High-frequency conversion to a "fluffy" developmental phenotype in Aspergillus spp. by 5-azacytidine treatment: evidence for involvement of a single nuclear gene.

Transient exposure of mycelia from Aspergillus niger and Aspergillus nidulans to the cytidine analog 5-azacytidine, leading to no more than 0.3 to 0.5% substitution for cytosine by 5-azacytosine in A. nidulans DNA, resulted in the conversion of a high fraction of the cell population (more than 20%) to a mitotically and meiotically stable "fluffy" developmental phenotype. The phenotypic variants are characterized by the developmentally timed production of a profuse fluffy network of undifferentiated aerial hyphae that seem to escape signals governing vegetative growth. Genetic analysis with six different fluffy clones reveals that this trait is not cytoplasmically coded, is recessive in heterozygous diploids but codominant in heterokaryons, and exhibits a 1:1 Mendelian segregation pattern upon sexual sporulation of heterozygous diploids. Complementation and mitotic haploidization studies indicated that all variants are affected in the same gene, which can be tentatively located on chromosome VIII of A. nidulans. Molecular analysis to search for modified bases showed that DNA methylation is negligible in in both A. niger and A. nidulans and that no differences could be detected among DNAs from wild-type cells, fluffy clones, or mycelia exposed to 5-azacytidine. It thus appears that high-frequency conversion of fungal mycelia to a stable, variant developmental phenotype by 5-azacytidine is the result of some kind of target action on a single nuclear gene and that this conversion can occur in organisms virtually devoid of DNA methylation.     

5-Azacytidine inhibits aflatoxin biosynthesis in Aspergillus flavus

Aflatoxins, mainly produced by Aspergillus flavus and A. parasiticus, are a group of potent mycotoxins with carcinogenic, hepatotoxic, and immunosuppressive properties. Many studies have been devoted to investigating their biosynthesis mechanism since they were discovered half a century ago. 5-Azacytidine (5-AC), a derivative of the nucleoside cytidine and an inactivator of DNA methyltransferase, is widely used for studies in epigenetics and cancer biology, and has also been used for studying secondary metabolism in fungi. In this study, 5-AC was applied to investigate its effect on the development and aflatoxin biosynthesis of A. flavus. The results indicate that 5-AC inhibits the ability to produce aflatoxin and also causes a fluffy aconidial phenotype. Further studies revealed that 5-AC affects gene expression of A. flavus to a limited degree, and the unique homolog of DNA methyltransferase gene (DmtA) expressed constitutively during different developmental stages of A. flavus irrespective of 5-AC. This work may provide some basic data to elucidate the role of 5-AC in aflatoxin biosynthesis and the development of A. flavus.     

Fungal ammonia fermentation, a novel metabolic mechanism that couples the dissimilatory and assimilatory pathways of both nitrate and ethanol. Role of acetyl CoA synthetase in anaerobic ATP synthesis

Fungal ammonia fermentation is a novel dissimilatory metabolic mechanism that supplies energy under anoxic conditions. The fungus Fusarium oxysporum reduces nitrate to ammonium and simultaneously oxidizes ethanol to acetate to generate ATP (Zhou, Z., Takaya, N., Nakamura, A., Yamaguchi, M., Takeo, K., and Shoun, H. (2002) J. Biol. Chem. 277, 1892-1896). We identified the Aspergillus nidulans genes involved in ammonia fermentation by analyzing fungal mutants. The results showed that assimilatory nitrate and nitrite reductases (the gene products of niaD and niiA) were essential for reducing nitrate and for anaerobic cell growth during ammonia fermentation. We also found that ethanol oxidation is coupled with nitrate reduction and catalyzed by alcohol dehydrogenase, coenzyme A (CoA)-acylating aldehyde dehydrogenase, and acetyl-CoA synthetase (Acs). This is similar to the mechanism suggested in F. oxysporum except A. nidulans uses Acs to produce ATP instead of the ADP-dependent acetate kinase of F. oxysporum. The production of Acs requires a functional facA gene that encodes Acs and that is involved in ethanol assimilation and other metabolic processes. We purified the gene product of facA (FacA) from the fungus to show that the fungus acetylates FacA on its lysine residue(s) specifically under conditions of ammonia fermentation to regulate its substrate affinity. Acetylated FacA had higher affinity for acetyl-CoA than for acetate, whereas non-acetylated FacA had more affinity for acetate. Thus, the acetylated variant of the FacA protein is responsible for ATP synthesis during fungal ammonia fermentation. These results showed that the fungus ferments ammonium via coupled dissimilatory and assimilatory mechanisms.     

Asexual sporulation signalling regulates autolysis of Aspergillus nidulans via modulating the chitinase ChiB production

Aims:  Elucidation of the regulation of ChiB production in Aspergillus nidulans .
Methods and Results:  Mutational inactivation of the A. nidulans chiB gene resulted in a nonautolytic phenotype. To better understand the mechanisms controlling both developmental progression and fungal autolysis, we examined a range of autolysis-associated parameters in A. nidulans developmental and/or autolytic mutants. Investigation of disorganization of mycelial pellets, loss of biomass, extra-/intracellular chitinase activities, ChiB production and chiB mRNA levels in various cultures revealed that, in submerged cultures, initialization of autolysis and stationary phase-induced ChiB production are intimately coupled, and that both processes are controlled by the FluG-BrlA asexual sporulation regulatory pathway. ChiB production does not affect the progression of apoptotic cell death in the aging A. nidulans cultures.
Conclusions:  The endochitinase ChiB plays an important role in autolysis of A. nidulans , and its production is initiated by FluG-BrlA signalling. Despite the fact that apoptosis is an inseparable part of fungal autolysis, its regulation is independent to FluG-initiated sporulation signalling.
Significance and Impact of the Study:  Deletion of chiB and fluG homologues in industrial filamentous fungal strains may stabilize the hyphal structures in the autolytic phase of growth and limit the release of autolytic hydrolases into the culture medium.     

Developmental decisions in Aspergillus nidulans are modulated by Ras activity.

To better understand how Ras controls development of multicellular organisms, we have chosen Aspergillus nidulans as a model system. When grown on solid medium, this fungus follows a well-defined program of development, sequentially giving rise to several cell types which produce three distinct structures: vegetative hyphae, aerial hyphae, and the conidiophore structure. Here we describe a ras homolog found in this fungus (Aras) and demonstrate that it is an essential gene that regulates the ordered program of development. We created dominant alleles of this gene and expressed them to different levels in order to vary the ratio of GTP-bound (active) to GDP-bound (inactive) A-Ras protein. When the amount of active Ras is large, nuclear division proceeds, but further development is inhibited at the early step of germ tube formation. At an intermediate level of active Ras, aerial hypha formation is inhibited, while at a low level, conidiophore formation is inhibited. Maintenance of an even lower level of the active Ras is essential for initiation and progression of conidiophore formation, the final stage of development. When the level of active Ras is artificially lowered, each stage of development is initiated prematurely except germination, the initial stage of development. Therefore, the progression of the ordered developmental pathway of A. nidulans is dependent upon an initial high level of active Ras followed by its gradual decrease. We propose that several concentration threshold exist, each of which allows development to proceed to a certain point, producing the proper cell type while inhibiting further development.     

Maize ribosome-inactivating protein inhibits normal development of Aspergillus nidulans and Aspergillus flavus

The abundant maize kernel ribosome-inactivating protein 1 (RIP1) was tested for antifungal activity against Aspergillus nidulans and Aspergillus flavus. A microculture assay was developed to monitor fungal growth and development after treatment of conidia with RIP1 or control proteins. A striking decrease in hyphal proliferation was observed when conidia of A. nidulans, a genetically well-characterized nonpathogenic species, were treated with RIP1 protein. Treatment with a RIP1 mutant protein that lacked enzymatic ribosome-inactivating activity caused no observable effects. RIP1 treatment of conidia from the maize pathogen A. flavus resulted in increased hyphal branching. Examination of the branched hyphae after Congo red staining revealed only one growing hyphal tip per conidium. These results indicate that both fungi were affected by RIP1 treatment, but the lysis seen with treatment of A. nidulans was apparently avoided by A. flavus. A developmental time course revealed that both fungal species were affected by RIP1 at the postdivisional growth stage. The inhibitory activity of RIP1 against normal fungal growth is consistent with a biological function to protect the seed from fungal invasion.     

Use of green fluorescent protein to quantify the growth of Colletotrichum during infection of tobacco

To develop a quantitative assay of fungal growth inside plant tissues, strains of Colletotrichum destructivum and Colletotrichum orbiculare were transformed with a modified green fluorescent protein (GFP) gene fused with a glyceraldehyde-3-phosphate dehydrogenase promoter from Aspergillus nidulans. Transformants expressed GFP in culture and had the same growth rate and general appearance as the wild type. GFP was observed in all fungal structures during infection of leaves of Nicotiana benthamiana, except for the melanized appressoria and setae. The timing and appearance of the fungal structures in the host appeared to be identical to that of the wild type. GFP accumulation in inoculated leaves of N. benthamiana was quantified in leaf extracts using a fluorescence microplate reader, and the quantity of fluorescence was strongly correlated with the growth of the fungus as measured by the amount of fungal actin gene expression using Northern blot hybridizations. These results demonstrated that assaying green fluorescence levels from a GFP-transformed fungus is an accurate, fast and easy means of quantifying fungal growth inside host plant cells.     

Isolation of a phytoalexin-detoxification gene from the plant pathogenic fungus Nectria haematococca by detecting its expression in Aspergillus nidulans

Detoxification of the pea phytoalexin pisatin via demethylation, mediated by a cytochrome P-450 monooxygenase, is thought to be important for pathogenicity of the fungus Nectria haematococca on pea. To isolate a fungal gene encoding pisatin demethylating activity (pda), we transformed Aspergillus nidulans with a genomic library of N. haematococca DNA constructed in a cosmid which carried the A. nidulans trpC gene. Transformants were selected for Trp+ and then screened for pda. One transformant among 1250 tested was Pda+ and was less sensitive to pisatin in culture than Pda- A. nidulans. The cosmid containing the gene (PDA) conferring this activity was recovered by phage lambda packaging of transformant genomic DNA. When A. nidulans was transformed with the cloned cosmid, 98% of the Trp+ transformants were Pda+. RNA blots probed with a 3.35 kb subclone carrying PDA indicated that the gene is expressed constitutively in A. nidulans but is inducible by pisatin in N. haematococca.     

Development of a homologous transformation system for the opportunistic human pathogen Aspergillus fumigatus based on the sC gene encoding ATP sulfurylase

The development of a homologous transformation system for the opportunistic human pathogenic fungus Aspergillus fumigatus is described. The system is based on the sC gene encoding ATP sulfurylase. Several A. fumigatus sC mutant strains were readily isolated by strong selection for selenate resistance. The coding region plus upstream and downstream regulatory sequences of the A. fumigatus sC gene were cloned by inverse PCR and then sequenced. Sequencing of the sC cDNA revealed the presence of five introns located within the first half of the gene. The A. fumigatus sC gene encodes a protein of 574 amino acids which is highly similar to ATP sulfurylases from the filamentous fungal species Aspergillus nidulans, Aspergillus terreus and Penicillium chrysogenum. By contrast, ATP sulfurylases from the yeasts Saccharomyces cerevisiae and Schizosaccharomyces pombe lack the C-terminal adenosine-5'-phosphosulfate kinase-like domain present in the filamentous fungal orthologues. A 3.8-kb DNA fragment amplified by PCR and containing the sC gene plus 5' and 3' flanking regions was cloned into pUC19 to give the vector pSCFUM. Transformation of two different sC mutant isolates with the plasmid pSCFUM established the functionality of this new homologous transformation system. Molecular analysis of sC+ transformants showed that up to 44% of transformed clones contained one or more copies of the entire plasmid integrated at the sC locus. This result also demonstrates the utility of the sC marker for targeting specific genetic constructs to the A. fumigatus sC locus, facilitating studies of gene regulation and function.     

Differential Esterase Expression in Developmental Mutants of Aspergillus nidulans

Esterase isozymes were used to detect substrate-preference polymorphism in five strains of Aspergillus nidulans, and to show differential gene expression in developmental mutants in response to 5-azacytidine treatment. The medusa mutants B116, SM23, and M25 were selected in the presence of 5-azacytidine (5AC); also the G839 bristle mutant obtained in the absence of 5AC as well as the UT196 master strain and the normal segregant SM24 were used for the esterase studies. The esterase isozyme patterns of the A. nidulans strains observed with 4-methylumbelliferyl esters and alpha- and beta-naphthyl esters indicated a total of 18 isoesterases. Substrate preference for either 4-methylumbelliferyl esters and alpha- or beta-naphthyl esters was observed. Similarity between the different A. nidulans genotypes was 84.4-100%. The genomic similarity of the B116, SM23, and M25 mutant strains (100%) supports previous observations that specific DNA sequences might be targets for 5AC action in this filamentous fungus, and the differential expression of the Est-4 isozyme in the medusa developmental mutant and the Est-2 isozyme specifically detected in the bristle mutant G839 seems to indicate esterase isozymes as possible markers of biochemical differences among different developmental mutants of A. nidulans.     

veA is required for toxin and sclerotial production in Aspergillus parasiticus

It was long been noted that secondary metabolism is associated with fungal development. In Aspergillus nidulans, conidiation and mycotoxin production are linked by a G protein signaling pathway. Also in A. nidulans, cleistothecial development and mycotoxin production are controlled by a gene called veA. Here we report the characterization of a veA ortholog in the aflatoxin-producing fungus A. parasiticus. Cleistothecia are not produced by Aspergillus parasiticus; instead, this fungus produces spherical structures called sclerotia that allow for survival under adverse conditions. Deletion of veA from A. parasiticus resulted in the blockage of sclerotial formation as well as a blockage in the production of aflatoxin intermediates. Our results indicate that A. parasiticus veA is required for the expression of aflR and aflJ, which regulate the activation of the aflatoxin gene cluster. In addition to these findings, we observed that deletion of veA reduced conidiation both on the culture medium and on peanut seed. The fact that veA is necessary for conidiation, production of resistant structures, and aflatoxin biosynthesis makes veA a good candidate gene to control aflatoxin biosynthesis or fungal development and in this way to greatly decrease its devastating impact on health and the economy.     

RNA silencing in Aspergillus nidulans is independent of RNA-dependent RNA polymerases

The versatility of RNA-dependent RNA polymerases (RDRPs) in eukaryotic gene silencing is perhaps best illustrated in the kingdom Fungi. Biochemical and genetic studies of Schizosaccharomyces pombe and Neurospora crassa show that these types of enzymes are involved in a number of fundamental gene-silencing processes, including heterochromatin regulation and RNA silencing in S. pombe and meiotic silencing and RNA silencing in N. crassa. Here we show that Aspergillus nidulans, another model fungus, does not require an RDRP for inverted repeat transgene (IRT)-induced RNA silencing. However, RDRP requirements may vary within the Aspergillus genus as genomic analysis indicates that A. nidulans, but not A. fumigatus or A. oryzae, has lost a QDE-1 ortholog, an RDRP associated with RNA silencing in N. crassa. We also provide evidence suggesting that 5' --> 3' transitive RNA silencing is not a significant aspect of A. nidulans IRT-RNA silencing. These results indicate a lack of conserved kingdom-wide requirements for RDRPs in fungal RNA silencing.     

The Neurospora rca-1 gene complements an Aspergillus flbD sporulation mutant but has no identifiable role in Neurospora sporulation.

The Aspergillus nidulans flbD gene encodes a protein with a Myb-like DNA-binding domain that is proposed to act in concert with other developmental regulators to control initiation of conidiophore development. We have identified a Neurospora crassa gene called rca-1 (regulator of conidiation in Aspergillus) based on its sequence similarity to flbD. We found that N. crassa rca-1 can complement the conidiation defect of an A. nidulans flbD mutant and that induced expression of rca-1 caused conidiation in submerged A. nidulans cultures just as was previously observed for overexpression of flbD. Thus, the N. crassa gene appears to be a functional homologue of A. nidulans flbD and this is the first demonstration of functional complementation of an A. nidulans sporulation defect using a gene from an evolutionarily distant fungus. However, deletion of the rca-1 gene in N. crassa had no major effect on growth rate, macroconidiation, microconidiation, or ascospore formation. The only phenotype displayed by the rca-1 mutant was straight or counterclockwise hyphal growth rather than the clockwise spiral growth observed for wild type. Thus, if rca-1 is involved in N. crassa development, its role is subtle or redundant.     

5-azacytidine induces micronuclei in and morphological transformation of Syrian hamster embryo fibroblasts in the absence of unscheduled DNA synthesis.

It is known that 5-azacytidine (5-AC) induces tumors in several organs of rats and mice. The mechanisms of these effects are still poorly understood although it is known that 5-AC can be incorporated into DNA. Furthermore, it can inhibit DNA methylation. The known data on its clastogenic and/or gene mutation-inducing potential are still controversial. Therefore, we have investigated the kinds of genotoxic effects caused by 5-AC in Syrian hamster embryo (SHE) fibroblasts. Three different endpoints (micronucleus formation, unscheduled DNA synthesis (UDS) and cell transformation) were assayed under similar conditions of metabolism and dose at target in this cell system. 5-AC induces morphological transformation of SHE cells, but not UDS. Therefore, 5-AC does not seem to cause repairable DNA lesions. Furthermore, our studies revealed that 5-AC is a potent inducer of micronuclei in the SHE system. Immunocytochemical analysis revealed that a certain percentage of these contain kinetochores indicating that 5-AC may induce both clastogenic events and numerical chromosome changes.     

Fungal S-adenosylmethionine synthetase and the control of development and secondary metabolism in Aspergillus nidulans

The filamentous fungus Aspergillus nidulans carries a single gene for the S-adenosylmethionine (SAM) synthetase SasA, whereas many other organisms possess multiple SAM synthetases. The conserved enzyme catalyzes the reaction of methionine and ATP to the ubiquitous methyl group donor SAM. SAM is the main methyl group donor for methyltransferases to modify DNA, RNA, protein, metabolites, or phospholipid target substrates. We show here that the single A. nidulans SAM synthetase encoding gene sasA is essential. Overexpression of sasA, encoding a predominantly cytoplasmic protein, led to impaired development including only small sterile fruiting bodies which are surrounded by unusually pigmented auxiliary Hülle cells. Hülle cells are the only fungal cell type which does not contain significant amounts of SasA. Sterigmatocystin production is altered when sasA is overexpressed, suggesting defects in coordination of development and secondary metabolism. SasA interacts with various metabolic proteins including methionine or mitochondrial metabolic enzymes as well as proteins involved in fungal morphogenesis. SasA interaction to histone-2B might reflect a putative epigenetic link to gene expression. Our data suggest a distinct role of SasA in coordinating fungal secondary metabolism and development.