Dong-Min Han

The veA gene activates sexual development in Aspergillus nidulans.

The previously isolated gene complementing the veA1 mutation was confirmed to be the veA gene. The determined nucleotide sequence of the gene demonstrated that there is an open reading frame (ORF) of a 573 amino acid polypeptide. The nucleotide sequence matched some clones of which functions were not assigned yet and the amino acid sequence matched that of Neurospora crassa VeA-related protein with 61% similarity. The nucleotide sequence of the veA1 mutant gene differed from that of the wild type gene by only one nucleotide and the nucleotide G in the initiation codon ATG of the VeA ORF was mutated to the nucleotide T. Then, the mutant ORF may use the 37th methionine codon of the wild type one as a new initiation codon. The veA transcript was present in the conidia and in mycelia cultured for up to 14h and expressed almost constitutively at an increased level throughout the asexual and sexual developmental processes, suggesting that it may act from a relatively early developmental stage. Null mutants of the gene never formed sexual structures, even under conditions where sexual development preferentially occurs in wild types. Over-expressors of the gene formed larger numbers of sexual structures with a much reduced number of conidial heads than a control strain (a veA1 mutant), even under conditions where wild type strains form little sexual structure but form conidial heads very well, such as in the presence of a salt at high concentration. Furthermore, over-expressors could form Hülle cells and cleistothecia, even in a liquid culture. These results indicated that the veA gene is a positive regulator in sexual development and simultaneously a negative one in asexual development.

The nsdD gene encodes a putative GATA‐type transcription factor necessary for sexual development of Aspergillus nidulans

The ability to reproduce both sexually and asexually is one of the characteristics of the homothalic ascomycete Aspergillus nidulans. Unlike the other Aspergillus species, A. nidulans undergoes sexual development that seems to be regulated by internal and external stimuli. To begin to understand the sexual reproduction of A. nidulans we previously isolated and characterized several NSD (never in sexual development) mutants that failed to produce any sexual reproductive organs, and identified four complementation groups, nsdA, nsdB, nsdC, and nsdD. The nsdD gene has been isolated, and it is predicted to encode a GATA‐type transcription factor with the type IVb zinc finger DNA‐binding domain. The mRNA of the nsdD gene started to accumulate in the early phase of vegetative growth, and the level increased as sexual development proceeded. However, it decreased during asexual sporulation and no nsdD mRNA was detected in conidia. Deletion of nsdD resulted in no cleistothecia (fruiting bodies) formation, even under the conditions that preferentially promoted sexual development, indicating that nsdD is necessary for sexual development. In contrast, when the nsdD gene was over‐expressed, sexual‐specific organ (Hülle cell) was formed even in submerged culture, which normally completely blocked sexual development, and the number of cleistothecia was also dramatically increased on solid medium. These results lead us to propose that the nsdD gene functions in activating sexual development of A. nidulans. Multiple copies of the nsdD gene could suppress nsdB5 and veA1, indicating that either nsdD acts downstream of these genes or possibly functions in overlapping pathway(s).

The GanB Gα-Protein Negatively Regulates Asexual Sporulation and Plays a Positive Role in Conidial Germination in Aspergillus nidulans

We isolated the ganB gene encoding the Gα-protein homolog from Aspergillus nidulans. To investigate the cellular function of GanB, various mutant strains were isolated. Deletion of constitutively inactive ganB mutants showed conidiation and derepressed brlA expression in a submerged culture. Constitutive activation of GanB caused a reduction in hyphal growth and a severe defect in asexual sporulation. We therefore propose that GanB may negatively regulate asexual sporulation through the BrlA pathway. In addition, deletion or constitutive inactivation of GanB reduced germination rate while constitutive activation led to precocious germination. Furthermore, conidia of a constitutively active mutant could germinate even without carbon source. Taken together, these results indicated that GanB plays a positive role during germination, possibly through carbon source sensing, and negatively regulates asexual conidiation in A. nidulans.

The nsdC Gene Encoding a Putative C2H2-Type Transcription Factor Is a Key Activator of Sexual Development in Aspergillus nidulans

The formation of the Aspergillus nidulans fruiting body is affected by a number of genetic and environmental factors. Here, the nsdC (never in sexual development) gene—encoding a putative transcription factor carrying a novel type of zinc-finger DNA-binding domain consisting of two C2H2s and a C2HC motif that are highly conserved in most fungi but not in plants or animals—was investigated. Two distinct transcripts of 2.6 and 3.0 kb were generated from nsdC. The 2.6-kb mRNA accumulated differentially in various stages of growth and development, while the level of the 3.0-kb mRNA remained relatively constant throughout the life cycle. While the deletion of nsdC resulted in the complete loss of fruiting body formation under all conditions favoring sexual development, overexpression of nsdC not only enhanced formation of fruiting bodies (cleistothecia) but also overcame inhibitory effects of certain stresses on cleistothecial development, implying that NsdC is a key positive regulator of sexual development. Deletion of nsdC also retarded vegetative growth and hyperactive asexual sporulation, suggesting that NsdC is necessary not only for sexual development but also for regulating asexual sporulation negatively. Overexpression of veA or nsdD does not rescue the failure of fruiting body formation caused by nsdC deletion. Furthermore, nsdC expression is not affected by either VeA or NsdD, and vice versa, indicating that NsdC regulates sexual development independently of VeA or NsdD.

Membrane-bound methyltransferase complex VapA-VipC-VapB guides epigenetic control of fungal development.

Epigenetic and transcriptional control of gene expression must be coordinated in response to external signals to promote alternative multicellular developmental programs. The membrane-associated trimeric complex VapA-VipC-VapB controls a signal transduction pathway for fungal differentiation. The VipC-VapB methyltransferases are tethered to the membrane by the FYVE-like zinc finger protein VapA, allowing the nuclear VelB-VeA-LaeA complex to activate transcription for sexual development. Once the release from VapA is triggered, VipC-VapB is transported into the nucleus. VipC-VapB physically interacts with VeA and reduces its nuclear import and protein stability, thereby reducing the nuclear VelB-VeA-LaeA complex. Nuclear VapB methyltransferase diminishes the establishment of facultative heterochromatin by decreasing histone 3 lysine 9 trimethylation (H3K9me3). This favors activation of the regulatory genes brlA and abaA, which promote the asexual program. The VapA-VipC-VapB methyltransferase pathway combines control of nuclear import and stability of transcription factors with histone modification to foster appropriate differentiation responses.

The MpkB MAP kinase plays a role in post-karyogamy processes as well as in hyphal anastomosis during sexual development in Aspergillus nidulans.

Two genes encoding MAP kinase homologs, designated as mpkB and mpkC, were isolated from Aspergillus nidulans by PCR with degenerate primers. Deletion and over-expression mutants of mpkC showed no detectable phenotypes under any external stress tested. Deletion of mpkB caused pleiotropic phenotypes including a failure in forming cleistothecia under any induction conditions for sexual development, increased Hülle cell production, slow hyphal growth and aberrant conidiophore morphology. Over-expression of mpkB led to increased cleistothecium production. While the transcripts of mpkB and mpkC were constitutively synthesized through the entire life cycle, their size and amount differed with developmental stages. An outcross test using fluorescent protein reporters showed that the mpkB deletion mutant could not form heterokaryons with wild type. Protoplast fusion experiments showed that the fusant of the mpkB mutant with wild type could undergo normal sexual development. However, heterokaryotic mycelia that were produced from a fusant between two mpkB deletion mutants could not form cleistothecia, although they did appear to form diploid nuclei. These results suggest that the MpkB MAP kinase is required for some post-karyogamy process as well as at the hyphal anastomosis stage to accomplish sexual development successfully.

The veA gene is necessary for the inducible expression by fructosyl amines of the Aspergillus nidulans faoA gene encoding fructosyl amino acid oxidase (amadoriase, EC 1.5.3)

Abstract. The faoA gene encoding fructosyl amino acid oxidase (FAOD, EC 1.5.3) was isolated from Aspergillus nidulans and characterized. The complete nucleotide sequence of the faoA (fructosyl amino acid oxidase) gene and its cDNA revealed that the faoA gene encodes a 441-amino-acid polypeptide interrupted by five introns. Expression of the A. nidulans faoA gene was inducible by fructosyl propylamine and fructosyl lysine, as is the case for the gene encoding FAOD in other organisms. The faoA gene was not induced by these fructosyl amines in a null mutant of the veA gene, which has been identified as an activator of sexual development and as an inhibitor of asexual development; the faoA gene was induced greatly in a veA+ wild-type. However, veA gene expression was not affected by fructosyl amines. Even in the absence of fructosyl propylamine, synthesis of the faoA transcript was higher in the veA+ background than in a veA-null mutation background. These results indicated that faoA gene expression is inducible by fructosyl amines and by the veA gene, and that the veA gene is necessary for full induction of faoA gene expression by fructosyl amines. Thus, the faoA gene is the first gene whose expression is dependent on the veA gene. Furthermore, the faoA gene, present in a single copy, seems to be dispensable for development and growth, since the faoA-null mutant grew normally and developed as many conidia and sexual structures as the wild-type.

Expression of the mnpA gene that encodes the mannoprotein of Aspergillus nidulans is dependent on fadA and flbA as well as veA.

The single copy mnpA gene that encodes a mannoprotein of Aspergillus nidulans and its cDNA were isolated from the genomic and cDNA libraries, respectively. The determined nucleotide sequences of the genomic DNA and its cDNA revealed that the gene has an open-reading frame of 261 amino acids without introns. The deduced amino acid sequence showed a 60% identity to that of Aspegillus fumigatus galactomannoprotein MP1. The mnpA gene was expressed more abundantly in the wild-type than in the veA-null mutant. It was expressed at a lower level in fadA-null mutants, veA(+) or veA1 (regardless of their genetic background), than in the fadA(+) strain. However, the expression level was slightly higher in the veA(+) DeltafadA strain than in the veA1 DeltafadA strain. Furthermore, the amount of the mnpA transcript was higher in the flbA(+) strain than in the flbA-null mutant. These results indicate that the fadA and flbA genes in addition to the veA gene are necessary for the mnpA expression. The mnpA gene was expressed highly in vegetative mycelia and at a reduced level in sexual structures, but not in conidia. Its expression was almost constitutive during asexual development up to 18h after the transfer of mycelial balls onto a solid medium, and decreased thereafter. During sexual development, its expression reached its maximum 0-20h after the induction of sexual development, and then decreased thereafter. The mnpA-null mutant, that was still viable, showed no phenotypic difference in development, growth rate, protein secretion, and germination of both the ascospores and conidia from the wild-type. This suggests that the mannoprotein that is encoded by the mnpA gene is dispensable.

The MpkB MAP kinase plays a role in autolysis and conidiation of Aspergillus nidulans

The mpkB gene of Aspergillus nidulans encodes a MAP kinase homologous to Fus3p of Saccharomyces cerevisiae which is involved in conjugation process. MpkB is required for completing the sexual development at the anastomosis and post-karyogamy stages. The mpkB deletion strain could produce conidia under the repression condition of conidiation such as sealing and even in the submerged culture concomitant with persistent brlA expression, implying that MpkB might have a role in timely regulation of brlA expression. The submerged culture of the deletion strain showed typical autolytic phenotypes including decrease in dry cell mass (DCM), disorganization of mycelial balls, and fragmentation of hyphae. The chiB, engA and pepJ genes which are encoding cell wall hydrolytic enzymes were transcribed highly in the submerged culture. Also, we observed that the enzyme activity of chitinase and glucanase in the submerged culture of mpkB deletion strain was much higher than that of wild type. The deletion of mpkB also caused a precocious germination of conidia and reduction of spore viability. The expression of the vosA gene, a member of velvet gene family, was not observed in the mpkB deletion strain. These results suggest that MpkB should have multiple roles in germination and viability of conidia, conidiation and autolysis through regulating the expression of vosA and brlA.

A putative APSES transcription factor is necessary for normal growth and development of Aspergillus nidulans.

The nsdD gene encoding a GATA type transcription factor positively controls sexual development in Aspergillus nidulans. According to microarray data, 20 genes that were upregulated by deleting nsdD during various life cycle stages were randomly selected and deleted for functional analysis. None of the mutants showed apparent changes in growth or development compared with those of the wild-type except the AN3154 gene that encodes a putative APSES transcription factor and is an ortholog of Saccharomyces cerevisiae swi4. Deleting AN3154 resulted in retarded growth and development, and the gene was named rgdA (retared growth and development). The rgdA deletion mutant developed a reduced number of conidia even under favorable conditions for asexual development. The retarded growth and development was partially suppressed by the veA1 mutation. The conidial heads of the mutant aborted, showing reduced and irregular shaped phialides. Fruiting body development was delayed compared with that in the wild-type. The mutant did not respond to various nutritional or environmental factors that affected the development patterns. The rgdA gene was expressed at low levels throughout the life cycle and was not significantly affected by several regulators of sexual and asexual development such as nsdD, veA, stuA, or brlA. However, the rgdA gene affected brlA and abaA expression, which function as key regulators of asexual sporulation, suggesting that rgdA functions upstream of those genes.