veA Gene Research Articles

Ochratoxin A Defective Aspergillus carbonarius Mutants as Potential Biocontrol Agents.

Aspergillus carbonarius is one of the main species responsible for wine, coffee and cocoa toxin contamination. The main mycotoxin produced by this fungus, ochratoxin A (OTA), is a secondary metabolite categorized as a possible carcinogen because of its significant nephrotoxicity and immunosuppressive effects. A polyketide synthase gene (otaA) encodes the first enzyme in the OTA biosynthetic pathway. It is known that the filamentous fungi, growth, development and production of secondary metabolites are interconnected processes governed by global regulatory factors whose encoding genes are generally located outside the gene clusters involved in the biosynthesis of each secondary metabolite, such as the veA gene, which forms part of the VELVET complex. Different fungal strains compete for nutrients and space when they infect their hosts, and safer non-mycotoxigenic strains may be able to outcompete mycotoxigenic strains during colonization. To determine the possible utility of biopesticides based on the competitive exclusion of mycotoxigenic strains by non-toxigenic ones, we used A. carbonarius ΔotaA and ΔveA knockout mutants. Our results showed that during both in vitro growth and infection of grapes, non-mycotoxigenic strains could outcompete the wild-type strain. Additionally, the introduction of the non-mycotoxigenic strain led to a drastic decrease in OTA during both in vitro growth and infection of grapes.

Overexpression of the global regulator FnVeA upregulates antitumor substances in endophytic Fusarium nematophilum.

The special niche of endophytic fungi promotes their potential to produce antitumor compounds with novel structure and significant bioactivity for screening of new antitumor drugs. In our previous studies, we isolated a Fusarium strain from the roots of the medicinal plant Nothapodytes pittosporoides and identified it as Fusarium nematophilum. We found that the crude extract of F. nematophilum had significant antitumor activity on A549 cancer cells, and overexpressing the global regulatory factor FnVeA (the VeA gene of the fungus F. nematophilum) resulted in a significant increase in the antitumor activity, which was approximately fivefold higher than wild strain for relative inhibition rate. In FnVeAOE, the accumulation of indole, alkene, alkaloid, steroid, and flavonoid metabolites with potential antitumor activity was significantly upregulated compared with wild type via metabolomic analysis. Moreover, the transcriptome analysis showed that 134 differential genes were considered to be closely related to the biosynthesis of antitumor substances, of which 59 differential genes were considered as candidate key genes, and related to tryptophan dimethylallyltransferase, cytochrome P450 monooxygenase, polyketide synthases, and transcription factors. Taken together, we suggest that FnVeA may regulate the biosynthesis of antitumor substances by mediating the expression of genes related to secondary metabolic pathways in F. nematophilum.

Expression of Concern: Comparative proteomic analysis reveals the regulatory network of the veA gene during asexual and sexual spore development of Aspergillus cristatus

The Editorial Office has been made aware of potential issues surrounding the scientific validity of this paper, hence has issued an expression of concern to notify readers whilst the Editorial Office investigates.

VeA Gene Acts as a Positive Regulator of Conidia Production, Ochratoxin A Biosynthesis, and Oxidative Stress Tolerance in Aspergillus niger.

The veA gene is a key regulator governing morphogenetic development and secondary metabolism in many fungi. Here, we characterized and disrupted a veA orthologue in an ochratoxigenic Aspergillus niger strain. Morphological development, ochratoxin A (OTA) biosynthesis, and oxidative stress tolerance in the wild-type and veA disruption strains were further analyzed. Accordingly, the link between the veA gene and development of specific gene brlA, OTA biosynthesis key gene pks, and oxidative-stress-tolerance-related gene cat was explored. Results demonstrated that the veA gene acts as a positive regulator of conidia production, OTA biosynthesis, and oxidative stress tolerance in A. niger, regardless of light conditions. Darkness promoted conidial production and OTA biosynthesis in the A. niger wild-type strain. Our results contribute to a better understanding of the veA regulatory mechanism and suggest the veA gene as a potential target for developing control strategies for A. niger infection and OTA biosynthesis.

Comparative proteomic analysis reveals the regulatory network of the veA gene during asexual and sexual spore development of Aspergillus cristatus.

Aspergillus cristatus is the predominant fungal population during fermentation of Chinese Fuzhuan brick tea, and belongs to the homothallic fungal group that undergoes a sexual stage without asexual conidiation under hypotonic conditions, while hypertonic medium induces initiation of the asexual stage and completely blocks sexual development. However, the veA deletion mutant only produces conidia in hypotonic medium after a 24-h culture, but both asexual and sexual spores are observed after 72 h. The veA gene is one of the key genes that positively regulates sexual and negatively regulates asexual development in A. cristatus. To elucidate the molecular mechanism of how VeA regulates asexual and sexual spore development in A. cristatus, 2D electrophoresis (2-DE) combined with MALDI-tandem ToF MS analysis were applied to identify 173 differentially expressed proteins (DEPs) by comparing the agamotype (24 h) and teleomorph (72 h) with wild-type (WT) A. cristatus strains. Further analysis revealed that the changed expression pattern of Pmk1-MAPK and Ser/Thr phosphatase signaling, heat shock protein (Hsp) 90 (HSP90), protein degradation associated, sulphur-containing amino acid biosynthesis associated, valine, leucine, isoleucine, and arginine biosynthesis involved, CYP450 and cytoskeletal formation associated proteins were involved in the production of conidia in agamotype of A. cristatus. Furthermore, the deletion of veA in A. cristatus resulted in disturbed process of transcription, translation, protein folding, amino acid metabolism, and secondary metabolism. The carbohydrate and energy metabolism were also greatly changed, which lied in the suppression of anabolism through pentose phosphate pathway (PPP) but promotion of catabolism through glycolysis and tricarboxylic acid (TCA) cycle. The energy compounds produced in the agamotype were mainly ATP and NADH, whereas they were NADPH and FAD in the teleomorph. These results will contribute to the existing knowledge on the complex role of VeA in the regulation of spore development in Aspergillus and provide a framework for functional investigations on the identified proteins.

The role of the veA gene in adjusting developmental balance and environmental stress response in Aspergillus cristatus

veA belongs to the velvet regulatory system that regulates the development and secondary metabolism of many fungi. To identify the function of veA in Aspergillus cristatus, veA deletion mutants were constructed by homologous recombination via Agrobacterium tumefaciens-mediated transformation. Deletion of veA led to increased conidial production and reduced sexual sporulation. The regulatory role of veA in A. cristatus was not light-dependent, and this differed from its role in other Aspergilli. Furthermore, veA deletion mutants were more sensitive to environmental stressors, including salt, osmotic pressure, temperature and pH. In contrast, deletion of veA resulted in increased resistance to oxidative stress. veA also affected aerial vegetative growth. Transcriptomic analysis of the veA-null mutant and wild type indicated that most asexual and sexual development genes were upregulated and downregulated, respectively. These findings confirmed that veA has a positive effect on sexual development but represses conidial formation. Overall, these results suggested that the veA gene plays a critical role in maintaining a developmental balance between asexual and sexual sporulation and is involved in vegetative growth and environmental stress response in A. cristatus.

Distinct Roles of Velvet Complex in the Development, Stress Tolerance, and Secondary Metabolism in Pestalotiopsis microspora, a Taxol Producer.

The velvet family proteins have been shown to play critical roles in fungal secondary metabolism and development. However, variations of the roles have been observed in different fungi. We report here the observation on the role of three velvet complex components VeA, VelB, and LaeA in Pestalotiopsis microspora, a formerly reported taxol-producing fungus. Deletion of individual members led to the retardation of vegetative growth and sporulation and pigmentation, suggesting critical roles in these processes. The mutant strain △velB appeared hypersensitive to osmotic stress and the dye Congo red, whereas △veA and △laeA were little affected by the pressures, suggesting only velB was required for the integrity of the cell wall. Importantly, we found that the genes played distinct roles in the biosynthesis of secondary metabolites in P. microspora. For instance, the production of pestalotiollide B, a previously characterized polyketide, required velB and laeA. In contrast, the veA gene appeared to inhibit the pestalotiollide B (PB) role in its biosynthesis. This study suggests that the three components of the velvet complex are important global regulators, but with distinct roles in hyphal growth, asexual production, and secondary metabolism in P. microspora. This work provides information for further understanding the biosynthesis of secondary metabolism in the fungus.

A phosphorylation code of the Aspergillus nidulans global regulator VelvetA (VeA) determines specific functions.

The velvet protein VeA is a global fungal regulator for morphogenetic pathways as well as for the control of secondary metabolism. It is found exclusively in filamentous fungi, where it fulfills conserved, but also unique functions in different species. The involvement of VeA in various morphogenetic and metabolic pathways is probably due to spatially and timely controlled specific protein-protein interactions with other regulators such as phytochrome (FphA) or velvet-like proteins (VelB). Here we present evidence that Aspergillus nidulans VeA is a multi-phosphorylated protein and hypothesize that at least four specific amino acids (T167, T170, S183 and Y254) undergo reversible phosphorylation to trigger development and sterigmatocystin biosynthesis. Double mutation of T167 to valine and T170 to glutamic acid exerted the largest effects with regards to sexual development and veA gene expression. In comparison with wild-type VeA, which shuttles out of the nuclei after illumination this VeA variant showed stronger nuclear accumulation than the wild type, independent of the light conditions. The interaction between VeA and VelB or FphA, respectively, was affected in the T167V-T170E mutant. Our results suggest complex regulation of the phosphorylation status of the VeA protein.

Real-Time PCR Assay Targeting the veA Gene for Quantification of Aspergillus carbonarius in Grapes

In this work, a SYBR Green I real-time PCR method has been developed for the detection and quantification of Aspergillus carbonarius in grapes by targeting the veA gene with a primer pair (veAF4/veAR4) that specifically amplifies a 91-bp PCR product. The quantification of the fungal DNA was performed by generation of standard curves for two A. carbonarius strains, using spectrophotometrically measured DNA quantities (Log) with a linearity range from 50 to 5 × 10(-4) ng of DNA. A high positive correlation (R(2) > 0.99) between exponential increases of DNA and real-time PCR threshold cycles showed a high amplification efficiency for the assay (E values 100.06 and 101.51%, respectively). Quantification of the fungal genomic DNA in grape samples artificially inoculated with A. carbonarius conidia was successfully performed with a minimum threshold of 10(4) conidia per g of grape berry. The assay developed would allow reliable, specific, and efficient detection and quantification of A. carbonarius in grapes.

Transcriptome Analysis of Aspergillus flavus Reveals veA-Dependent Regulation of Secondary Metabolite Gene Clusters, Including the Novel Aflavarin Cluster.

The global regulatory veA gene governs development and secondary metabolism in numerous fungal species, including Aspergillus flavus. This is especially relevant since A. flavus infects crops of agricultural importance worldwide, contaminating them with potent mycotoxins. The most well-known are aflatoxins, which are cytotoxic and carcinogenic polyketide compounds. The production of aflatoxins and the expression of genes implicated in the production of these mycotoxins are veA dependent. The genes responsible for the synthesis of aflatoxins are clustered, a signature common for genes involved in fungal secondary metabolism. Studies of the A. flavus genome revealed many gene clusters possibly connected to the synthesis of secondary metabolites. Many of these metabolites are still unknown, or the association between a known metabolite and a particular gene cluster has not yet been established. In the present transcriptome study, we show that veA is necessary for the expression of a large number of genes. Twenty-eight out of the predicted 56 secondary metabolite gene clusters include at least one gene that is differentially expressed depending on presence or absence of veA. One of the clusters under the influence of veA is cluster 39. The absence of veA results in a downregulation of the five genes found within this cluster. Interestingly, our results indicate that the cluster is expressed mainly in sclerotia. Chemical analysis of sclerotial extracts revealed that cluster 39 is responsible for the production of aflavarin.

Aspergillus fumigatus에서 Methyltransferase 유전자 AfuvipB와 AfuvipC의 분리 및 분석

Department of Pharmaceutical Engineering, Woosuk University, Wanju 565-701, KoreaABSTRACT: In filamentous fungi, velvet complex associated with the veA gene plays pivotal roles in development and secondarymetabolism. In a model fungus Aspergillus nidulans, many proteins that can interact with VeA, including two methyltransferasesVipB and VipC, have been isolated and characterized. In this study, we isolated homologs of the vipB and vipC genes in thehuman opportunistic pathogenic fungus Aspergillus fumigatus and named AfuvipB and AfuvipC. The AfuvipB gene, annotated asAfu3g14920 in the Aspergillus Genome Database (AspGD) database, consists of 1,510 bp interrupted with 10 introns yielding 336amino acid-long putative methyltransferase protein. Similarly, AfuvipC, which is Afu8g01930, has 10 introns and encodes apolypeptide with 339 amino acids having a methyltransferase domain in the middle of the protein. To characterize the functionof the genes in A. fumigatus, knock-out mutants were generated and the phenotypes were observed. Deletion of AfuvipB genecaused no obvious phenotypic change on point inoculation but showed smaller colony than wild-type when the mutant wassubjected to culture on single spore-driven culture condition. However, AfuvipC deletion mutant demonstrated no phenotypicdifference from wild type both in point inoculation and streaking cultures. These results indicate that the two methyltransfereasesmight have a redundant role and could be dispensable in normal culture conditions.KEYWORDS :

The Velvet Underground Emerges

The Velvet Underground Emerges

The Fumagillin Gene Cluster, an Example of Hundreds of Genes under veA Control in Aspergillus fumigatus

Aspergillus fumigatus is the causative agent of invasive aspergillosis, leading to infection-related mortality in immunocompromised patients. We previously showed that the conserved and unique-to-fungi veA gene affects different cell processes such as morphological development, gliotoxin biosynthesis and protease activity, suggesting a global regulatory effect on the genome of this medically relevant fungus. In this study, RNA sequencing analysis revealed that veA controls the expression of hundreds of genes in A. fumigatus, including those comprising more than a dozen known secondary metabolite gene clusters. Chemical analysis confirmed that veA controls the synthesis of other secondary metabolites in this organism in addition to gliotoxin. Among the secondary metabolite gene clusters regulated by veA is the elusive but recently identified gene cluster responsible for the biosynthesis of fumagillin, a meroterpenoid known for its anti-angiogenic activity by binding to human methionine aminopeptidase 2. The fumagillin gene cluster contains a veA-dependent regulatory gene, fumR (Afu8g00420), encoding a putative C6 type transcription factor. Deletion of fumR results in silencing of the gene cluster and elimination of fumagillin biosynthesis. We found expression of fumR to also be dependent on laeA, a gene encoding another component of the fungal velvet complex. The results in this study argue that veA is a global regulator of secondary metabolism in A. fumigatus, and that veA may be a conduit via which chemical development is coupled to morphological development and other cellular processes.

Transcriptomic profiling of Aspergillus flavus in response to 5-azacytidine

Aspergillus flavus is a common saprophyte and opportunistic pathogen producing aflatoxin (AF) and many other secondary metabolites. 5-Azacytidine (5-AC), a derivative of the nucleoside cytidine, is widely used for studies in epigenetics and cancer biology as an inactivator of DNA methyltransferase and is also used for studying secondary metabolism in fungi. Our previous studies showed that 5-AC affects development and inhibits AF production in A. flavus, and that A. flavus lacks DNA methylation. In this study, an RNA-Seq approach was applied to explore the mechanism of 5-AC’s effect on A. flavus. We identified 240 significantly differentially expressed (Q-value<0.05) genes after 5-AC treatment, including two backbone genes respectively in secondary metabolite clusters #27 and #35. These two clusters are involved in development or survival of sclerotia. GO functional enrichment analysis showed that these significantly differentially expressed genes were mainly involved in catalytic activity and proteolytic functions. The expressed transcripts of most genes in the AF biosynthetic gene cluster in A. flavus showed no significant changes after treatment with 5-AC and were expressed at low levels, and the transcription regulator genes aflR and aflS in this cluster did not show differential expression relative to the sample without 5-AC treatment. We found that the veA gene, which encodes protein bridges VelB and LaeA, decreased profoundly the expressed transcripts, and brlA, which encodes an early regulator of development, increased its transcripts in A. flavus after 5-AC treatment. Our data support a model whereby 5-AC affects development through increasing the expression of brlA by depressing the expression of veA and AF production through suppressing veA expression and dysregulating carboxypeptidase activity, which then prevents the aflatoxisomes (vesicles) from performing their normal function in AF formation. Furthermore, the suppressed veA expression weakens or even interrupts the connection between VelB and LaeA, leading to dysregulation of the expression pattern of genes involved in development and secondary metabolism in A. flavus. The RNA-seq data presented in this work were also served to improve the annotation of the A. flavus genome. This work provides a comprehensive view of the transcriptome of A. flavus responsive to 5-AC and supports the conclusion that fungal development and secondary metabolism are co-regulated.

The veA gene of the pine needle pathogen Dothistroma septosporum regulates sporulation and secondary metabolism

Fungi possess genetic systems to regulate the expression of genes involved in complex processes such as development and secondary metabolite biosynthesis. The product of the velvet gene veA, first identified and characterized in Aspergillus nidulans, is a key player in the regulation of both of these processes. Since its discovery and characterization in many Aspergillus species, VeA has been found to have similar functions in other fungi, including the Dothideomycete Mycosphaerella graminicola. Another Dothideomycete, Dothistroma septosporum, is a pine needle pathogen that produces dothistromin, a polyketide toxin very closely related to aflatoxin (AF) and sterigmatocystin (ST) synthesized by Aspergillus spp. Dothistromin is unusual in that, unlike most other secondary metabolites, it is produced mainly during the early exponential growth phase in culture. It was therefore of interest to determine whether the regulation of dothistromin production in D. septosporum differs from the regulation of AF/ST in Aspergillus spp. To begin to address this question, a veA ortholog was identified and its function analyzed in D. septosporum. Inactivation of the veA gene resulted in reduced dothistromin production and a corresponding decrease in expression of dothistromin biosynthetic genes. Expression of other putative secondary metabolite genes in D. septosporum such as polyketide synthases and non-ribosomal peptide synthases showed a range of different responses to loss of Ds-veA. Asexual sporulation was also significantly reduced in the mutants, accompanied by a reduction in the expression of a putative stuA regulatory gene. The mutants were, however, able to infect Pinus radiata seedlings and complete their life cycle under laboratory conditions. Overall this work suggests that D. septosporum has a veA ortholog that is involved in the control of both developmental and secondary metabolite biosynthetic pathways.

The velvet gene, FgVe1, affects fungal development and positively regulates trichothecene biosynthesis and pathogenicity in Fusarium graminearum

Trichothecenes are a group of toxic secondary metabolites produced mainly by Fusarium graminearum (teleomorph: Gibberella zeae) during the infection of crop plants, including wheat, maize, barley, oats, rye and rice. Some fungal genes involved in trichothecene biosynthesis have been shown to encode regulatory proteins. However, the global regulation of toxin biosynthesis is still enigmatic. In addition to the production of secondary metabolites belonging to the trichothecene family, F. graminearum produces the red pigment aurofusarin. The gene regulation underlying the production of aurofusarin is not well understood. The velvet gene (veA) is conserved in various genera of filamentous fungi. Recently, the veA gene from Aspergillus nidulans has been shown to be the key component of the velvet complex regulating development and secondary metabolism. Using blast analyses, we identified the velvet gene from F. graminearum, FgVe1. Disruption of FgVe1 causes several phenotypic effects. However, the complementation of this mutant with the FgVe1 gene restores the wild-type phenotypes. The in vitro phenotypes include hyperbranching of the mycelium, suppression of aerial hyphae formation, reduced hydrophobicity of the mycelium and highly reduced sporulation. Our data also show that FgVe1 modulates the production of the aurofusarin pigment and is essential for the expression of Tri genes and the production of trichothecenes. Pathogenicity studies performed on flowering wheat plants indicate that FgVe1 is a positive regulator of virulence in F. graminearum.

Simple identification of veA1 mutation in Aspergillus nidulans

The veA gene plays an important role in development of a homothallic filamentous fungus Aspergillus nidulans. The veA1 phenotype can be difficult to distinguish from the wild-type veA. Despite the importance of the veA allele, no efficient identification method has been reported besides DNA sequencing. Here, we present simple physiological and molecular biological ways to distinguish between the veA wild-type and veA1 allele. The novel approaches, which involve incubation in the presence of oxalic acid, polymerase chain reaction using double mismatched primers, and BstXI enzyme digestion, are simpler, faster and more cost-efficient than genome sequencing.

The veA gene is necessary for the negative regulation of the veA expression in Aspergillus nidulans

The veA gene is one of the key genes in regulating sexual development of Aspergillus nidulans. During the study on the veA gene, it was observed that the veA expression level is slightly higher in a veA1 mutant than in a wild type at 37 degrees C, suggesting that the wild type veA gene is necessary for the negative regulation of the veA expression. In the veA1 mutant, the veA expression was higher than in a wild type grown at 42 degrees C but equal at 30 degrees C. Furthermore, in a veA deletion mutant having its own promoter and the N-terminus of the VeA ORF, expression of the N-terminus by the veA promoter was highly up-regulated, supporting the possibility that the veA gene is important for the negative regulation of the veA expression. Analyses of the lacZ transcript and the beta-galactosidase activity from the reporter strains in the veA1 background, which were constructed by transformation of the lacZ reporter plasmids containing the lacZ gene under the control of the intact or the truncated veA promoters from the -943 to +262 bp region, showed that the truncated promoters produced more veA transcript and higher beta-galactosidase activity than the intact one at 30 degrees C, but equal at 42 degrees C. In addition, the serial-deletion analysis of the veA promoter identified a crucial region in the promoter from -943 to -740 bp for this derepression of the veA expression. Taken together, these results indicated that the veA gene is necessary for the negative regulation of the veA expression. Moreover, the veA expression was derepressed in the light-illuminated condition, where the VeA protein is hardly transported into the nucleus.

The Role of veA in Aspergillus flavus Infection of Peanut, Corn and Cotton

The aflatoxin-producing fungus Aspergillus flavus is a causal agent of preharvest contamination of food commodities such as oil seed crops worldwide. Peanut, corn and cottonseed are among the oil seeds that are susceptible to aflatoxin contamination during invasion of these crops by A. flavus. Contamination of agricultural commodities with aflatoxins can result in serious economic hardships to producers and adverse health impacts in both humans and domestic animals. It is therefore of great importance to determine ways to control A. flavus dissemination, survival and toxin formation. In this study we demonstrate the role of the veA regulatory gene in the contamination of peanut, corn and cotton by A. flavus. Virulence of A. flavus on peanut and corn seeds was reduced in the absence of the veA gene product. Generation of air-borne asexual spores was reduced and production of aflatoxin and sclerotia in peanut seeds, viable or non-viable, or in viable corn seed was completely blocked when infected with the A. flavus veA mutant (� veA). In planta inoculation of cotton bolls also showed that conidiation was decreased in bolls inoculated with theveA strain and spread of theveA strain to seed in locules adjacent to the inoculated locule was less than observed with the wild-type veA strain. As observed in peanut and corn, no aflatoxin was produced in seed harvested from cotton bolls inoculated with theveA strain while aflatoxin was present in seed from wild-type veA inoculated bolls.

FvVE1 Regulates Biosynthesis of the Mycotoxins Fumonisins and Fusarins in Fusarium verticillioides

The veA gene positively regulates sterigmatocystin production in Aspergillus nidulans and aflatoxin production in Aspergillus parasiticus and Aspergillus flavus . Whether veA homologues have a role in regulating secondary metabolism in other fungal genera is unknown. In this study, we examined the role of the veA homologue, FvVE1, on the production of two mycotoxin families, fumonisins and fusarins, in the important corn pathogen Fusarium verticillioides . We found that FvVE1 deletion completely suppressed fumonisin production on two natural substrates, corn and rice. Furthermore, our results revealed that FvVE1 is necessary for the expression of the pathway-specific regulatory gene FUM21 and structural genes in the fumonisin biosynthetic gene (FUM) cluster. FvVE1 deletion also blocked production of fusarins. The effects of FvVE1 deletion on the production of these toxins were found to be the same in two separate mating types. Our results strongly suggest that FvVE1 plays an important role in regulating mycotoxin production in F. verticillioides .