Histone deacetylase SirE regulates development, DNA damage response and aflatoxin production in Aspergillus flavus.

Aspergillus flavus is a ubiquitous saprotrophic soil-borne pathogenic fungus that causes crops contamination with the carcinogen aflatoxins. Although sirtuin E (SirE) is known to be a NAD-dependent histone deacetylase involved in global transcriptional regulation. Its biological functions in A. flavus are not fully understood. To explore the effects of SirE, we found that SirE was located in the nucleus and increased the level of H3K56 acetylation. The ΔsirE mutant had the most severe growth defect in the sirtuin family. The RNA-Seq revealed that sirE was crucial for secondary metabolism production as well as genetic information process and oxidation-reduction in A. flavus. Further analysis revealed that the ΔsirE mutant increased aflatoxin production. Both the sirE deletion and H3K56 mutants were highly sensitive to DNA damage and oxidative stresses, indicating that SirE was required for DNA damage and redox reaction by the H3K56 locus. Furthermore, the ΔsirE mutant displayed high sensitivity to osmotic stress and cell wall stress, but they may not be associated with the H3K56. Finally, the catalytic activity site N192 of SirE was required for regulating growth, deacetylase function and aflatoxin production. Together, SirE is essential for histone deacetylation and biological function in A. flavus.

Histone acetyltransferases MystA and MystB contribute to morphogenesis and aflatoxin biosynthesis by regulating acetylation in fungus Aspergillus flavus.

Myst family is highly conserved histone acetyltransferases in eukaryotic cells and is known to play crucial roles in various cellular processes; however, acetylation catalysed by acetyltransferases is unclear in filamentous fungi. Here, we identified two classical nonessential Myst enzymes and analysed their functions in Aspergillus flavus, which generates aflatoxin B1, one of the most carcinogenic secondary metabolites. MystA and MystB located in nuclei and cytoplasm, and mystA could acetylate H4K16ac, while mystB acetylates H3K14ac, H3K18ac and H3K23ac. Deletion mystA resulted in decreased conidiation, increased sclerotia formation and aflatoxin production. Deletion of mystB leads to significant defects in conidiation, sclerotia formation and aflatoxin production. Additionally, double-knockout mutant (ΔmystA/mystB) display a stronger and similar defect to ΔmystB mutant, indicating that mystB plays a major role in regulating development and aflatoxin production. Both mystA and mystB play important role in crop colonization. Moreover, catalytic domain MOZ and the catalytic site E199/E243 were important for the acetyltransferase function of Myst. Notably, chromatin immunoprecipitation results indicated that mystB participated in oxidative detoxification by regulating the acetylation level of H3K14, and further regulated nsdD to affect sclerotia formation and aflatoxin production. This study provides new evidences to discover the biological functions of histone acetyltransferase in A. flavus.

The Fungi-specific histone Acetyltransferase Rtt109 mediates morphogenesis, Aflatoxin synthesis and pathogenicity in Aspergillus flavus by acetylating H3K9.

Aspergillus flavus is a common saprophytic filamentous fungus that produces the highly toxic natural compound aflatoxin during its growth process. Synthesis of the aflatoxins, which can contaminate food crops causing huge losses to the agricultural economy, is often regulated by epigenetic modification, such as the histone acetyltransferase. In this study, we used Aspergillus flavus as an experimental model to construct the acetyltransferase gene rtt109 knockout strain (△rtt109) and its complementary strain (△rtt109·com) by homologous recombination. The growth of △rtt109 was significantly suppressed compared to the wild type (WT) strain and the △rtt109·com strain. The sclerotium of △rtt109 grew smaller, and the amount of sclerotia generated by △rtt109 was significantly reduced. The number of conidiums of △rtt109 was significantly reduced, especially on the yeast extract sucrose (YES) solid medium. The amount of aflatoxins synthesized by △rtt109 in the PDB liquid medium was significantly decreased We also found that the △rtt109 strain was extremely sensitive to DNA damage stress. Through the maize seed infection experiment, we found that the growth of △rtt109 on the surface of affected corn was largely reduced, and the amount of aerial mycelium decreased significantly, which was consistent with the results on the artificial medium. We further found that H3K9 was the acetylated target of Rtt109 in A. flavus. In conclusion, Rtt109 participated in the growth, conidium formation, sclerotia generation, aflatoxin synthesis, environmental stress response, regulation of infection of A. flavus. The results from this study of rtt109 showed data for acetylation in the regulation of life processes and provided a new thought regarding the prevention and control of A. flavus hazards.

Gas Chromatography-Mass Spectrometry Profiling of Volatile Compounds Reveals Metabolic Changes in a Non-Aflatoxigenic Aspergillus flavus Induced by 5-Azacytidine.

Aspergillus flavus is one of the most opportunistic pathogens invading many important oilseed crops and foodstuffs with such toxic secondary metabolites as aflatoxin (AF) and Cyclopiazonic acid. We previously used the DNA methylation inhibitor 5-azacytidine to treat with an AF-producing A. flavus A133 strain, and isolated a mutant (NT) of A. flavus, which displayed impaired abilities of AF biosynthesis and fungal development. In this study, gas chromatography-mass spectrometry (GC-MS) analysis was used to reveal the metabolic changes between these two strains. A total of 1181 volatiles were identified in these two strains, among which 490 volatiles were found in these two strains in vitro and 332 volatiles were found in vivo. The NT mutant was found to produce decreasing volatile compounds, among which most of the fatty acid-derived volatiles were significantly downregulated in the NT mutant compared to the A133 strain, which are important precursors for AF biosynthesis. Two antioxidants and most of the amino acids derived volatiles were found significantly upregulated in the NT mutant. Overall, our results reveal the difference of metabolic profiles in two different A. flavus isolates, which may provide valuable information for controlling infections of this fungal pathogen.

The HosA Histone Deacetylase Regulates Aflatoxin Biosynthesis Through Direct Regulation of Aflatoxin Cluster Genes.

Histone deacetylases (HDACs) always function as corepressors and sometimes as coactivators in the regulation of fungal development and secondary metabolite production. However, the mechanism through which HDACs play positive roles in secondary metabolite production is still unknown. Here, classical HDAC enzymes were identified and analyzed in Aspergillus flavus, a fungus that produces one of the most carcinogenic secondary metabolites, aflatoxin B1 (AFB1). Characterization of the HDACs revealed that a class I family HDAC, HosA, played crucial roles in growth, reproduction, the oxidative stress response, AFB1 biosynthesis, and pathogenicity. To a lesser extent, a class II family HDAC, HdaA, was also involved in sclerotia formation and AFB1 biosynthesis. An in vitro analysis of HosA revealed that its HDAC activity was considerably diminished at nanomolar concentrations of trichostatin A. Notably, chromatin immunoprecipitation experiments indicated that HosA bound directly to AFB1 biosynthesis cluster genes to regulate their expression. Finally, we found that a transcriptional regulator, SinA, interacts with HosA to regulate fungal development and AFB1 biosynthesis. Overall, our results reveal a novel mechanism by which classical HDACs mediate the induction of secondary metabolite genes in fungi.

Set3 Is Required for Asexual Development, Aflatoxin Biosynthesis, and Fungal Virulence in Aspergillus flavus.

Aspergillus flavus is an opportunistic pathogenic fungus for both plant and animal that produces carcinogenic toxins termed aflatoxins (AFs). To identify possible genetic targets to reduce AF contamination, in this study, we have characterized a novel A. flavus Set3, and it shares sequence homology with the yeast protein Set3. The set3 deletion mutants present no difference in growth rate but alterations in asexual development and secondary metabolite production when compared to the A. flavus wild type. Specifically, deletion of set3 gene decreases conidiophore formation and conidial production through downregulating expression of brlA and abaA genes. In addition, normal levels of set3 are required for sclerotial development and expression of sclerotia-related genes nsdC and sclR. Further analyses demonstrated that Set3 negatively regulates AF production as well as the concomitant expression of genes in the AF gene cluster. Importantly, our results also display that A. flavus Set3 is involved in crop kernel colonization. Taking together, these results reveal that a novel Set3 plays crucial roles in morphological development, secondary metabolism, and fungal virulence in A. flavus.

Cyclase-Associated Protein Cap with Multiple Domains Contributes to Mycotoxin Biosynthesis and Fungal Virulence in Aspergillus flavus.

In Aspergillus, the cyclic adenosine monophosphate (cAMP) signaling modulates asexual development and mycotoxin biosynthesis. Here, we characterize the cyclase-associated protein Cap in the pathogenic fungus Aspergillus flauvs. The cap disruption mutant exhibited dramatic reduction in hyphal growth, conidiation, and spore germination, while an enhanced production of the sclerotia was observed in this mutant. Importantly, the cap gene was found to be important for mycotoxin biosynthesis and virulence. The domain deletion study demonstrated that each domain played an important role for the Cap protein in regulating cAMP/protein kinase A (PKA) signaling, while only P1 and CARP domains were essential for the full function of Cap. The phosphorylation of Cap at S35 was identified in A. flavus, which was found to play a negligible role for the function of Cap. Overall, our results indicated that Cap with multiple domains engages in mycotoxin production and fungal pathogenicity, which could be designed as potential control targets for preventing this fungal pathogen.

Investigation of Aspergillus flavus in animal virulence.

Aspergillus flavus is a common fungal pathogen of plants, animals and humans. Recently, many genes of A. flavus have been reported involving in regulation of pathogenesis in crops, but whether these genes are involved in animal virulence is still unknown. Here, we used a previous easy-to-use infection model for A. flavus based on mouse model by intravenous inoculation of A. flavus conidia. The outcome of infections in mice model showed that A. flavus NRRL3357 and laboratory strain CA14 PTS were both in dose dependent manner and highly reproducible. The progress of disease could be monitored by mice survival and histology analysis. Fungal burden analysis indicated it was gradually decreased within 7 days after infection. Moreover, aspergillosis caused by A. flavus significantly up-regulated gene expression levels of immune response mediators, including INF-γ, TNF-α, Dectin-1 and TLR2. Furthermore, the defined deletion A. flavus strains that previously displayed virulence in crop infection were also determined in this mouse model, and the results showed comparable degrees of infection in mice. Our results suggested that intravenous inoculation of conidia could be a suitable model for testing different A. flavus mutants in animal virulence. We hope to use this model to determine distinct A. flavus strains virulence in animals and study novel therapeutic methods to help control fungus diseases in the future.

The Putative Histone Methyltransferase DOT1 Regulates Aflatoxin and Pathogenicity Attributes in Aspergillus flavus.

Lysine methyltransferases transfer methyl groups in specific lysine sites, which regulates a variety of important biological processes in eukaryotes. In this study, we characterized a novel homolog of the yeast methyltransferase DOT1 in A. flavus, and observed the roles of dot1 in A. flavus. Deletion of dot1 showed a significant decrease in conidiation, but an increase in sclerotia formation. A change in viability to multiple stresses was also found in the Δdot1 mutant. Additionally, aflatoxin (AF) production was found severely impaired in the Δdot1 mutant. Further analysis by qRT-PCR revealed that the transcription of AF structural genes and their regulator gene aflS were prominently suppressed in the Δdot1 mutant. Furthermore, our data revealed that Dot1 is important for colonizing maize seeds in A. flavus. Our research indicates that Dot1 is involved in fungal development, aflatoxin biosynthesis and fungal virulence in A. flavus, which might provide a potential target for controlling A. flavus with new strategies.

The Aspergillus flavus Histone Acetyltransferase AflGcnE Regulates Morphogenesis, Aflatoxin Biosynthesis, and Pathogenicity.

Histone acetyltransferases (HATs) help regulate fungal development and the production of secondary metabolites. In this study, we determined that the HAT AflGcnE influenced morphogenesis and aflatoxin biosynthesis in Aspergillus flavus. We observed that AflGcnE localized to the nucleus and cytoplasm during the conidial production and germination stages, while it was located mainly in the nucleus during the hyphal development stage. Deletion of AflgcnE inhibited the growth of A. flavus and decreased the hydrophobicity of the cell surface. The ΔAflgcnE mutant exhibited a lack of asexual sporulation and was unable to generate sclerotia. Additionally, AflgcnE was required to maintain cell wall integrity and genotoxic stress responses. Importantly, the ΔAflgcnE mutant did not produce aflatoxins, which was consistent with a significant down-regulation of aflatoxin gene expression levels. Furthermore, our data revealed that AflgcnE is a pathogenicity factor required for colonizing maize seeds. In summary, we revealed that A. flavus AflGcnE is crucial for morphological development, aflatoxin biosynthesis, stress responses, and pathogenicity. Our findings help clarify the functional divergence of GcnE orthologs, and may provide a possible target for controlling A. flavus infections of agriculturally important crops.

The DmtA methyltransferase contributes to Aspergillus flavus conidiation, sclerotial production, aflatoxin biosynthesis and virulence.

DNA methylation is essential for epigenetic regulation of gene transcription and development in many animals, plants and fungi. We investigated whether DNA methylation plays a role in the development and secondary metabolism of Aspergillus flavus, identified the DmtA methyltransferase from A. flavus, and produced a dmtA knock-out mutant by replacing the dmtA coding sequence with the pyrG selectable marker. The A. flavus dmtA null mutant lines produced white fluffy mycelium in liquid medium, and displayed a slightly flavescent conidial pigmentation compared with the normal yellow of the wild-type strain when grown on agar. The ΔdmtA lines exhibited decreased conidiation and aflatoxin (AF) biosynthesis, compared with the wild-type line, suggesting that the DmtA knock-out affected the transcriptional level of genes in the AF cluster. In particular, sclerotia development and host colonization were altered in the dmtA null mutants. Green fluorescent protein tagging at the C-terminus of DmtA showed that DmtA localized to the nucleus and cytoplasm. DNA methylation content measurements in the dmtA mutants revealed no widespread DNA methylation in the mutants or wild-type lines. Thus, our findings suggest that DmtA, apart from being a C-5 cytosine methyltransferase in A. flavus, contributes to asexual development, aflatoxin biosynthesis, sclerotial production and virulence.

Adenylate Cyclase AcyA Regulates Development, Aflatoxin Biosynthesis and Fungal Virulence in Aspergillus flavus.

Aspergillus flavus is one of the most important opportunistic pathogens of crops and animals. The carcinogenic mycotoxin, aflatoxins produced by this pathogen cause a health problem to human and animals. Since cyclic AMP signaling controls a range of physiological processes, like fungal development and infection when responding to extracellular stimuli in fungal pathogens, in this study, we investigated the function of adenylate cyclase, a core component of cAMP signaling, in aflatoxins biosynthesis and virulence on plant seeds in A. flavus. A gene replacement strategy was used to generate the deletion mutant of acyA that encodes the adenylate cyclase. Severe defects in fungal growth, sporulation and sclerotia formation were observed in the acyA deletion mutant. The defect in radical growth could be partially rescued by exogenous cAMP analog. The acyA mutant was also significantly reduced in aflatoxins production and virulence. Similar to the former studies in other fungi, The acyA mutant showed enhancing tolerance to oxidative stress, but more sensitive to heat stress. Overall, the pleiotropic defects of the acyA deletion mutant indicates that the cAMP-PKA pathway is involved in fungal development, aflatoxins biosynthesis and plant seed invasion in A. flavus.