Comparative acetylome analysis reveals the potential roles of lysine acetylation for DON biosynthesis in Fusarium graminearum.
ABSTRACT: BACKGROUND: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. RESULTS: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. CONCLUSIONS: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.
Project description:Deoxynivalenol (DON) is a mycotoxin produced in cereal crops infected with Fusarium graminearum. DON poses a serious threat to human and animal health, and is a critical virulence factor. Various environmental factors, including reactive oxygen species (ROS), have been shown to interfere with DON biosynthesis in this pathogen. The regulatory mechanisms of how ROS trigger DON production have been investigated extensively in F. graminearum. However, the role of the endogenous ROS-generating system in DON biosynthesis is largely unknown. In this study, we genetically analysed the function of leucine zipper-EF-hand-containing transmembrane 1 (LETM1) superfamily proteins and evaluated the role of the mitochondrial-produced ROS in DON biosynthesis. Our results show that there are two Letm1 orthologues, FgLetm1 and FgLetm2, in F. graminearum. FgLetm1 is localized to the mitochondria and is essential for mitochondrial integrity, whereas FgLetm2 plays a minor role in the maintenance of mitochondrial integrity. The ΔFgLetm1 mutant demonstrated a vegetative growth defect, abnormal conidia and increased sensitivity to various stress agents. More importantly, the ΔFgLetm1 mutant showed significantly reduced levels of endogenous ROS, decreased DON biosynthesis and attenuated virulence in planta. To our knowledge, this is the first report showing that mitochondrial integrity and endogenous ROS production by mitochondria are important for DON production and virulence in Fusarium species.
Project description:Protein lysine acetylation is a post-translational modification that alters the charge, conformation, and stability of proteins. A number of genome-wide characterizations of lysine-acetylated proteins, or acetylomes, in bacteria have demonstrated that lysine acetylation occurs on proteins with a wide diversity of functions, including central metabolism, transcription, chemotaxis, and cell size regulation. Bacillus subtilis is a model organism for studies of sporulation, motility, cell signaling, and multicellular development (or biofilm formation). In this work, we investigated the role of global protein lysine acetylation in multicellular development in B. subtilis. We analyzed the B. subtilis acetylome under biofilm-inducing conditions and identified acetylated proteins involved in multicellularity, specifically, swarming and biofilm formation. We constructed various single and double mutants of genes known to encode enzymes involved in global protein lysine acetylation in B. subtilis. Some of those mutants showed a defect in swarming motility while others demonstrated altered biofilm phenotypes. Lastly, we picked two acetylated proteins known to be important for biofilm formation, YmcA (also known as RicA), a regulatory protein critical for biofilm induction, and GtaB, an UTP-glucose-1-phosphate uridylyltransferase that synthesizes a nucleotide sugar precursor for biosynthesis of exopolysaccharide, a key biofilm matrix component. We performed site-directed mutagenesis on the acetylated lysine codons in ymcA and gtaB, respectively, and assayed cells bearing those point mutants for biofilm formation. The mutant alleles of ymcA(K64R), gtaB(K89R), and gtaB(K191R) all demonstrated a severe biofilm defect. These results indicate the importance of acetylated lysine residues in both YmcA and GtaB. In summary, we propose that protein lysine acetylation plays a global regulatory role in B. subtilis multicellularity.
Project description:Fusarium graminearum, the main pathogenic fungus causing Fusarium head blight (FHB), produces deoxynivalenol (DON), a key virulence factor, which is synthesized in the endoplasmic reticulum (ER). Sey1/atlastin, a dynamin-like GTPase protein, is known to be required for homotypic fusion of ER membranes, but the functions of this protein are unknown in pathogenic fungi. Here, we characterized Sey1/atlastin homologue FgSey1 in F. graminearum Like Sey1/atlastin, FgSey1 is located in the ER. The FgSEY1 deletion mutant exhibited significantly reduced vegetative growth, asexual development, DON biosynthesis, and virulence. Moreover, the ?Fgsey1 mutant was impaired in the formation of normal lipid droplets (LDs) and toxisomes, both of which participate in DON biosynthesis. The GTPase, helix bundle (HB), transmembrane segment (TM), and cytosolic tail (CT) domains of FgSey1 are essential for its function, but only the TM domain is responsible for its localization. Furthermore, the mutants FgSey1K63A and FgSey1T87A lacked GTPase activity and failed to rescue the defects of the ?Fgsey1 mutant. Collectively, our data suggest that the dynamin-like GTPase protein FgSey1 affects the generation of LDs and toxisomes and is required for DON biosynthesis and pathogenesis in F. graminearum IMPORTANCE Fusarium graminearum is a major plant pathogen that causes Fusarium head blight (FHB) of wheats worldwide. In addition to reducing the plant yield, F. graminearum infection of wheats also results in the production of deoxynivalenol (DON) mycotoxins, which are harmful to humans and animals and therefore cause great economic losses through pollution of food products and animal feed. At present, effective strategies for controlling FHB are not available. Therefore, understanding the regulation mechanisms of fungal development, pathogenesis, and DON biosynthesis is important for the development of effective control strategies of this disease. In this study, we demonstrated that a dynamin-like GTPase protein Sey1/atlastin homologue, FgSey1, is required for vegetative growth, DON production, and pathogenicity in F. graminearum Our results provide novel information on critical roles of FgSey1 in fungal pathogenicity; therefore, FgSey1 could be a potential target for effective control of the disease caused by F. graminearum.
Project description:Post-translational modifications of chromatin structure by histone acetyltransferase (HATs) play a central role in the regulation of gene expression and various biological processes in eukaryotes. Although HAT genes have been studied in many fungi, few of them have been functionally characterized. In this study, we identified and characterized four putative HATs (FgGCN5, FgRTT109, FgSAS2, FgSAS3) in the plant pathogenic ascomycete Fusarium graminearum, the causal agent of Fusarium head blight of wheat and barley. We replaced the genes and all mutant strains showed reduced growth of F. graminearum. The ?FgSAS3 and ?FgGCN5 mutant increased sensitivity to oxidative and osmotic stresses. Additionally, ?FgSAS3 showed reduced conidia sporulation and perithecium formation. Mutant ?FgGCN5 was unable to generate any conidia and lost its ability to form perithecia. Our data showed also that FgSAS3 and FgGCN5 are pathogenicity factors required for infecting wheat heads as well as tomato fruits. Importantly, almost no Deoxynivalenol (DON) was produced either in ?FgSAS3 or ?FgGCN5 mutants, which was consistent with a significant downregulation of TRI genes expression. Furthermore, we discovered for the first time that FgSAS3 is indispensable for the acetylation of histone site H3K4, while FgGCN5 is essential for the acetylation of H3K9, H3K18, and H3K27. H3K14 can be completely acetylated when FgSAS3 and FgGCN5 were both present. The RNA-seq analyses of the two mutant strains provide insight into their functions in development and metabolism. Results from this study clarify the functional divergence of HATs in F. graminearum, and may provide novel targeted strategies to control secondary metabolite expression and infections of F. graminearum.
Project description:The lysine acetylation of proteins is a reversible post-translational modification that plays a critical regulatory role in both eukaryotes and prokaryotes. Mycobacterium tuberculosis is a facultative intracellular pathogen and the causative agent of tuberculosis. Increasing evidence shows that lysine acetylation may play an important role in the pathogenesis of M. tuberculosis. However, only a few acetylated proteins of M. tuberculosis are known, presenting a major obstacle to understanding the functional roles of reversible lysine acetylation in this pathogen. We performed a global acetylome analysis of M. tuberculosis H37Ra by combining protein/peptide prefractionation, antibody enrichment, and LC-MS/MS. In total, we identified 226 acetylation sites in 137 proteins of M. tuberculosis H37Ra. The identified acetylated proteins were functionally categorized into an interaction map and shown to be involved in various biological processes. Consistent with previous reports, a large proportion of the acetylation sites were present on proteins involved in glycolysis/gluconeogenesis, the citrate cycle, and fatty acid metabolism. A NAD(+)-dependent deacetylase (MRA_1161) deletion mutant of M. tuberculosis H37Ra was constructed and its characterization showed a different colony morphology, reduced biofilm formation, and increased tolerance of heat stress. Interestingly, lysine acetylation was found, for the first time, to block the immunogenicity of a peptide derived from a known immunogen, HspX, suggesting that lysine acetylation plays a regulatory role in immunogenicity. Our data provide the first global survey of lysine acetylation in M. tuberculosis. The dataset should be an important resource for the functional analysis of lysine acetylation in M. tuberculosis and facilitate the clarification of the entire metabolic networks of this life-threatening pathogen.
Project description:Agmatine and other putrescines are known for being strong inducers of deoxynivalenol (DON) production in Fusarium graminearum. Other important species produce DON and/or other trichothecene type B toxins (3 acetylated DON, 15 acetylated DON, Fusarenon-X, Nivalenol), such as F. culmorum and F. poae. In order to verify whether the mechanism of the regulation of trichothecene type B induction by agmatine is shared by different species of Fusarium, we tested the hypothesis on 19 strains belonging to 3 Fusarium species (F. graminearum, F. culmorum, F. poae) with diverse genetic chemotypes (3ADON, 15ADON, NIV) by measuring trichothecene B toxins such as DON, NIV, Fusarenon-X, 3ADON and 15ADON. Moreover, we tested whether other toxins like zearalenone were also boosted by agmatine. The trichothecene type B boosting effect was observed in the majority of strains (13 out of 19) in all the three species. Representative strains from all three genetic chemotypes were able to boost toxin production after agmatine treatment. We identified the non-responding strains to the agmatine stimulus, which may contribute to deciphering the regulatory mechanisms that link toxin production to agmatine (and, more generally, polyamines).
Project description:Protein lysine acetylation is a prevalent post-translational modification that plays pivotal roles in various biological processes in both prokaryotes and eukaryotes. Aspergillus flavus, as an aflatoxin-producing fungus, has attracted tremendous attention due to its health impact on agricultural commodities. Here, we performed the first lysine-acetylome mapping in this filamentous fungus using immune-affinity-based purification integrated with high-resolution mass spectrometry. Overall, we identified 1383 lysine-acetylation sites in 652 acetylated proteins, which account for 5.18% of the total proteins in A. flavus. According to bioinformatics analysis, the acetylated proteins are involved in various cellular processes involving the ribosome, carbon metabolism, antibiotic biosynthesis, secondary metabolites, and the citrate cycle and are distributed in diverse subcellular locations. Additionally, we demonstrated for the first time the acetylation of fatty acid synthase ? and ? encoded by aflA and aflB involved in the aflatoxin-biosynthesis pathway (cluster 54), as well as backbone enzymes from secondary metabolite clusters 20 and 21 encoded by AFLA_062860 and AFLA_064240, suggesting important roles for acetylation associated with these processes. Our findings illustrating abundant lysine acetylation in A. flavus expand our understanding of the fungal acetylome and provided insight into the regulatory roles of acetylation in secondary metabolism.
Project description:Lysine acetylation of proteins, a major post-translational modification, plays a critical regulatory role in almost every aspects in both eukaryotes and prokaryotes. Yarrowia lipolytica, an oleaginous yeast, is considered as a model for bio-oil production due to its ability to accumulate a large amount of lipids. However, the function of lysine acetylation in this organism is elusive. Here, we performed a global acetylproteome analysis of Y. lipolytica ACA-DC 50109. In total, 3163 lysine acetylation sites were identified in 1428 proteins, which account for 22.1% of the total proteins in the cell. Fifteen conserved acetylation motifs were detected. The acetylated proteins participate in a wide variety of biological processes. Notably, a total of 65 enzymes involved in lipid biosynthesis were found to be acetylated. The acetylation sites are distributed in almost every type of conserved domains in the multi-enzymatic complexes of fatty acid synthetases. The provided dataset probably illuminates the crucial role of reversible acetylation in oleaginous microorganisms, and serves as an important resource for exploring the physiological role of lysine acetylation in eukaryotes.
Project description:Nε-Acetylation of lysine residues represents a frequently occurring post-translational modification widespread in bacteria that plays vital roles in regulating bacterial physiology and metabolism. However, the role of lysine acetylation in cyanobacteria remains unclear, presenting a hurdle to in-depth functional study of this post-translational modification. Here, we report the lysine acetylome of Synechococcus sp. PCC 7002 (hereafter Synechococcus) using peptide prefractionation, immunoaffinity enrichment, and coupling with high-precision liquid chromatography-tandem mass spectrometry analysis. Proteomic analysis of Synechococcus identified 1653 acetylation sites on 802 acetylproteins involved in a broad range of biological processes. Interestingly, the lysine acetylated proteins were enriched for proteins involved in photosynthesis, for example. Functional studies of the photosystem II manganese-stabilizing protein were performed by site-directed mutagenesis and mutants mimicking either constitutively acetylated (K99Q, K190Q, and K219Q) or nonacetylated states (K99R, K190R, and K219R) were constructed. Mutation of the K190 acetylation site resulted in a distinguishable phenotype. Compared with the K190R mutant, the K190Q mutant exhibited a decreased oxygen evolution rate and an enhanced cyclic electron transport rate in vivo Our findings provide new insight into the molecular mechanisms of lysine acetylation that involved in the negative regulation of oxygen evolution in Synechococcus and creates opportunities for in-depth elucidation of the physiological role of protein acetylation in photosynthesis in cyanobacteria.
Project description:Lysine acetylation is a dynamic and reversible post-translational modification that plays an important role in diverse cellular processes. Botrytis cinerea is the most thoroughly studied necrotrophic species due to its broad host range and huge economic impact. However, to date, little is known about the functions of lysine acetylation in this plant pathogen. In this study, we determined the lysine acetylome of B. cinerea through the combination of affinity enrichment and high-resolution LC-MS/MS analysis. Overall, 1582 lysine acetylation sites in 954 proteins were identified. Bioinformatics analysis shows that the acetylated proteins are involved in diverse biological functions and show multiple cellular localizations. Several particular amino acids preferred near acetylation sites, including K(ac)Y, K(ac)H, K(ac)***R, K(ac)F, FK(ac) and K(ac)***K, were identified in this organism. Protein interaction network analysis demonstrates that a variety of interactions are modulated by protein acetylation. Interestingly, 6 proteins involved in virulence of B. cinerea, including 3 key components of the high-osmolarity glycerol pathway, were found to be acetylated, suggesting that lysine acetylation plays regulatory roles in pathogenesis. These data provides the first comprehensive view of the acetylome of B. cinerea and serves as a rich resource for functional analysis of lysine acetylation in this plant pathogen.