Aspergillus SteA (sterile12-like) is a homeodomain-C2/H2-Zn+2 finger transcription factor required for sexual reproduction.

Saccharomyces cerevisiae Ste12p plays a key role in coupling signal transduction through MAP kinase modules to cell-specific or morphogenesis-specific gene expression required for mating and pseudohyphal (PH)/filamentous growth (FG). Ste12p homologues in the pathogenic yeasts Candida albicans and Filobasidiela neoformans apparently play similar roles during dimorphic transitions. Here we report the isolation and characterization of the first Ste12 protein from a true filamentous fungus. Aspergillus nidulans steA encodes a protein with a homeodomain 63-75% identical to those of other Ste12 proteins, with greatest similarity to FnSte12alphap. SteAp and Ste12alphap lack the pheromone induction domain found in budding yeast Ste12p, but have C-terminal C2/H2-Zn+2 finger domains not present in the other Ste12 proteins. A DeltasteA strain is sterile and differentiates neither ascogenous tissue nor fruiting bodies (cleistothecia). However, the development of sexual cycle-specific Hülle cells is unaffected. Filamentous growth, conidiation and the differentiation of PH-like asexual reproductive cells (metulae and phialides) are normal in the deletion strain. Northern analysis of key regulators of the asexual and sexual reproductive cycles support the observation that although SteAp function is restricted to the sexual cycle, cross regulation between the two developmental pathways exists. Our results further suggest that while several classes of related proteins control similar morphogenetic events in A. nidulans and the dimorphic yeasts, significant differences must exist in the regulatory circuitry.

Functional analysis of DNA sequences required for conidium-specific expression of the SpoC1-C1C gene of Aspergillus nidulans.

The SpoC1-C1C gene is centrally located within the A. nidulans conidium-specific SpoC1 gene cluster. With one exception, the 14 genes within the cluster are coordinately regulated. C1C transcript is first detected late in conidiation, coincidental with the appearance of mature conidia, and accumulates approximately 1000-fold in conidia. We show that C1C expression is restricted to conidia, with mRNA abundance decreasing immediately after induction of germination. C1C transcription and translation are not temporally separated and, similar to C1C RNA abundance, a C1C::beta-galactosidase fusion protein is first detected with the appearance of mature conidia and decreases after induction of germination. Cell-specific C1C expression requires both a position-dependent mechanism of regulation, responsible for repression in hyphae, and a position-independent mechanism of regulation, responsible for developmental expression. We show by functional analysis of upstream DNA sequences that a 10-bp sequence and two adjacent 6-bp direct repeats are necessary for position-independent, condium-specific expression of both the intact C1C gene and the reporter gene. At least one repeat (CAACAT) is required for normal levels of expression. We find that the C1C gene is not a direct target of the BrlAp and AbaAp developmental regulators, but of a yet unidentified conidium-specific transcriptional activator.

Suppression and enhancement of the Aspergillus nidulans medusa mutation by altered dosage of the bristle and stunted genes.

Asexual reproduction in Aspergillus nidulans is characterized by the orderly differentiation of multicellular reproductive structures (conidiophores) and chains of uninucleate conidia (spores). Mutations in the developmental modifier medusa (medA) result in aberrant conidiophores with branching chains of reiterated reproductive cells (metulae), delayed conidial differentiation and frequent reinitiation of secondary conidiophores. We show that incorrect morphology is in part a consequence of modified bristle (brlA) and abacus (abaA) expression, key regulators of the core genetic pathway directing conidial differentiation. First, correct temporal expression of both brlA transcripts (brlA alpha and brlA beta) requires MedAp. Second, MedAp functions as a coactivator required for normal levels of abaA expression. Finally, we show that wild-type morphology results from a finely tuned balance in the expression of brlA, medA and the developmental modifier stunted (stuA). One extra copy of brlA suppresses medA mutations and restores normal abaA mRNA abundance. In contrast, an extra copy of stuA in a medA- strain results in an enhanced medusoid phenotype with extensive metulae proliferation and nearly complete absence of conidia. abaA and brlA alpha transcription are completely repressed in these strains. In general, low stuA:brlA ratios promoted conidiation while high ratios caused proliferation of unicellular sterigmata and inhibited conidiation.

StuA is required for cell pattern formation in Aspergillus.

The stunted (stuA) gene product is required for the orderly differentiation and spatial organization of cell types of the Aspergillus nidulans conidiophore. Expression of the stuA gene is complex. Two transcripts, stuA alpha and stuA beta, are initiated from separate promoters. Transcription of both RNAs increases approximately 50-fold during the establishment of developmental competence. Induction-dependent transcriptional and post-transcriptional regulatory mechanisms further enhance expression 15-fold. Consistent with the latter observation, both transcripts have structural features characteristic of RNAs subject to translational control. Conidiophore morphogenesis requires regulatory interactions between the products of the stuA, bristle (brlA), and abacus (abaA) genes. Enhanced stunted expression is cell type specific and dependent on a functional BrlA protein. StuA affects the spatial localization of AbaA by acting directly, or indirectly, to repress abaA expression.

Isolation and transcriptional characterization of a morphological modifier: the Aspergillus nidulans stunted (stuA) gene.

The functions of at least four potential regulatory genes are known to overlap temporally during elaboration of the multicellular asexual reproductive apparatus (conidiophore) of Aspergillus nidulans. One of these, the stuA (stunted) gene, has been previously classified as a morphological modifier essential for correct spatial organization of the conidiophore. The gene was cloned by complementation of a strain carrying the stuA1 mutation and has been localized to a 5.0 kb KpnI fragment that encodes a 3.3 kb mRNA. The stuA mRNA was detected at very low levels in mature conidia and in somatic hyphae that had not established developmental competence. A dramatic increase in the abundance of this mRNA occurred coincidentally with the establishment of competence, but prior to the induction of conidiation. RNA abundance remained at this elevated level during conidiophore morphogenesis. These results are consistent with genetic data suggesting that stuA gene function is required from the very earliest events of asexual reproduction until completion of conidiophore development, but is not specifically required for differentiation of conidia. The expression of the stuA transcript was not affected by any of the other characterized developmental mutations. These latter results suggest that transcriptional activation at the onset of competence is mediated by an as yet unidentified genetic locus or loci.

Position-dependent and -independent mechanisms regulate cell-specific expression of the SpoC1 gene cluster of Aspergillus nidulans.

Many genes that are expressed specifically in the differentiating asexual spores (conidia) of Aspergillus nidulans are organized into clusters. We investigated the effects of altered chromosomal position on expression of a gene from the conidiation-specific SpoC1 gene cluster. The gene became deregulated when integrated at nonhomologous chromosomal sites, in that transcript levels were elevated in vegetative cells (hyphae) and variably altered in conidia. We also investigated the effects on expression of insertion of the nonregulated argB gene into the SpoC1 region. Levels of argB transcripts were markedly reduced in hyphae. The results suggest that a cis-acting regional regulatory mechanism represses transcription of SpoC1 genes in hyphae. They also indicate that expression of individual SpoC1 genes is modulated during conidiation by trans-acting factors. We propose that the two types of regulation act together to produce the major differences in transcript levels observed in hyphae versus conidia.

Direct and indirect gene replacements in Aspergillus nidulans.

We performed three sets of experiments to determine whether cloned DNA fragments can be substituted for homologous regions of the Aspergillus nidulans genome by DNA-mediated transformation. A linear DNA fragment containing a heteromorphic trpC+ allele was used to transform a trpC- strain to trpC+. Blot analysis of DNA from the transformants showed that the heteromorphic allele had replaced the trpC- allele in a minority of the strains. An A. nidulans trpC+ gene was inserted into the argB+ gene, and a linear DNA fragment containing the resultant null argB allele was used to transform a trpC- argB+ strain to trpC+. Approximately 30% of the transformants were simultaneously argB-. The null argB allele had replaced the wild-type allele in a majority of these strains. The A. nidulans SpoC1 C1-C gene was modified by removal of an internal restriction fragment and introduced into a trpC- strain by transformation with a circular plasmid. A transformant containing a tandem duplication of the C1-C region separated by plasmid DNA was self-fertilized, and trpC- progeny were selected. All of these had lost the introduced plasmid DNA sequences, whereas about half had retained the modified C1-C gene and lost the wild-type copy. Thus, it is possible with A. nidulans to replace chromosomal DNA sequences with DNA fragments that have been cloned and modified in vitro by using either one- or two-step procedures similar to those developed for Saccharomyces cerevisiae.

Structure and regulated expression of the SpoC1 gene cluster from Aspergillus nidulans.

We have previously described the organization of a 13.3 kb region of the Aspergillus nidulans genome, designated SpoC1, coding for multiple poly(A)+ RNAs that accumulate in asexual spores but not in somatic cells. We have determined the limits of the SpoC1 gene cluster by investigating the transcriptional features of 53 kb of chromosomal DNA. This segment of the genome codes for at least 19 poly(A)+ RNAs, some of which are transcribed from overlapping regions. The area of developmental regulation is approximately 38 kb in length and is delimited by 1.1-kb direct repeats. With one exception, RNAs transcribed from the central part of the cluster appear late during conidiophore development and accumulate specifically in spores. The exceptional transcript appears earlier during development and accumulates specifically in cells of the conidiophore. In contrast, RNAs encoded at the borders of the cluster occur in both somatic cells and spores. The results indicate that if a chromatin-level control mechanism operates to regulate expression of the SpoC1 gene cluster, as previously suggested by us, additional levels of regulation must also exist.