Magnaporthe oryzae is the main causative agent of rice blast outbreaks and has severely restricted rice yield in all rice-producing countries worldwide (Shahriar et al., 2020). Blast causes a 10-30% loss in rice yield annually, an amount that is enough to feed more than 60 million people (Sella et al., 2021). Due to the widespread distribution, destructive power and strong adaptability of rice blast fungus, various control methods, such as the use of fungicides or the cultivation of resistant varieties, have been used to eliminate this fungus. However, this pathogen rapidly develops adaptive mutations that render fungicides and resistant varieties ineffective. The “tactical” response or escape strategy of rice blast fungus has long been the focus of research efforts.

The reasons for the variation in the fungal response to adverse stress and plant host resistance have been extensively studied by various techniques, and epigenetic modifications have been shown to be involved in this process (Jeon et al., 2015; Dubey and Jeon, 2017; He et al., 2020). DNA methylation, including cytosine methylation (5mC) and adenine methylation (6mA), is a common DNA epigenetic modification that has crucial effects on gene activity (Moore et al., 2013). A study on Metarhizium robertsii showed that the DNA 5mC level was 0.38%-0.42%, and differential DNA methylation may contribute to the temporal and spatial regulation of gene expression and the development of mycelia and conidia in this organism (Li et al., 2017). A study on M. oryzae showed that 5mC accounted for 0.22% of all genomic cytosines in mycelia, which underwent global reprogramming during fungal development, and this process occurred in and around genes as well as transposable elements, contributing to the silencing of transposable elements and transcript abundance of genes (Jeon et al., 2015). In Saccharomyces cerevisiae, Schizosaccharomyces pombe, Caenorhabditis elegans, and Tetrahymenather mophila, 5mC is absent; however, 6mA is present in these organisms and is involved in the regulation of DNA replication, repair, transposition, and transcription (Hattman, 2005; Liang et al., 2018; Xiao et al., 2018a). Mondo et al. reported that 6mA is widespread in fungi, with levels as high as 2.8% in some fungi. It mainly appears symmetrically in the ApT sequence at the transcription initiation point (Mondo et al., 2017). Additional studies have shown that 6mA may be a common epigenetic marker in eukaryotes, including humans, pigs, mice, fishes, frogs, flies, worms, ciliates, Chlamydomonas and fungi, and potentially functions in transcriptional activation or silencing, chromatin regulation, and the stress response (Fu et al., 2015; Greer et al., 2015; Mondo et al., 2017; Liang et al., 2018; Xiao et al., 2018b).

Although much progress has been made in understanding the adaptation of M. oryzae to fungicides (Kim and Kim, 2009; Bohnert et al., 2019; Xu et al., 2020; Zhang et al., 2020), the contribution of epigenetic factors, such as DNA methylation, to this enigmatic process remains to be studied. As shown by Jeon et al. (2015), 5mC could be a dynamic epigenetic factor contributing to fungal development and genome defense in M. oryzae. In this work, we evaluated the gene expression response of M. oryzae to the fungicide pyraclostrobin and its relationship to changes in 5mC and 6mA methylation.

Pyraclostrobin is an exoquinone inhibitor that can bind to ubiquinone site of the cytochrome bc1 complex to prevent electron transport on the inner mitochondrial membrane. This interferes with the process of oxidative phosphorylation, resulting in insufficient production of cellular energy ATP and ultimately leading to fungal death. M. oryzae strain is sensitive to pyraclostrobin, with half maximal effective concentration (EC50) of 0.0012-0.0128 μg/mL (Ruan et al., 2022a). The sensibility was higher than other popular fungicides such as tebuconazole, carbendazim, propiconazole (Ruan et al., 2022a). However, the rapid development of resistance in M. oryzae to the fungicide and consequent control failure has become increasingly problematic. The main mechanism conferring resistance involves mutations in the cytochrome b gene CYTB, causing the substitution of glycine by alanine or serine at position 143 (G143A/S) (Li et al., 2022; Peng et al., 2022; Ruan et al., 2022b). The overexpression of alternative oxidase gene (AOX) in the alternative oxidation pathway and efflux transporter ATP-binding cassette (ABC) gene MoABC-R1 are also likely to contribute to the resistance (Gisi et al., 2002; Fernández-Ortuño et al., 2008; Hu et al., 2023). This study will enhance the understanding for potential mechanism of quinone inhibitor resistance, which will facilitate the development of drug targets as well as feasible strategies for keeping the high efficacy of this kind of fungicide.

M. oryzae has been reported to have high pathogenic variation with respect to host range and variety specificity. Pathogenic variation is the main reason why rice blast fungus breaks through the resistance barrier of rice plants or makes fungicides ineffective. M. oryzae has evolved sophisticated strategies to attach and subsequently infect its hosts, processes that often involve unique epigenetic changes. Clarifying the metabolism and molecular changes associated with fungicide action is beneficial for understanding the mechanism of resistance of rice blast fungus to drugs, as well as the requisite for the development of effective disease control strategies. In this work, RNA-Seq was used to analyze genome-wide changes in gene expression in response to the popular fungicide pyraclostrobin, and several critical molecules were shown to play important roles in this process.

Chaperones are central to homeostasis in eukaryotic cells and play essential roles in protein quality control in the ER and in ER-associated degradation (Nishikawa et al., 2005; Hartl et al., 2011). Hsp70 is considered a sentinel chaperone that plays an essential role in aberrant protein degradation in the ubiquitin−proteasome system by cooperating with other cellular chaperones to form dynamic and functionally versatile complexes (Fernandez-Fernandez et al., 2017; Rosenzweig et al., 2019). For example, CHIP, a cochaperone of Hsp70, competes for binding to the C-terminus of Hsp70 and ubiquitinates Hsp70-bound substrates, thereby directing the substrates to the proteasome for degradation (Stankiewicz et al., 2010; Rosenzweig et al., 2019). These chaperones cooperate with cellular degradation machinery to guard cells from the deleterious effects of various proteotoxic stresses. Although the functions of the identified DEGs are only recognized at the level of integrated methylome and transcriptome analysis in this study, it is well known that when organisms face large-scale gene damage, SOS repair is initiated, which inevitably leads to many mutations and the generation of many abnormal functional proteins. These abnormal proteins need to be degraded through ER-associated degradation and ubiquitin-mediated proteolysis pathways. In this study, the expression of genes related to these two pathways increased, which is beneficial for cell survival because the accumulation of abnormal proteins is toxic to organisms. If certain mutations allow the organism to survive, then these mutations will be passed on, and the organism will become resistant to the drug. Therefore, the susceptibility of M. oryzae to developing drug resistance may be related to its strong ability to degrade abnormal proteins. This prevents cells that have favorable mutations from dying due to the accumulated toxicity of abnormal proteins.

Ribonucleotide reductases, including tryparedoxin and trypanothione, can catalyze the biosynthesis of deoxyribonucleotides from the corresponding ribonucleotides using thioredoxin as H donors ( Figure 4 ) (Krauth-Siegel et al., 2003; Comini et al., 2007). The downregulation of RRM1 expression contributes to high concentrations of trypanothione and tryparedoxin. It has been reported that trypanothione and tryparedoxin are involved in diverse cellular functions, including maintenance of thiol redox activity, oxidant defenses, defense against xenobiotics, ascorbate homeostasis, sequestration of heavy metals, drug resistance and modulation of the host immune response (Fairlamb and Cerami, 1992; Schmidt and Krauth-Siegel, 2002; Comini et al., 2007; Suman et al., 2018; Gonzalez-Chavez et al., 2019). Moreover, ribonucleotide reductase is responsible for the de novo conversion of ribonucleoside diphosphates to deoxyribonucleoside diphosphates, providing the precursors necessary for DNA synthesis. The reduction in enzyme activity inhibited DNA replication and repair, decreasing the fidelity of the DNA-coding genetic information, consistent with the inhibition of DNA replication and repair, as shown in Table 3 .

Cardiolipin is one of the main phospholipids constituting the inner mitochondrial membrane, improving the fluidity of the inner mitochondrial membrane and facilitating the lateral diffusion of respiratory chain complexes in the membrane lipid bilayer (Houtkooper and Vaz, 2008; Paradies et al., 2014). Moreover, cardiolipin is involved in the formation of all enzyme complexes of the respiratory chain, including complexes I, II, III, IV and V and flexible cytochrome c (Fry and Green, 1981; Eble et al., 1990; Robinson et al., 1990; Ott et al., 2002; Yankovskaya et al., 2003). It has been shown that abnormal cardiolipin can dysregulate respiratory chain complex I and mitochondrial quality control (Anzmann et al., 2021). Studies have also revealed that cardiolipin and phosphatidic acid control mitochondrial division and fusion and coordinate the balance between these dynamic processes (Kameoka et al., 2018). Therefore, inhibition of the expression of the AYR1, PLD1 and CRLS genes not only disrupts the structure of the mitochondrial inner membrane and electron transport but also leads to an imbalance of phosphatidic acid and cardiolipin, which interferes with mitochondrial fusion and division. The dynamic balance of these two processes is vital for maintaining the functional competence and quality of mitochondria (Youle and van der Bliek, 2012; Ban-Ishihara et al., 2013; Roy et al., 2015; Shirihai et al., 2015).

An increase in alternative splicing events will generate multiple transcripts from a single pre-mRNA, which contributes to the gain or loss of some protein functional domains. This produces protein isoforms that respond to the demands associated with pathogenicity and environmental pressures such as fungicides in fungi as shown by Gehrmann et al. (2016) and Fang et al. (2020). Several gene knockout experiments have confirmed that alternative splicing, which likely generates protein isoforms, is directly involved in the resistance mechanism in M. oryzae. For example, the removal of the MoHMT1 gene, which causes genome‐wide alternative splicing, resulted in increased pathogenicity in M. oryzae (Li et al., 2020). Alternative splicing of MoPTEN contributes to conidium and appressorium development and invasive hyphal growth of M. oryzae in plant cells (Wang et al., 2021). Alternatively spliced SMN orthologs in M. oryzae are required for stress resistance and disease development (Liang et al., 2015). Deletion of the splicing factor MoSrp1 verified that alternative splicing participated in mycelial growth, conidiation, and virulence in M. oryzae (Shi et al., 2022).

In this study, the expression of several cell cycle-related genes was downregulated or alternatively spliced. Mei2 is considered an RNA-binding protein that can form a complex with a specific RNA species to promote meiosis (Yamamoto, 1996). One study showed that mei2 gene expression is regulated by cAMP (Watanabe et al., 1988). Moreover, cAMP is generated from ATP, and this process is blocked by pyraclostrobin. This may be one of the reasons for the decreased expression of mei2. Additionally, mcm7 (MGG_09300) and ANAPC1 (MGG_03314), two other meiosis-related genes, were downregulated 2.17-fold and 1.46-fold, respectively. MCM7 is a DNA replication licensing factor that is involved in the initiation of replication by loading onto DNA replication origins (Cvetic and Walter, 2006; Evrin et al., 2014). ANAPC1 is one of the subunits of the anaphase-promoting complex (APC), which is an E3 ubiquitin ligase that targets cell cycle regulatory proteins for degradation by the proteasome and functions in cell cycle transition (Castro et al., 2005). These results showed that pyraclostrobin may disrupt cell cycle progression by inhibiting cell cycle-related proteins. MGG_04006 is a Rho-GAP domain-containing protein. Ye et al. (2014) reported that there are eight putative Rho GAP proteins in M. oryzae. Some Rho GAP proteins, such as MoRga1 and MoLrg1, may play important roles in vegetative growth, conidiation, conidial morphology, appressorium formation and pathogenicity. However, MGG_04006 did not affect fungal development of virulence. Nevertheless, exon skipping occurred in this gene under pyraclostrobin stress, suggesting that it may be needed for fungal survival. However, further research is needed to study the function of this gene.

Emerging evidence has shown that DNA methylation in transcribed regions is involved in the regulation of alternative splicing through two possible mechanisms. First, DNA methylation modulates the elongation rate of RNA polymerase II via CCCTC-binding factor (CTCF) and methyl-CpG binding protein 2 (MeCP2), allowing some weak splicing signals to be identified as involved in splicing (Shukla et al., 2011; Maunakea et al., 2013). Second, DNA methylation-dependent heterochromatin protein 1 (HP1) can recruit splicing factors to alternative exons that contribute to alternative splicing (Yearim et al., 2015). Further study confirmed that DNA methylation of exon-encoding regions is directly involved in the regulation of alternative splicing (Shayevitch et al., 2018). However, additional underlying mechanisms remain to be elucidated (Lev Maor et al., 2015).

Additionally, the distributions of 5mC and m6A were negatively correlated. In regions with high levels of 5mC, the 6mA level was low, and in regions with low levels of 5mC, the 6mA level was high. A negative correlation between the distributions of 5mC and 6mA in diverse fungi has been reported by Mondo et al. (2017). After pyraclostrobin treatment, the levels of both 5mC and 6mA increased, indicating that pyraclostrobin treatment can increase the methylation rate in the gene body and 2 kb upstream and 2 kb downstream flanking regions. The methylation sites alter the binding of certain proteins, affecting the efficiency of RNA polymerase or the binding of splicing factors, thereby affecting transcription and alternative splicing. Studies have shown that 5mC and 6mA in the gene body are associated with enhanced gene expression (Arechederra et al., 2018; Zhang et al., 2018). Yang et al. (2014) reported that there is a causal relationship between C methylation in gene body and gene expression. 6mA has been reported to be associated with active genes, especially RNA polymerase II-transcribed genes (Mondo et al., 2017; Wang et al., 2017). Further studies suggested that the main function of gene body methylation is not to modulate expression during development or respond to the environment but to stabilize gene expression by preventing aberrant transcription from internal cryptic promoters and enhance splicing efficiency to reduce expression variability by excluding the histone variant H2A.Z (Kim and Zilberman, 2014; Bewick and Schmitz, 2017; Zilberman, 2017).

Although DNA methylation can change the functional state of regulatory regions, the connectivity between DNA methylation and gene expression is complex and poorly understood (Schubeler, 2015; Angermueller et al., 2016). Li et al. (2011) showed that 5mC in the promoter of Pib plays a positive role in inducing the expression of Pib in M. grisea. Partial demethylation by 5-azacytidine treatment reduced Pib expression and compromised blast disease resistance. Singh and Vinod (2020) also reported that only a small proportion of differentially hypermethylated genes with 5mC showed downregulated expression. These results suggest that aberrant DNA methylation may play both a positive and negative role in regulating gene expression (Zhu et al., 2016). This study is helpful for understanding the adaptation and variation mechanisms of rice blast fungus, and promoting the control of rice blast disease.

Under pyraclostrobin stress, proteolysis, glutathione metabolism, and alternative splicing were enhanced in M. oryzae, while DNA replication, DNA damage repair, and lipid metabolism were inhibited. These changes may be related to the elevated levels of 5mC and 6mA in the gene body. This work enriches the understanding for potential mechanism of quinone fungicides resistance. Meanwhile, it reflects the complexity of the adaptation mechanism of M. oryzae to quinone fungicides and the necessity for ongoing research in this field.

Both transcriptome and methylome data presented in the study have been deposited in the BioProject database of NCBI. The accession number is PRJNA1096937 (URL: http://www.ncbi.nlm.nih.gov/bioproject/1096937).

SF: Data curation, Writing – original draft, Formal analysis, Visualization. HW: Data curation, Writing – original draft. KQ: Data curation, Writing – original draft. YP: Formal analysis, Writing – original draft. CL: Data curation, Writing – original draft. XL: Conceptualization, Writing – review & editing.