Comparative acetylomic analysis reveals differentially acetylated proteins regulating fungal metabolism in hypovirus‐infected chestnut blight fungus

Abstract Cryphonectria parasitica, the chestnut blight fungus, and hypoviruses are excellent models for examining fungal pathogenesis and virus–host interactions. Increasing evidence suggests that lysine acetylation plays a regulatory role in cell processes and signalling. To understand protein regulation in C. parasitica by hypoviruses at the level of posttranslational modification, a label‐free comparative acetylome analysis was performed in the fungus with or without Cryphonectria hypovirus 1 (CHV1) infection. Using enrichment of acetyl‐peptides with a specific anti‐acetyl‐lysine antibody, followed by high accuracy liquid chromatography–tandem mass spectrometry analysis, 638 lysine acetylation sites were identified on 616 peptides, corresponding to 325 unique proteins. Further analysis revealed that 80 of 325 proteins were differentially acetylated between C. parasitica strain EP155 and EP155/CHV1‐EP713, with 43 and 37 characterized as up‐ and down‐regulated, respectively. Moreover, 75 and 65 distinct acetylated proteins were found in EP155 and EP155/CHV1‐EP713, respectively. Bioinformatics analysis revealed that the differentially acetylated proteins were involved in various biological processes and were particularly enriched in metabolic processes. Differences in acetylation in C. parasitica citrate synthase, a key enzyme in the tricarboxylic acid cycle, were further validated by immunoprecipitation and western blotting. Site‐specific mutagenesis and biochemical studies demonstrated that the acetylation of lysine‐55 plays a vital role in the regulation of the enzymatic activity of C. parasitica citrate synthase in vitro and in vivo. These findings provide a valuable resource for the functional analysis of lysine acetylation in C. parasitica, as well as improving our understanding of fungal protein regulation by hypoviruses from a protein acetylation perspective.


| INTRODUC TI ON
Protein acetylation is a dynamic and highly conserved posttranslational modification in prokaryotes and eukaryotes (Hart & Ball, 2013). Histone acetylation was first found to be closely related to transcriptional regulation (Allfrey et al., 1964). Acetylation is also found in many nonhistone proteins such as transcription factors, nuclear-related proteins, hormone receptors, cell metabolismrelated proteins, and cancer-related proteins (Batta et al., 2007;Spange et al., 2009). Through the reversible addition of an acetyl group to lysine residues, protein acetylation regulates enzymatic activity, protein localization, protein stability, and protein-protein and protein-nucleic acid interactions (Arif et al., 2010). Recent advances in high-affinity purification of lysine-acetylated peptides and high-resolution mass spectrometry have promoted the development of the lysine acetylome in many eukaryotes and prokaryotes. Proteome-wide analyses have led to the discovery of various cellular functions of lysine acetylation, especially those associated with central metabolic pathways (Henriksen et al., 2012;Nie et al., 2015;Weinert et al., 2011;Wu et al., 2011;Xie et al., 2015;Zhang et al., 2013).
Recent evidence suggests that lysine acetylation is common in fungi. In Candida albicans, 477 of 9038 (5.28%) proteins were found to be acetylated. This first study of acetylated proteins in human-pathogenic fungi provides an important basis for further studies on the functional analysis of acetylated proteins in pathogenic fungi (Zhou et al., 2016). Further knowledge of lysine acetylation in human-pathogenic fungi was obtained from Histoplasma capsulatum (Xie et al., 2016), Trichophyton rubrum (Xu et al., 2018), Cryptococcus neoformans (Brandao et al., 2018), and Aspergillus fumigatus (Lin et al., 2020). In plant-pathogenic fungi, systematic analyses of the lysine acetylome have been performed in Fusarium graminearum (Zhou & Wu, 2019), Phytophthora sojae , Botrytis cinerea , Magnaporthe oryzae (Liang et al., 2018), and Aspergillus flavus (Yang et al., 2019). These reports demonstrate that lysine acetylation not only regulates important cellular processes, such as enzyme activity, signal transduction, cell division, and metabolism, but also morphological transition, stress response, biofilm formation, and other processes, thus regulating the fungal life cycle Kim et al., 2015;Narita et al., 2019). However, the relationship between the specific mechanism of acetylation and fungal virulence regulation is yet to be determined.
The ascomycete fungus Cryphonectria parasitica is the causal agent of chestnut blight disease, which destroys billions of American chestnut trees (Rigling & Prospero, 2018). The fungus hosts a wide range of viruses and serves as a useful model to examine virus-host interactions and fungal pathogenesis (Eusebio-Cope et al., 2015).
Wild-type C. parasitica strain EP155 is an orange-pigmented, virulent, hypovirus-free strain that can induce large cankers on chestnut stems. Fungal virulence is attenuated when infected with hypoviruses, a group of single-stranded, positive-sense RNA viruses (Dawe & Nuss, 2001). Furthermore, hypovirus infection alters the phenotypic traits of fungi, such as reduced growth rate and pigmentation, loss of asexual sporulation, and suppression of female sterility (Nuss, 2005). Many efforts have been made to understand the mechanism of hypovirulence and comparative transcriptomic, proteomic, metabolomic, and methylomic research has revealed a wide range of hypovirus-regulated and virulence-related proteins (Allen et al., 2003;Chun et al., 2020;Dawe et al., 2009;Li et al., 2018;Shang et al., 2008;Wang et al., 2014). Nevertheless, the detailed regulatory mechanisms of protein expression, particularly at the posttranslational level, are unexplored.
In this study, the impact of hypoviral infection on lysine acetylation in C. parasitica was investigated by generating and comparing two fungal strain acetylomes using label-free quantitative proteomics. The different acetylation levels of C. parasitica citrate synthase (CpCS), a key enzyme in the tricarboxylic acid (TCA) cycle, were further verified by immunoprecipitation and western blotting. The functional significance of the lysine acetylation (Kac) site on CpCS was confirmed by site-specific mutagenesis and biochemical studies.
Our study provides extensive data about lysine acetylation in C. parasitica for the first time and reveals novel insights into the relationship between hypovirulence and protein acetylation.

| Hypovirus infection affects the global acetylome of C. parasitica
To investigate whether the global lysine acetylated protein level of C. parasitica was influenced by hypovirus infection, western blotting was performed using an anti-acetyl-lysine antibody. The acetylation patterns of total fungal proteins were different between the wild-type strain EP155 and its isogenic Cryphonectria hypovirus 1 (CHV1)-infected strain EP155/CHV1-EP713, suggesting that the lysine acetylome of host proteins changed in response to hypovirus infection (Figure 1a). We therefore used a label-free quantitative proteomic approach combined with immunoaffinity enrichment and liquid chromatography-tandem mass spectrometry (LC-MS/ MS) to identify the acetylome of EP155 and EP155/CHV1-EP713 ( Figure 1b). To validate the MS data, the mass errors of all identified peptides were checked. The distribution of mass error was near 0 and most were less than 6 ppm, showing that the MS data conformed with the requirement ( Figure S1). The results revealed 638 nonredundant Kac sites on 616 peptides, distributed in 325 unique proteins (Table S2). Most peptides were 7−18 amino acids in length (Figure 1c), which is consistent with the property of tryptic peptides. Among the 325 acetylated proteins, 198 contained one Kac site and most of the other 127 acetylated proteins contained two to four Kac sites (Figure 1d). A total of 75 and 65 proteins were distinctively acetylated in EP155 and EP155/CHV1-EP713, respectively (Table S3). Further quantitative analysis of acetylated peptides was conducted between EP155 and EP155/CHV1-EP713. A significant difference in acetylation is defined as changes in acetyl levels of more than 2-fold and p values less than 0.05. The results showed that hypovirus infection induced 66 up-regulated Kac sites in 43 proteins and 42 down-regulated Kac sites in 37 proteins (Table S4).
Based on western blot analysis and proteomics screening, hypovirus infection induced a wide change in global fungal proteins at the lysine acetylation level.

| Conserved motif analysis of lysine acetylation sites
To identify possible motifs around the Kac sites, the sequences of all acetylated peptides were analysed to identify potential motifs using the Motif-X software. The preferences for amino acid profiles were observed from positions −7 to +7 around the Kac sites. Nine enriched Kac site motifs were identified in the C. parasitica acetylome ( Figure 2a and Table S5). In particular, motifs A*Kac, A****Kac, and EKac occupied the highest proportion (Kac represents acetylated lysine and * represents a random amino acid residue). The acetylated peptides with these motifs were 71, 56, and 53, which account for 20.2%, 16%, and 15% of all the identified peptides, respectively ( Figure 2b and Table S4). Importantly, although most of the acetylation motifs identified in C. parasitica were also found in other eukaryotes Guo et al., 2017;Liu et al., 2016;Zhu et al., 2016), the significantly conserved motifs (A*Kac, A****Kac,  and Ekac) were rarely identified in other organisms. Because the acetylated peptides with the three motifs account for 51.2% of all identified peptides, it is likely that proteins with these motifs have some special function in C. parasitica.

| Bioinformatics analysis of differentially lysine acetylated proteins
To investigate the functions and features of the differentially acetylated proteins, Gene Ontology (GO) functional classification and subcellular location prediction were performed. The first two distributed biological processes were metabolic process and cellular process on hypovirus infection ( Figure S2). The results for the molecular function category showed that binding-and catalytic activity-related proteins were preponderant because their percentages reached 49% and 43%, respectively. For the cellular component category, most of the differentially acetylated proteins belonged to the cell, organelle, and macromolecular complex. Subcellular location prediction results showed that differentially acetylated proteins were distributed in diverse subcellular locations, with the nucleus (31%), cytoplasmic matrix (29%), and mitochondria (27%) being prominent ( Figure S3).
To further elucidate the functionality of lysine acetylation of proteins regulated by hypovirus, enrichment analysis based on GO and Kyoto Encyclopedia of Genes and Genomes (KEGG) pathway was performed ( Figure 3). In the up-regulated proteins ( Figure 3a and Table S6), metabolic processes and biosynthetic processes were enriched in the biological process category. Consistently, results for molecular function showed that many up-regulated proteins were associated with binding and catalytic activity. In the cellular component category, The results of KEGG analysis revealed that the most enriched pathways for the up-regulated proteins were glycolysis or gluconeogenesis, amino sugar and nucleotide sugar metabolism, and carbon metabolism ( Figure 3c and Table S8). However, proteins with downregulated acetylation levels were enriched for carbon fixation in photosynthetic organisms ( Figure 3d and Table S9). Changes in the most significantly enriched pathway, glycolysis or gluconeogenesis are shown in Figure 4. As indicated in the map, 13 proteins in this pathway were up-regulated at the lysine acetylation level, including phosphogluconate dehydrogenase (Pgls), phosphoglucose isomerase (Pgi), fructose-bisphosphate aldolase (Fba), triose-phosphate isomerase (Tpi), glyceraldehyde-3-phosphate dehydrogenase (GAPDH), phosphoglycerate kinase (Pgk), phosphoglycerate mutase (Pgam), phosphopyruvate hydratase (Eno), pyruvate kinase (PK), pyruvate decarboxylase (Pdc), alcohol dehydrogenase (Adh), dihydrolipoamide acetyltransferase (DLAT), and dihydrolipoyl dehydrogenase (DLD). Based on these results, it appears that the differentially acetylated proteins are closely linked to metabolism in C. parasitica.

| Hypovirus infection increases CpCS acetylation
MS data revealed that citrate synthase (CpCS) has an acetylated peptide (Figure 5a), and its acetylation level in EP155/CHV1-EP713 was 5.1-fold higher than that in EP155. To validate the acetylome results, immunoprecipitation and western blotting was performed using CpCS from EP155 and EP155/CHV1-EP713 (Figure 5b,c). The results revealed no significant differences in the level of CpCS expression between the two samples, but the acetylation status of CpCS was up-regulated 6.1-fold in EP155/CHV1-EP713. The result indicates that the acetylation level of CpCS was increased by hypovirus infection.

| Lysine acetylation of CpCS is important for its function in C. parasitica
According to the result shown in Figure 5a, a reliable acetylation site (lysine-55) was identified on CpCS. Additional amino acid sequence alignment results showed that lysine-55 of CpCS was F I G U R E 4 Schematic representation of the differentially expressed acetylated proteins involved in glycolysis or gluconeogenesis. Cryphonectria parasitica wild-type strain EP155 was set as reference. The average change (Cryphonectria hypovirus 1-infected strain EP155/CHV1-EP713 versus EP155) were obtained from three independent experiments. A value of 10 e means that the protein acetylation appears only in EP155/CHV1-EP713 and a value of −10 e means that the protein acetylation appears only in EP155. The proteins in red are up-regulated and those in green are down-regulated by hypovirus infection. Proteins in blue are expressed without significant differences. Hxk, hexokinase; Pgls, phosphogluconate dehydrogenase; Pgi, phosphoglucose isomerase; PfkA, phosphofructokinase; Fba, fructosebisphosphate aldolase; Tpi, triose-phosphate isomerase; GAPDH, glyceraldehyde-3-phosphate dehydrogenase; Pgk, phosphoglycerate kinase; Pgam, phosphoglycerate mutase; Eno, phosphopyruvate hydratase; PK, pyruvate kinase; Pdc, pyruvate decarboxylase; Adh, alcohol dehydrogenase; DLAT, dihydrolipoamide acetyltransferase; DLD, dihydrolipoyl dehydrogenase.
highly conserved in C. parasitica orthologues, indicating that this residue may be necessary for the conserved function of CpCS ( Figure S4). Recombinant CpCS was overexpressed in Escherichia coli ( Figure 6a) and purified to test whether the lysine-55 acetylation would affect the enzymatic activity of CpCS. The modified residue was mutated to glutamine (K55Q) and arginine (K55R) to mimic the acetylation and deacetylation state on lysine as previous studies, respectively (Guan et al., 2021;You et al., 2019).
The successful construction of both mutant plasmids was verified by DNA sequencing results ( Figure S5). Compared with wild-type (WT) protein, CpCS-K55Q displayed significantly reduced enzyme activity. Meanwhile, the replacement of lysine-55 with arginine resulted in a decrease in the acetylation level of CpCS and a significant increase in its enzyme activity (Figures 6 and S6). These data indicate that the acetylation of lysine-55 is critical for CpCS enzyme activity in vitro.
To further examine the potential function of lysine acetylation on CpCS in C. parasitica, we first constructed the CpCS null mutant using replacement with the hygromycin-resistance gene (hph). The single-spored transformants were screened by PCR and confirmed by Southern blotting ( Figure S7). When the CpCS null mutant was grown on a potato dextrose agar (PDA) plate, it displayed a defect in growth rate and had an irregular colony margin relative to the wildtype strain EP155 and parent strain KU80 (Figure 7a). In addition, the deletion of CpCS resulted in a remarkable reduction in virulence and sporulation. The complemented strain ∆CpCS-com restored the abnormal phenotypes compared with the parent strain (Figure 7b-d).
Next, the sequencing-confirmed CpCS lysine-55 mutated genes F I G U R E 5 Verification of acetylated citrate synthase (CpCS) in Cryphonectria parasitica wild-type strain EP155 and Cryphonectria hypovirus 1-infected strain EP155/CHV1-EP173. (a) MS/MS spectra of acetyl-peptide. The peptide FAELLPEKIEEIK(ac)ALR is from CpCS. The acetylation site is indicated by ac. (b) CpCS was immunoprecipitated from C. parasitica cell lysate and detected by western blotting using an anti-acetyl-lysine antibody (anti-Ack) or antibody specific for CpCS. Cell lysate without immunoprecipitation was used as the input. (c) Quantification of CpCS acetylation level. Average levels of CpCS acetylation were quantified using ImageJ and normalized relative to the value obtained in EP155, based on the acetylome data and western blotting (IP-WB). The IP-WB assays were repeated in triplicate. The error bars represent standard deviations. The asterisks (**) indicate a statistically significant difference from EP155 (p ≤ 0.01, t test).
were introduced into the CpCS null mutant using pCPXG418-CpCS (K55Q) and pCPXG418-CpCS (K55R). Compared with the wild-type and ∆CpCS-com strains, the site-specific mutant ∆CpCS-com (K55Q) grew much more slowly on PDA plates and exhibited reduced pigment, virulence, and conidial spores. In contrast, the colony morphology and growth rate of the site-specific mutant ∆CpCS-com (K55R) were similar to the wild-type and ∆CpCS-com strains. However, obvious decreases in sporulation and virulence occurred in the mutant.
Mutation of K55R had less impact on virulence and sporulation than mutation of K55Q (Figure 7). This finding suggests that the acetylation of lysine-55 is important in regulating CpCS function in vivo.

| DISCUSS ION
Protein lysine acetylation is one of the most ubiquitous posttranslational modifications that fine-tunes the major cellular processes of many life forms (Narita et al., 2019), but little is known about how this modification functions in fungi. In this study, the lysine acetylome of C. parasitica was determined to examine the effects of CHV1 infection on protein lysine acetylation profiles. To our knowledge, this study is the first comprehensive analysis of lysine acetylated proteins in response to a hypovirus infection covering the entire acetylome of a fungus.
A total of 616 unique acetylation peptides, encompassing 638 high-confidence Kac sites, were identified from 325 C. parasitica proteins. Most proteins were acetylated at a single lysine site. However, some acetylated proteins, including histone H2B (11 sites), GAPDH (nine sites), Eno (nine sites), acetyl-CoA acetyltransferase (ACAT1, nine sites), and PK (nine sites), contained multiple modification sites. These results suggest that lysine acetylation is abundant in these proteins. Emerging evidence has shown that nearly all major metabolic enzymes undergo acetylation, demonstrating the existence of a common mechanism of acetylation involved in metabolic regulation Huang et al., 2014;Wang et al., 2010). Consistent with these findings, a large proportion of metabolic enzymes involved in central metabolism were found to be acetylated, such as enzymes involved in glycolysis or gluconeogenesis, the TCA cycle, and fatty acid metabolism, implying that lysine acetylation may regulate cellular metabolic processes in C. parasitica. Additionally, protein acetylation occurs in proteins involved in the ribosome, mitochondrion, and protein translation and folding, which is consistent with previous studies . In this study, some modification enzymes, including methyltransferase (protein ID: 226763, 263,282) and N-acetyltransferase (protein ID: 103287, 247,896, 248,339, 258,510), were found to be acetylated. In agreement with these observations, AflO-a key O-methyltransferase for aflatoxin synthesis in A. flavus-was found to be acetylated at K241 and K384. Lysine acetylation plays a vital role in the regulation of the enzymatic activity of AflO (Yang et al., 2019). Some proteomic studies have also observed lysine acetyltransferases among acetylated proteins, which can acetylate specific lysine residues in histones or other proteins Vogelauer et al., 2012). Notably, acetyltransferases were shown to play a critical role in morphogenetic hyphae growth, biofilm formation, drug resistance, and virulence of fungi (Dubey et al., 2019;Zhang et al., 2021). Overall, these findings suggest that lysine acetylation is widely distributed in C. parasitica and may play an important regulatory role in diverse biological processes. Previous studies have reported that human Borna disease virus infection alters the acetylome of host cells to increase energy levels and transporters . Comparative proteomic analysis of lysine acetylation in fish CIK cells suggested that cellular metabolism was greatly altered due to grass carp reovirus .
In silkworm cells, baculovirus infection globally impacted the acetylome of host cells. A total of 6.96% and 14.8% of 431 quantified proteins were significantly up-or down-regulated, respectively, and were mostly involved in metabolism and biosynthesis (Hu et al., 2018). Recently, Murray et al. (2018) reported that protein lysine acetylation regulates both virus replication and antiviral defence by switching on and off protein functions. To evaluate the impact of fungal viruses on host protein acetylation, intensive proteomic quantification analysis was used to generate lysine acetylation profiles of C. parasitica with or without CHV1 infection. Consistent with other reports, the lysine acetylome of host proteins was found to be changed in response to hypovirus infection (Figure 1a). Furthermore, many acetylated proteins affected by hypovirus infection were found to be involved in metabolic processes according to GO classification ( Figure S2). KEGG pathway enrichment analysis revealed significant enrichment of up-regulated proteins in glycolysis or gluconeogenesis, amino sugar and nucleotide sugar metabolism, and F I G U R E 7 Analysis of phenotypes, virulence, and sporulation of CpCS deletion mutants. (a) Mutant colony morphologies on potato dextrose agar plates. Photographs were taken 7 and 14 days after inoculation. The strains indicated are wild-type EP155, its Cryphonectria hypovirus 1-infected isogenic strain EP155/CHV1-EP713, the gene disruption strain KU80, CpCS deletion strain ∆CpCS, the complementation strain ∆CpCS-com, and the mutant complementation strains ∆CpCS-com (K55Q) and ∆CpCS-com (K55R). (b) Cankers induced by the tested strains on dormant stems of Chinese chestnut. The inoculated stems were kept at 26°C and cankers were measured and photographed 25 days after inoculation. (c) Canker size measurements of the tested strains. (d) Sporulation levels of the indicated strains. Spores were counted on day 14. The error bars represent standard deviations from three independent experiments. The asterisks (**) indicate a statistically significant difference from EP155 (p ≤ 0.01, t test). carbon metabolism (Figure 3c). Notably, most glycolytic enzymes from the CHV1-infected strain had higher levels of acetylation compared with enzymes from the wild-type strain, suggesting that this modification may modulate enzyme activity (Figure 4). GAPDH was one of the differentially acetylated enzymes. GAPDH is an obligatory enzyme in glycolysis. GAPDH acetylation increases its activity and promotes cell proliferation and tumour growth (Li et al., 2014).
Pgk is a crucial enzyme in glycolysis. The acetylation of Pgk1 (K323) reportedly promotes its enzyme activity and cancer cell metabolism . PK is involved in the last step of glycolysis. A recent study found that increasing PKM2 hyperacetylation may be closely related to hepatotoxicity by increasing its activity and intracellular lactate concentration (Na et al., 2021). In light of the critical role of glycolysis in cellular energy production, it is reasonable to speculate that CHV1 perturbs the energy balance by regulating the acetylation of metabolic enzymes. Consistent with our findings, a metabolomic analysis in another study found that CHV1 infection increased the metabolic rate of C. parasitica, resulting in greater glucose depletion (Dawe et al., 2009). Nevertheless, more research is needed on how hypovirus infection alters global cellular acetylation dynamically.
Comparison of this acetylome with previous reported proteomic results (Wang et al., 2013  Viral proteins are also reported to be acetylated by the host acetyltransferase, which may be relevant for viral replication (Kumar et al., 2020). For example, human immunodeficiency virus Tat acetylation serves as a critical step in its transcriptional activity (Ott et al., 2004). Lysine acetylation regulates the activity of influenza virus proteins, such as NP (K77, K113, and K229) and NS1 (K108), which are important for viral replication and growth (Giese et al., 2017;Ma et al., 2020). Murray et al. (2018) demonstrated that the acetylation of human cytomegalovirus transcriptional activator pUL26 inhibits virus production. Recently, 39 Kac sites were identified in 22 acetylated Bombyx mori nucleopolyhedroviral proteins (Hu et al., 2018). In this study, no viral proteins were found to be acetylated based on the acetylome of EP155/CHV1-EP713. It is possible that the regulatory mechanism of mycoviruses differs significantly from that of animal viruses. However, how posttranslational modification functions in mycovirus replication remains unclear.
Among all metabolic pathway enzymes, TCA cycle enzymes are acetylated and involved in the final common oxidative pathway for carbohydrates, fats, and amino acids in other organisms (Wang et al., 2010). This study found that six proteins involved in the TCA cycle were acetylated, suggesting a potentially conserved role of acetylation in regulating the pathway. Citrate synthase catalyses the first reaction of the TCA cycle, playing an important role in central metabolism and amino acid biosynthesis (Weitzman, 1980).
The data of this study show that CpCS contains an acetylated peptide, and its acetylation was 5.1-fold greater in EP155/ CHV1-EP713 than in EP155. We hypothesized that lysine acetylation may be important for regulating fungal phenotypic traits by affecting CpCS function. To investigate the role of lysine acetylation in CpCS enzyme activity, the protein was expressed in E. coli and proteins containing point mutations were constructed. While the K55Q mutation decreased the enzyme activity of CpCS, the K55R mutation increased it ( Figure 6). Consistently, the enzyme activity of eukaryotic type I citrate synthase was reported to be significantly reduced when acetylation increased (Cui et al., 2017).
A similar observation was reported in E. coli type II citrate synthase (Venkat et al., 2019). Furthermore, in this study, CpCS was deleted and point mutant strains were constructed to investigate the role of lysine acetylation on the growth, sporulation, and virulence of C. parasitica. K55Q and K55R mutants showed a significant decrease in sporulation and virulence (Figure 7). Thus, it is proved that the lysine acetylation of CpCS could affect the enzyme activity and fungal phenotypic traits in C. parasitica.
In summary, these results represent the first extensive data on lysine acetylation in C. parasitica with or without CHV1 infection.
Although these findings improve our understanding of fungal protein regulation by a hypovirus from a lysine acetylation perspective, further experiments will be needed to investigate this interesting phenomenon.

| Fungal strains and growth conditions
The C. parasitica WT strain EP155 (ATCC 38755), its isogenic strain EP155/CHV1-EP713 (EP155 infected with synthetic hypovirus CHV1-EP713, ATCC 52571) (Chen et al., 1994), and a highly efficient gene disruption strain KU80 (∆ku80 of EP155)  were used in this study. For morphological characterization and DNA and RNA extraction, the strains were grown on PDA at 26°C with a 12 h light/dark cycle (Hillman et al., 1990). For protein extraction, the strains were cultured in EP complete liquid medium for 3 days with shaking at 100 rpm (Puhalla & Anagnostakis, 1971).

| Protein extraction
Fungal proteins were extracted based on a protocol modified from Wang et al. (2014). Briefly, fungal mycelia were ground into powder in liquid nitrogen, resuspended in five-fold volumes of prechilled trichloroacetic acid:acetone (1:9), incubated at −20°C overnight, and centrifuged at 7000 g for 15 min at 4°C. The supernatant was discarded and the precipitate was washed three times with precooled acetone. The precipitate was air dried and resuspended in lysis buffer (8 M urea, 50 mM Tris-HCl pH 8.0, 80 mM dithiothreitol, 2% protease inhibitor). The suspension was ultrasonicated 10 times at 100 W (for 10 s, with 10 s intervals) and centrifuged at 15,000 g for 20 min. Protein concentration was quantified with the Bradford assay (Bio-Rad) and the protein was stored at −80°C until use.

| Trypsin digestion and affinity enrichment of acetylated proteins
Protein digestion was performed with trypsin, as described, with some minor modifications . In brief, the protein sample was diluted with 100 mM NH 4 CO 3 to a urea concentration of about 1 M. Then, trypsin (Promega) was added at 1:50 trypsin: protein mass ratio overnight at 37°C. Finally, the peptides of each sample were desalted on the C18-SD extraction disk cartridge and vacuum-dried.
Samples of three biological triplicates were subjected to lysine-acetylated peptide enrichment (Rappsilber et al., 2007). The

| LC-MS/MS analysis
The enriched peptides were analysed with the EASY-nLC1000 system (Thermo Finnigan) connected to a Q Exactive mass spectrometer (Thermo Finnigan). Briefly, peptide samples were dissolved in 0.1% formic acid, directly loaded onto a reverse-phase precolumn (Thermo EASY column SC200, 150 μm × 100 mm). A reverse-phase analytical column (Thermo EASY column SC001 traps, 150 μm × 20 mm) was used for peptide separation, as described previously (Li, Sun, et al., 2016). The resulting peptides were evaluated with the Q Exactive mass spectrometer for 120 min. The mass spectrometer was operated in positive ion mode. MS data were acquired using a data-dependent procedure, dynamically choosing the most abundant precursor ions from the survey scan (350-1800 m/z) for high-energy collision dissociation fragmentation. Survey scans were acquired at a resolution of 70,000 at m/z 200 and the resolution for high-energy collision dissociation spectra was set to 17,500 at m/z 200. Automatic gain control was used to prevent overfilling of the ion trap and 5 × 10 4 ions were accumulated for generating the MS/MS spectra. Each LC-MS/MS analysis was repeated three times to reduce technical variation.

| Data analysis
The resulting MS/MS data were processed using the MaxQuant search engine (v. 1.5.2.8). Tandem mass spectra were searched against the C. parasitica database (http://genome.jgi-psf.org/Crypa 2/Crypa2.home.html) concatenated with the reverse decoy database. Trypsin was specified as a cleavage enzyme, allowing up to four missing cleavages. Mass tolerance for precursor ions was set at 20 ppm in the first search and 6 ppm in the main search. Mass tolerance for fragment ions was set as 0.02 Da. Carbamidomethyl on Cys was specified as a fixed modification, and oxidation on Met, acetylation on Lys, and acetylation on protein N-terminal were specified as variable modifications. False discovery rate thresholds were specified at 1%. All the other parameters in MaxQuant were set to default values. The quantitative value of each acetylated peptide was calculated based on intensity information derived from LC-MS data as previously described . Then the ratio of the average value between the two samples was calculated.

| Bioinformatics analysis
Proteins were classified using GO database annotation (Hulsegge et al., 2009), based on three categories: biological process, cellular component, and molecular function. KEGG (Moriya et al., 2007) was used to annotate protein pathways. The WoLF PSORT software was used to analyse subcellular localization (Horton et al., 2007). Functional enrichment analysis was performed using the DAVID Bioinformatics Resources 6.7 (Huang et al., 2007). The motif-x software was used to analyse the model of sequences constituted with amino acids in specific positions of acetyl-15-mers (seven amino acids upstream and downstream of the site) in all protein sequences (Chou & Schwartz, 2011). Functional interaction network analysis was performed using the publicly available program STRING (http://string-db.org/).

| Immunoprecipitation and western blotting
To verify the acetylation of CpCS, immunoprecipitation was performed as reported (Mo et al., 2015). Equal amounts (50 μg total protein) were incubated with 1 μg of the anti-CpCS antibody (preserved in our laboratory) in a microcentrifuge tube overnight at 4°C.
Then, the antibody-lysate sample was added to 20 μL of protein A/G plus agarose in the spin column with gentle end-over-end mixing for 1 h. The conjugated resin was washed three times with the immunoprecipitation lysis/wash buffer to remove unbound proteins. Bound proteins were boiled with SDS loading buffer for 10 min, separated using 12% SDS-PAGE, and transferred to a polyvinylidene fluoride membrane (Millipore) using the Hoefer TE 77 semidry transfer unit (Hoefer). For western blotting, the membranes were blocked with 5% nonfat milk in Tris-buffered saline with 0.1% Tween 20 (TBST) for 1 h. Primary anti-acetyl-lysine antibody (Cell Signalling Technology) or CpCS antibody were diluted at 1:1000 in TBST. The membranes were incubated overnight at 4°C with the antibodies, washed with TBST, incubated with a horseradish peroxidase-conjugated secondary antibody (1:5000) for 1 h at 37°C, and detected according to the manufacturer's instructions.

| Measurement of CpCS activity
For expressing recombinant CpCS in E. coli, a cDNA fragment of CpCS was amplified by PCR using primers CpCS-Ex-F and CpCS-Ex-R (Table S1). The PCR product was cloned into the prokaryotic expression vector pGEX-4T-1 (GE Healthcare) to generate pGEX-4T-1-CpCS.

| Generation of fungal mutants
CpCS deletion mutants were generated using the KU80 strain and homologous recombination method . Sequences upstream and downstream of CpCS in the genomic DNA were amplified using the primers listed in Table S1. The hygromycin B resistance (hph) marker was amplified using plasmid pCPXHY2 as template with primers Hyg-F and Hyg-R (Chen et al., 2011). The upstream and downstream DNA sequences and hph were joined by overlapping PCR. The purified PCR products were transformed into KU80 protoplasts. Transformants were selected on PDA supplemented with hygromycin. Putative CpCS deletion mutants were confirmed using PCR (primers CpCS-all-F and CpCS-all-R; Table S1) and Southern blotting, and purified to nuclear homogeneity by single-spore isolation (Sambrook & Russell, 2001). To generate the complementation strain ΔCpCS-com, the complete open reading frame of CpCS (including the promoter sequence) was amplified and inserted into the transformation vector pCPXG418 (containing the G418 resistance marker) (Chen et al., 2011), generating the wild-type gene complementation plasmid pCPXG418-CpCS. The resulting plasmid was used for the generation of the K55Q and K55R mutant plasmids using the Mut Express II fast mutagenesis kit V2 (Vazyme Biotech). Each mutant plasmid was confirmed by DNA sequencing.
pCPXG418-CpCS, pCPXG418-CpCS (K55Q) and pCPXG418-CpCS (K55R) were transformed into the knockout strain ΔCpCS, respectively. Complementation was selected on PDA supplemented with hygromycin and G418, and confirmed by PCR with primers CpCSall-F and CpCS-all-R. All primer sequences used in the construction and verification of fungal mutants were shown in Table S1.

| Virulence assay
For the fungal virulence analysis, dormant stems of Chinese chestnut (Castanea mollissima) were inoculated and incubated in a plastic bag at 26°C to allow lesion development, as described (Liao et al., 2012).
Canker size was observed and analysed 25 days after inoculation. The infection experiments were repeated three times for each fungal strain.

ACK N O WLE D G E M ENTS
This work was supported by the National Natural Science Foundation of China (grant 31760498).

DATA AVA I L A B I L I T Y S TAT E M E N T
The data that support the findings of this study are available from the corresponding author upon reasonable request.