supplemented with hygromycin was used to select the resistant transformants. The positive single-spored ΔCpALKBH deletion mutants were verified using PCR, reverse transcription PCR (RT-PCR), and Southern blotting. Subsequently, single-spore isolation was performed to purify these mutants (54). To construct the complementation strain, ΔCpALKBH-com, the open reading frame (ORF) and promoter of CpALKBH, were amplified and cloned into the vector pCPXG418 (55), which includes the resistance G418 marker. After validation through DNA sequencing, the vector pCPXG418-CpALKBH was introduced into the ∆CpALKBH deletion strain protoplast. The transformants that exhibited resistance to both G418 and hygromycin were selected, and subjected to PCR screening of the complementation construct, which was subsequently validated through Southern blotting.

To generate strains that express a fusion protein of CpALKBH with a 3 × Flag tag at the N-terminus, the CpALKBH ORF was inserted into a modified pCPXG418-3 × Flag vector. The pCPXG418-3 × Flag-CpALKBH plasmid was introduced into the EP155 strain. PCR and Western blot analysis were used to confirm the expression of CpALKBH. The gene deletion, complementation, and overexpression constructs of CpZap1 were generated by similar methods as described above. Table S6 lists all primer sequences used for constructing and verifying fungal mutants.

Fungal phenotype and virulence analysis

As a comparison to EP155, KU80, and EP155/CHV1-EP713, the phenotypic characteristics and virulence of the constructed mutants were analyzed. Phenotypic alterations in pigmentation, growth rate, and conidiation were assessed (56). After 14 days of culturing fungi on PDA, conidia were eluted with sterile water and counted using a hemacytometer. Fungal virulence was measured with the strain samples by inoculating the dormant Chinese chestnut (Castanea mollissima) stems and red Fuji apple. Incubation of the stems and apples at 26°C for 25 and 10 days, respectively. The canker size was observed and quantified according to Shi et al (57).

RNA extraction and quantitative real-time PCR

The MiniBEST plant RNA extraction kit (Takara) was used to extract total RNA from C. parasitica, and the HiScript III RT supermix (Vazyme Biotech, R323) was used to prepare cDNA. Using SYBR green PCR master mix (Takara), qRT-PCR was carried out on the LightCycler480II real-time PCR system (Roche). Following the completion of the reactions, relative gene expression levels were quantified based on the 2^−ΔΔCt method. 18S rRNA served as a reference gene (58). A list of the primers used for qRT-PCR can be found in Table S6.

Measurement of m⁶A RNA methylation levels

The colorimetric EpiQuik m⁶A RNA methylation quantification kit (EpiGentek, USA) was used to quantify the m⁶A RNA methylation level based on the manufacturer’s instructions. The assay wells were coated with 200 ng RNA or the m⁶A standard and then capture and detection antibodies were added. m⁶A levels were determined by measuring the absorbance at 450 nm (OD₄₅₀) in each well. Subsequently, the relative m⁶A RNA methylation levels were quantified.

Methylated RNA immunoprecipitation coupled with qRT-PCR

The methylated RNA immunoprecipitation (MeRIP) assay was conducted according to the manufacturer’s instructions using the m⁶A RNA enrichment kit (EpiGentek, USA). A total of 10 µg of RNA was extracted from each sample and subsequently fragmented into segments of 300 nucleotides or fewer. RNA samples were immunoprecipitated using magnetic beads coated with 10 µg of anti-m⁶A antibody or IgG. The product of IP or input assay was subsequently subjected to analysis via qRT-PCR utilizing special primers (Table S6).

RNA immunoprecipitation

The RNA immunoprecipitation (RIP) assay was performed using the RNA Immunoprecipitation (RIP) Kit (BerSinBio, Bes5101) according to the manufacturer’s instructions. In brief, fungal cells were collected and lysed by RIP lysis buffer on ice, and DNA contamination was removed by DNase treatment. Subsequently, the cell lysate was immunoprecipitated with anti-Flag antibodies (Abmart) at 4℃ for 16 h, followed by a 1 h incubation with protein A/G beads. Finally, qRT-PCR was used to quantify the co-precipitated RNA. Table S6 lists the primers used in this assay.

MazF RNA restriction enzyme assay

MazF (TaKaRa 2415A) was used to detect the methylation site on CpZap1 mRNA (59). Initially, a total of 12.5 µg of RNA was used for the isolation of poly(A) mRNA, employing VAHTS mRNA capture beads (Vazyme) in accordance with the manufacturer’s protocol. In a total volume of 20 µL, the isolated mRNA was combined with MazF buffer (4  µL), RNase inhibitor (0.5  µL), and MazF enzyme (1  µL) solution. Subsequently, RNA samples were resuspended in RNase-free water, and cDNA synthesis was conducted using the HiScript III RT supermix kit (Vazyme Biotech, R323). The resulting cDNA was used as a template for PCR with primers (Table S6), and the products were analyzed on a 1% agarose TBE gel.

Analysis of RNA-seq

Based on three biological replicates per strain, RNA-seq was performed on the C. parasitica strains KU80 and ΔCpALKBH. Total RNA was extracted; library was constructed and sequenced with Illumina HiSeqTM 4000 in Gene Denovo Biotechnology Co (Guangzhou, China). High-quality clean reads were obtained by using Fastp software (v0.18.0) to filter out reads with adapters, over 10% unknown nucleotides, and low-quality reads (Q-value ≤20) (60). With HISAT2 v2.4 software (61), clean data from each sample were mapped to the genome of C. parasitica (https://genome.jgi.doe.gov/portal/Crypa2/Crypa2.download.ftp.html). Differentially expressed genes (DEGs) were analyzed using DESeq2 (62). DEGs were considered significant if their log₂(fold change) >1 and their false discovery rate <0.05.

Western blot

Fungal proteins were extracted using NP40 lysis buffer (Solarbio) according to the previously described method (63). Subsequently, the protein was subjected to boiling with SDS loading buffer followed by separation with 12% SDS-PAGE and transfer onto a PVDF membrane (Millipore, USA). After the membrane was blocked, anti-Flag (Abmart) or anti-actin (ABclonal) antibodies were incubated overnight at 4°C. As a final step, ECL detection reagents (Coolaber, China) were used to identify the membrane.

RNA decay assay

The stability of CpZap1 mRNA in KU80 and ΔCpALKBH strains was detected using actinomycin D (GlpBio Technology, CA, USA). These strains were cultured in the EP liquid medium and then treated with 20 µM actinomycin D (resuspended in dimethyl sulfoxide). The strains inoculated in the same volume of dimethyl sulfoxide were used as controls. For RNA extraction, fungal samples were collected after 0, 8, and 24 h. qRT-PCR was used to measure the expression level of CpZap1 mRNA (64).

Statistical analysis

Analysis of variance (ANOVA) and student’s t-test were conducted using IBM SPSS Statistics 22 software. All data and error bars are presented as the mean ± SD from at least three independent experiments. Statistical significance was defined as a P-value less than 0.05. Unless otherwise noted, all experiments were conducted in triplicate.

DATA AVAILABILITY

All data necessary for the evaluation of the conclusions presented in the paper are included within the main text and the Supporting Information. RNA-seq raw data were submitted to the sequence read archive database (SRA) under accession number SRP531574.

SUPPLEMENTAL MATERIAL

The following material is available online at https://doi.org/10.1128/mbio.01844-24.

Supplemental Figures. mbio.01844-24-s0001.docx.

Figures S1 to S8.

Tables S1 to S4. mbio.01844-24-s0002.xlsx.

Raw data, and results for differentially expressed genes.

Table S5. mbio.01844-24-s0003.xlsx.

CpALKBH-up and CpZap1-up; CpALKBH-down and CpZap1-down.

Table S6. mbio.01844-24-s0004.xlsx.

Primers used in this study.

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