Comparative acetylome analysis reveals the potential roles of lysine acetylation for DON biosynthesis in Fusarium graminearum
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Fusarium graminearum is a destructive fungal pathogen of wheat, barley and other small grain cereals. During plant infection, the pathogen produces trichothecene mycotoxin deoxynivalenol (DON), which is harmful to human and livestock. FgGCN5 encodes a GCN5 acetyltransferase. The gene deletion mutant Fggcn5 failed to produce DON. We assumed that lysine acetylation might play a key regulatory role in DON biosynthesis in the fungus.
In this study, the acetylome comparison between Fggcn5 mutant and wild-type strain PH-1 was performed by using affinity enrichment and high resolution LC-MS/MS analysis. Totally, 1875 acetylated proteins were identified in Fggcn5 mutant and PH-1. Among them, 224 and 267 acetylated proteins were identified exclusively in Fggcn5 mutant and PH-1, respectively. Moreover, 95 differentially acetylated proteins were detected at a significantly different level in the gene deletion mutant:43 were up-regulated and 52 were down-regulated. GO enrichment and KEGG-pathways enrichment analyses revealed that acetylation plays a key role in metabolism process in F. graminearum.
Seeing that the gens playing critical roles in DON biosynthesis either in Fggcn5 mutant or PH-1. Therefore, we can draw the conclusion that the regulatory roles of lysine acetylation in DON biosynthesis in F. graminearum results from the positive and negative regulation of the related genes. The study would be a foundation to insight into the regulatory mechanism of lysine acetylation on DON biosynthesis.
KeywordsFusarium graminearum Deoxynivalenol Lysine acetylation Acetylome
Automatic gain control
Enzyme-linked immunosorbent assay
False discovery rate
Fusarium head blight
High Performance Liquid Chromatography
Kyoto Encyclopedia of Genes and Genomes
Potato dextrose agar
cAMP- dependent protein kinase
Fusarium graminearum is a disastrous fungal pathogen which causes Fusarium head blight (FHB) on wheat, barley and other small grain cereals [1, 2]. In addition to the severe yield loss and quality damage, the pathogen produces trichothecene-type mycotoxins, such as deoxynivalenol (DON) in the infected tissue. DON is a secondary metabolite, which contributes to the spread of the fungus in the spikelet and contaminates cereal grains and cereal-based products, resulting in a threat to the health of human and livestock [3, 4].
Lysine acetylation is a conserved post-translational modification (PTM) of proteins occurring both in eukaryotes and prokaryotes. The modification consists of two reversible reactions: the acetylation, in which the acetyl-groups were added to the lysine residues of target protein by lysine acetyltransferase (KAT); in contrast, the deacetylation is a reversed process to remove the acetyl-groups from the acetylated proteins by lysine deacetylase (KDAC) [5, 6]. The balance of acetylation/deacetylation status of proteins is dynamically regulated by KATs and KDACs in order to achieve their proper roles during numerous cellular processes such as cell morphology, metabolic pathways, protein synthesis [7, 8, 9]. The acetylation was first identified in histone proteins, whose acetylated form is responsible for the structure remodeling of the chromatin and activation of genes expression [10, 11]. In recent years, the protein acetylation has been widely studied by using advanced mass spectrometry based proteomics tool. Global analyses of acetylome have been successfully performed in plants [12, 13], fungi [14, 15], and prokaryotes [16, 17], revealing that acetylation contributes to diverse protein functions in living cells, including protein localization, enzymatic activity, protein-protein and protein-nucleic acids interaction [18, 19, 20].
The lysine acetylation also plays a crucial role in regulating central metabolism as the extensively acetylated enzymes responsible for metabolism have been found in both eukaryotes and prokaryotes [9, 17, 21]. For instance, most enzymes involved in glycolysis, the tricarboxylic (TCA) cycle, gluconeogenesis, the urea cycle, and fatty acid metabolism were acetylated in human liver tissue . A global acetylome analysis in Salmonella enterica revealed that about 90% of the enzymes of central metabolism were found to be acetylated . In addition, the protein acetylation is also involved in the secondary metabolism process, such as nonribosomal peptide synthesis, hydroxamate siderophore and phosphinic acid products biosynthesis .
The gene FgGCN5 (FGRAMPH1_01T00753) in F. graminearum PH-1 encodes a GCN5 acetyltransferase. The most attractive defect of the gene deletion mutant is the functional block in DON biosynthesis , indicating that the gene plays a crucial role in producing DON in the fungus. To reveal the potential roles of lysine acetylation in DON biosynthesis, we performed a global acetylome comparison between the gene deletion mutant Fggcn5 and the wild-type strain PH-1. Totally, 2626 acetylated lysine sites in 1875 proteins were identified in Fggcn5 mutant and PH-1.
Results and discussion
Difference of the acetylated proteins between the wild type and Fggcn5 deletion mutant
To identify proteins acetylated by FgGCN5, total proteins were isolated from PH-1 and Fggcn5 mutant. After digestion with trypsin, lysine-acetylated peptides were enriched with the anti-acetyl-lysine antibody and analyzed with LC-MS/MS as described . A total of 2626 lysine acetylation sites (Additional file 1: Table S1) were identified in 1875 proteins from PH-1 and Fggcn5 mutant (Additional file 2: Table S2). Among them, 95 proteins were differentially acetylated at a significant level of Ratio > + /− 2 (p < 0.05) in the Fggcn5 deletion mutant in comparison with PH-1. 43 proteins were up-regulated, 52 were down-regulated in the mutant (Additional file 3: Table S3). It is possible that the acetylation down-regulated proteins in the Fggcn5 mutant function in a positive slight role in the DON biosynthesis, while the up-regulated proteins play the opposite role.
Acetylated proteins specially detected in wild type strain PH-1
Rapamycin binding protein
Virulence, hyphae growth
DON, virulence, sexual and asexual
DON, virulence and development
Glycogen synthase kinase
DON, virulence and development
DON, virulence, sexual and asexual
Acetylated proteins specially detected in Fggcn5 mutant
DON, virulence and development
DON, virulence and development
DON, virulence and development
DON, virulence and development
Virulence and development
DON, virulence and development
Sexual and asexual development
Asexual and vegetative growth
The abundance of acetylated proteins detected in this study indicated that lysine acetylation is a common protein modification in F. graminearum similar to the observations in other living organisms [12, 13, 14, 15]. Approximately 14.24% of the acetylated proteins identified in this study were only detected in the wild type strain and are potential targets of FgGCN5 lysine acetyltransferase.
Functional annotation and enrichment analysis of the proteins differentially acetylated in PH-1 and the Fggcn5 mutant
Furthermore, the GO enrichment analysis was performed to identify the biological processes and molecular functions of the acetylated proteins (Fig. 2b, Additional file 5: Table S5). The results showed that the acetylated proteins identified in this study were significantly enriched in several GO biological processes, including monocarboxylic acid metabolism, pyridine nucleotide metabolism, nicotinamide nucleotide metabolism, pyruvate metabolic process, glucose 6-phosphate metabolic process, glyceraldehyde-3- phosphate metabolic process, and NADP metabolic process. In the GO molecular functions, most of the acetylated proteins were significantly enriched in structural molecular activity, structural constituent of ribosome, oxidoreductase activity. From the GO cellular compartment categories, we found that a great proportion of the identified acetylated proteins were in intracellular non-membrane-bounded organelles, ribonucleoprotein complexes, and ribosome.
Protein-protein interaction network analysis
Proteins acetylated in PH-1 involved in DON biosynthesis
Since the Fggcn5 gene deletion mutant was defective in DON biosynthesis, lysine acetylation manipulated by FgGCN5 likely plays important regulatory roles in DON biosynthesis in F. graminearum. In this study, we found that some proteins involved in DON production are the potential acetylation targets of FgGCN5.
GzBrom002 (FGSG_06291) encoding a transcription factor plays essential roles in DON production, virulence, asexual and sexual reproduction. The gene deletion mutant GzBrom002 completely lost virulence on wheat, ability in DON production and asexual and sexual spores production . A Homocysteine transferase gene (FGSG_10825) is also multifunctional in F. graminearum. Phenotype assays showed that the virulence and DON production were reduced in the gene deletion mutant Moreover, the mutant failed to produce perithecia and aerial mycelia . Another gene FGK3 (FGSG-07329), encodes a glycogen synthase kinase orthologous to mammalian GSK3. The gene deletion mutant Δfgk3 is defective in DON production . FgGCN5 might positively regulate DON biosynthesis through acetylating these proteins.
It has been well demonstrated that cAMP- dependent protein kinase (PKA) plays critical roles in DON biosynthesis in F. graminearum [46, 47]. In this study, PKR (FGSG_09908), the regulatory subunit of PKA, was found to be acetylated in PH-1 but not in the Fggcn5 mutant. The result indicates that the PKR may be one of the substrates of FgGCN5 acetyltransferase in F. graminearum. However, PKR acts as a negative factor in DON production as the DON content was increased in the gene deletion mutant of PKR . This suggests that the negative effect of PRK on DON biosynthesis may be limited by FgGCN5 through lysine acetylation.
Proteins acetylated in Fggcn5 mutant are associated with DON production
Proteins acetylated specifically in Fggcn5 mutant were identified as well, suggesting that the proteins are targets of other KATs rather than FgGCN5. Interestingly, some proteins were proved to be associated with DON biosynthesis.
It is well known on the functions of some TRI genes in DON biosynthesis. In this study, the acetylated TRI15 (FGSG_11205) was detected only in the Fggcn5 mutant. TRI15, encoding a Cys2-His2 zinc finger protein, acts as a negative regulator of the trichothecene biosynthetic genes [39, 48]. It is likely that TRI15 is activated by other KATs and thereby plays a negative role in DON production in Fggcn5 mutant.
Additionally, FgHXK1(FGSG_00500) encodes a rate-limiting enzyme in DON biosynthesis. DON production was severely inhibited in the gene deletion mutant. Moreover, the ΔFgHXK1 mutant is nonpathogenic on wheat, defective in hyphae growth and conidiation . Some transcription factors identified in this study were also characterized to play key roles in DON production and pathogen virulence including GzHMG002 (FGSG_00385), GzCCHC011 (FGSG_10716) and GzZC230 (FGSG_07133) . Recently, a ATP-binding cassette (ABC) transporte FgArb1 (FGSG_04181) was proved to function in pathogenesis and DON production in F. graminearum, as the virulence and DON production were dramatically reduced in the gene deletion mutant . It is likely that acetylation of these proteins by other KATs in the absence of FgGCN5 leads to the inactivation of the genes, and finally leads to the inhibition of the DON production in Fggcn5 mutant.
In summary, the acetylome comparison between Fggcn5 mutant and PH-1 was performed by high throughput proteomics analysis. The differentially acetylated proteins were identified. Our results indicate that genes play critical roles in DON production in Fggcn5 mutant or PH-1. Therefore, we can draw the conclusion that the DON biosynthesis in F. graminearum was properly regulated by lysine acetylation both in positive and negative ways. The study would be a foundation to insight into the regulatory mechanism of lysine acetylation on DON production.
Generation of Fggcn5 mutant
Primers for construction and identification of FgGCN5 gene deletion mutant
DON content measurement assay
DON content in LTB cultures [50, 51] was assayed with a competitive enzyme-linked immunosorbent assay (ELISA) based DON detection plate kit (Beacon Analytical Systems, Inc., USA) after incubation at 25 °C for 5 days, as described by Gardiner et al. . In the assay, the Tri5 deletion mutant was used as a negative control.
Strains culture conditions
The wild-type strain PH-1 and the gene deletion mutant Fggcn5 of F. graminearum were cultured on potato dextrose agar (PDA) at 25 °C for 4 d. The mycelia were harvested and ground with MiniBead Beater-16 (Biospec, USA) at 30 Hz for 30 s. The ground mycelium were then cultured in 100 mL liquid YEPD medium with shaking at 25 °C for 24 h. Subsequently, the mycelium was collected by filtration with sterile macrocloth and was then incubated in DON inducing medium in the dark with shaking at 25 °C for 4 d. The DON inducing medium was prepared as described by Gardiner et al. .
Protein extraction and trypsin digestion
The procedures of protein extraction and peptide digestion were modified from a previous report . In brief, the mycelia samples were harvested and ground into cell powder in liquid nitrogen. The resulting cell powder was then transferred into 5 × volume of TCA/acetone (1:9, containing 65 mM DTT) and mixed by vortex. After placed at − 20 °C overnight, the mixture was centrifuged at 7000×g for 20 min at 4 °C. The precipitate was washed with ice-cold acetone for three times and was air-dried at 4 °C. The dried precipitate was then resolved in UA buffer (8 M urea, 150 mM Tris-HCl, Ph8.0) and sonicated 10 times (10 s burst with a 15 s interval for each time) on ice using a high intensity ultrasonic processor (Scientz, Ningbo, China). After centrifuged at 14,000 g for 40 min, the supernatant was filtered with 0.45 μm filters. The filtrate was quantified with the BCA Protein Assay Kit (Bio-Rad, USA).
The resulting protein solution was reduced with 10 mM DTT for 1 h at 37 °C and alkylated with 20 mM iodoacetamide for 45 min at room temperature in darkness. For trypsin digestion, 100 mM (NH4)2CO3 was added to urea concentration less than 2 M. Finally, trypsin was added at an enzyme-to-substrate mass ratio of 1:50 overnight and additional trypsin was added at an enzyme-to-substrate mass ratio of 1:100 for 4 h to ensure complete digestion.
The sample was separated into fractions by high pH reverse-phase HPLC followed Zhou et al. . As a result, the tryptic peptides were separated into 6 fractions which were then dried by vacuum centrifuging.
The purification and enrichment of lysine acetylated peptides were performed as described [15, 52]. Briefly, tryptic peptides were re-dissolved in NETN buffer (100 mM NaCl, 1 mM EDTA, 50 mM Tris-HCl, 0.5% NP-40, pH 8.0). Subsequently, the pre-washed agarose-conjugated anti-acetyllysine antibody beads (Cat. No. 104, PTM Biolabs, Hangzhou, China) were added. The tryptic peptides were incubated at 4 °C overnight with gentle shaking. The beads were then washed four times with NETN buffer and twice with pure water. Finally, the bound peptides were eluted from the beads with 0.1% trifluoroacetic acid and were then vacuum-dried. The resulting peptides were cleaned with C18 ZipTips (Millipore, Billerica, MA) according to the manufacturer’s instructions before LC-MS/MS analysis.
LC-MS/MS analysis was performed as described  with modification. In the assay, a Q-Exactive mass spectrometer (Thermo Scientific) coupled to Easy nLC (Proxeon Biosystems, now Thermo Fisher Scientific) was used. The mass spectrometer was operated in positive ion mode. MS data was acquired using a data-dependent top10 method dynamically choosing the most abundant precursor ions from the survey scan (300–1800 m/z) for HCD fragmentation. Automatic gain control (AGC) target was set to 3e6, and maximum inject time to 10 ms. Dynamic exclusion duration was 40.0 s. Survey scans were acquired at a resolution of 70,000 at m/z 200 and resolution for HCD spectra was set to 17,500 at m/z 200, and isolation width was 2 m/z. Normalized collision energy was 30 eV and the underfill ratio, which specifies the minimum percentage of the target value likely to be reached at maximum fill time, was defined as 0.1%. The instrument was run with peptide recognition mode enabled.
The resulting MS/MS data was processed using MaxQuant with integrated Andromeda search engine (v.184.108.40.206). Tandem mass spectra were searched against UniProt_F. graminearum database concatenated with reverse decoy database. Trypsin was specified as the cleavage enzyme, the maximum missing cleavage was set as 4, and up to 5 modifications and 5 charges were allowed in each peptide. Mass error was set at 10 ppm for precursor ions and 0.02 Da for fragment ions. Carbamidomethylation on cysteine was specified as fixed modification and oxidation on methionine, acetylation on lysine and acetylation on protein N-terminal were specified as variable modifications. False discovery rate (FDR) thresholds for peptide and modification site were specified at 0.01. Minimum peptide length was set as 7. All the other parameters in MaxQuant were set to default values. The site localization probability was set as 0.75.
Acetylated protein annotation
Gene Ontology (GO) annotation of identified acetylated proteins was derived from the UniProt-GOA database (http://www.ebi.ac.uk/GOA/). Firstly, the identified protein ID was converted to UniProt ID, and then mapped to GO ID by protein ID. The proteins were classified by GO annotation based on three categories: biological process, cellular component and molecular function. The subcellular localization of the protein was predicted with WoLF PSORT (http://wolfpsort.seq.cbrc.jp/). Secondary structures of proteins were predicted by NetSurfP . Domain descriptions of identified protein were annotated by InterProScan5 based on protein sequence alignment, and the InterPro domain database (http://www.ebi.ac.uk/interpro/) was used. Kyoto Encyclopedia of Genes and Genomes (KEGG) database was used to annotate protein pathway . Functional annotation tool of DAVID bioinformatics resources 6.7 was used to identify GO terms, KEGG pathways and protein domains .
GO, KEGG pathway, domain and motif enrichment analysis
A two-tailed Fisher’s exact test was performed to examine the enrichment of the protein-acetylated entries against all proteins. Correction for multiple hypothesis testing was carried out using a previously described method . Any term with a corrected p < 0.05 was considered significant.
Acetylation protein–protein interaction network analysis
The protein–protein interaction was obtained from the STRING database following the methods [41, 25]. The protein–protein interaction network of the identified acetylated proteins was performed with Cytoscape software (version 3.2.1, www.cytoscape.org).
Significant differences between the mutant and wild type strain were calculated according to the peptide intensity using a one-way analysis of variance with SPSS16.05 version. The p values of 0.05 were considered to be statistically significant.
SZ designed the work, analyzed data and wrote the manuscript. CW conducted the experiments. Both authors have read and approved the final manuscript.
This study was supported by the open project of the State Key Laboratory of Crop Stress Biology for Arid Areas (CSBAA2016001). The funder has no role in the study.
Ethics approval and consent to participate
Consent for publication
The authors declare that they have no competing of interests.
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