Here we have characterized the putative Zn(II)₂Cys₆ transcription factor RosA from the filamentous fungus Aspergillus nidulans. The rosA gene encodes a protein of 713 aa, which shares 38% sequence similarity to Pro1 from Sordaria macrospora. In contrast to Pro1, which promotes the transition from protoperithecia to perithecia, RosA is a negative regulator of sexual development in A. nidulans. Transcript levels of rosA were usually very low and were only transiently upregulated upon carbon starvation and at 12 hr of asexual development. Deletion of rosA only slightly induced fruiting-body formation under standard culture conditions, but enabled sexual development under low-glucose and high-osmolarity conditions and the production of Hülle cells under submersed growth conditions. Stimulation of fruiting-body formation on agar surfaces was dependent on veA. In rosA strains, transcript levels of the sexual developmental regulators nsdD, veA, and stuA were increased. Overexpression of rosA led to a reduction of hyphal growth and to a fluffy phenotype. Post-transcriptional regulation of RosA, with a regulated accumulation in the nucleus, was shown using a RosA-GFP fusion protein. We propose that RosA represses sexual development upon integration of several environmental signals.

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SPORE formation is a common strategy of many fungi to cope with unfavorable conditions or to propagate in the environment and conquer new habitats. The mechanisms of fungal spore formation can be quite different among genera but usually involve the generation of specialized morphological structures. A model fungus to study spore formation at the molecular level is the euascomycete Aspergillus nidulans (ADAMS et al. 1998; FISCHER 2002). This mold grows vegetatively through hyphal extension and produces spore-bearing structures, called conidiophores, after a defined period of time. This asexual developmental program occurs at a water-air interface and requires the presence of red light. In addition to the asexual developmental program, A. nidulans is able to undergo sexual reproduction with or without prior mating with another strain (BRAUS et al. 2002). The first visible sign of sexual differentiation is the formation of a nest-like structure made of so-called Hülle cells. Underneath these thick-walled cells, primordia develop and mature to form a ripe cleistothecium, also known as the fruiting body. Within the cleistothecia nuclear fusion and subsequent meiosis and ascospore formation occurs (SOHN and YOON 2002).

Abiotic and biotic factors, such as a water-air interface, light, CO₂ concentration, nutritional status, and a fatty-acid-based and probably a peptide-based pheromone system, are responsible for the developmental decisions determining sexual or asexual reproduction (ZONNEVELD 1977; ECKERT et al. 1999; BRAUS et al. 2002; CALVO et al. 2002; DYER et al. 2003; LARA-ORTIZ et al. 2003; TSITSIGIANNIS et al. 2004). Recently, it was discovered that the concentration of reactive oxygen species (ROS) could be a critical factor (SCHERER et al. 2002). A NADPH oxidase (NoxA), which generates ROS in A. nidulans, was reported to be necessary for sexual development (LARA-ORTIZ et al. 2003).

All factors are integrated and transduced into physiological and morphological responses via G-protein-coupled receptor systems, a MAP kinase and cAMP-signaling cascades, and a number of putative transcription factors (HAN and PRADE 2002; KAWASAKI et al. 2002; WEI et al. 2003; HAN et al. 2004). Some of these signaling components were discovered through mutant analyses. Thus, stuA mutants display a strong defect in asexual sporulation, have shorter conidiophores, and fail to produce metulae and spore-producing phialides (MILLER et al. 1992). Instead, spores are directly produced from the vesicle. The StuA protein is a helix-loop-helix transcription factor, which appears to repress the conidiophore-specific regulators abaA (DUTTON et al. 1997) and brlA (BUSBY et al. 1996). In addition to the asexual phenotype, stuA strains fail to produce sexual reproduction structures and are therefore self-sterile. In this line, StuA has been found to be an activator of a catalase-peroxidase gene, cpeA, expressed in Hülle cells (SCHERER et al. 2002). Another regulator discovered after mutant analysis is the protein NsdD, a putative GATA-type transcription factor (CHAE et al. 1995; HAN et al. 2001). The nsdD deletion strains are unable to form cleistothecia, while their overexpression causes an increase of the number of fruiting bodies on solid medium as well as masses of Hülle cells in liquid culture. A gene with a key role in the regulation of A. nidulans development is veA, which mediates the light response (KäFER 1965; MOONEY and YAGER 1990). A. nidulans normally conidiates in the presence of red light while it undergoes sexual reproduction under permanent dark conditions. When veA is mutated, conidiophore development is enhanced and occurs even in the dark. Concomitantly, sexual reproduction is lowered. Likewise, veA overexpression causes the induction of the sexual cycle even under inappropriate conditions, such as high osmolarity or in liquid medium. This suggests a complex function for this regulator in both sexual and asexual development (KIM et al. 2002). A putative transcription factor that plays a role in both reproduction strategies is dopA. Corresponding mutants grow with abnormal vegetative hyphae and produce aberrant conidiophores. Conidiation is drastically reduced due to delayed and asynchronous initiation of asexual development. Sexual reproduction in this mutant is completely abolished (PASCON and MILLER 2000). The homeodomain C₂/H₂-Zn⁺² finger transcription factor SteA, with similarity to the Saccharomyces cerevisiae Ste12 protein, is also an important regulator of sexual reproduction in A. nidulans (VALLIM et al. 2000). Its deletion causes a developmental block of fruiting-body formation at the Hülle cell stage. Ascogenous hyphae or cleistothecia are not produced.

Recently, a role for the signalosome in sexual development was discovered (BUSCH et al. 2003). The COP9 signalosome is a conserved multiprotein complex involved in the regulation of development in mammals and plants. Deletion of two of the COP9-signalosome components, csnD and csnE, in A. nidulans showed the same specific block in an early stage of cleistothecial formation. However, this signaling module appears to be involved in several other morphogenetic pathways. Despite the accumulated knowledge regarding single signaling components regulating the developmental decision between sexual and asexual reproduction, the links among them are still missing.

Similarly, little is known about regulators involved after the onset of the sexual developmental program. A possible regulator of the process of cleistothecia maturation could be a homolog of the Zn(II)₂Cys₆ transcription factor, Pro1, from the homothallic fungus Sordaria macrospora (MASLOFF et al. 1999). Mutation of this regulator results in a specific block of perithecium development. We screened the genomic DNA database of A. nidulans and identified a gene, rosA, whose translation product shared sequence similarity to Pro1. We have characterized the gene and its protein and found, interestingly, that Pro1 and RosA serve different functions in the two ascomycetous fungi. We present evidence that RosA acts as a repressor of sexual development initiation under low-carbon conditions and in submersed culture.

Strains, plasmids, and culture conditions:

Supplemented minimal and complete media for A. nidulans were prepared as described, and standard strain construction procedures were used (HILL and KäFER 2001). A list of A. nidulans strains used in this study is given in Table 1. Standard laboratory Escherichia coli strains (XL-1 blue, Top 10 F') were used. Plasmids and cosmids are listed in Table 2.

View this table: In this window In a new window   TABLE 1 A. nidulans and E. coli strains used in this study

 

View this table: In this window In a new window   TABLE 2 Plasmids used in this study

 

Molecular techniques:

Standard DNA transformation procedures were used for A. nidulans (YELTON et al. 1984) and E. coli (SAMBROOK and RUSSEL 1999). For PCR experiments standard protocols were applied using a capillary rapid cycler (Idaho Technology, Idaho Falls, ID) for the reaction cycles. DNA sequencing was done commercially (MWG Biotech, Ebersberg, Germany). Genomic DNA was extracted from A. nidulans with the DNeasy plant mini kit (QIAGEN, Hilden, Germany). RNA was isolated with Trizol according to the manufacturer's protocols (GibcoBRL Life Technologies, Paisley, Scotland, UK). DNA and RNA analyses (Southern and Northern hybridizations) were performed as described (SAMBROOK and RUSSEL 1999).

Deletion of rosA:

The rosA open reading frame was replaced with the argB marker. The argB auxotrophic marker was cloned into BamHI-EcoRI sites of pKV15. This results in deletion of a 2.5-kb restriction fragment including the rosA open reading frame with only 100 bp upstream of the stop codon left in the subclone flanked by 1.2- and 2.5-kb border regions, respectively. The deletion construct (pKV18) was linearized with KpnI/XbaI and transformed into the arginine-auxotrophic A. nidulans strain SRF200. To verify the disruption event, we used the following three primers: primer 1, 5'-CCCAGCTCGACCATGCCG-3'; primer 2, 5'-GCCCTCGAGAAATATTAAAGCAGAAGATAGTGCC-3'; and primer 3, 5'-CTGAGAAATGATTCGTGAATG-3'.

GFP labeling of RosA:

For the N-terminal fusion construct, the rosA open reading frame (starting from ATG and ending 750 bp after the stop codon) was amplified from genomic DNA as template and Pfu ultra polymerase (Stratagene, La Jolla, CA). Primers used were RosA-for-AscI (5'-GGCGCGCCCGGGATGTCAGCTCTCGCCGGAC-3') with an AscI site added at the 5'-end and RosA-long-rev-PacI (5'-TTAATTAAGAATGTTTCAGACTCATGTAG-3'), introducing a PacI site at the 3'-end. The PCR product was incubated for 30 min at 72° with Taq-Polymerase to add a T at the 5'-end and afterward cloned into the TOPO-TA cloning vector pCR2.1 (Invitrogen, Karlsruhe, Germany). The clone was sequenced by MWG Biotech and the rosA was cloned in the pCMB17apx plasmid (kindly provided by V. Efimov, Piscataway, NJ) into AscI-PacI sites behind the alcA promoter and the GFP gene (pKV50). Truncated versions of GFP-RosA were obtained by digests with PstI (642 aa, pKV57) and BglII (449 aa, pKV58) and by religation. This results in an additional 18 aa for pKV58 and 33 aa for pKV57 due to translation of the polylinker of the plasmids. For the two short constructs (80 aa, pKV60 and 294 aa, pKV48), parts of the rosA open reading frame were amplified with genomic DNA as template, the same forward primer as above, RosA-for-AscI, and the reverse primers RosA-80-aa-rev (5'-TTAATTAACTATCGGTATTCGCAGCTGAC-3') or RosA-300-aa-rev (5'-TTAATTAATTAGTCGACAAAGTGATAGAGTAG-3'), both containing an additional PacI site and a stop codon. Cloning of the PCR products was the same as already described for pKV50. All GFP constructs were transformed into the two pyrG mutant strains SRF200 (veA1) and SKV103 (veA⁺).

Overexpression of rosA:

The rosA open reading frame (from 70 bp upstream of the ATG to the predicted stop codon) was amplified with Expand polymerase (Roche, Mannheim) and the primers RosA-5' (5'-TACCTCGAGCACCTGTCGCAGATATCTGG-3') and RosA-3' (5'-GCCCTCGAGAAATATTAAAGCAGAAGATAGTGCC-3'), both containing an additional XhoI site. The PCR product was first cloned into the TOPO-TA cloning vector pCR2.1 (Invitrogen) and from there into the XhoI site of the overexpression vector pKV8, containing the inducible A. nidulans alcA promoter and the auxotrophy marker argB. The rosA overexpression construct (pKV12) was transformed into the arginine-auxotrophic strain RMS011, and transformants were grown under repressing (glucose) and inducing (ethanol) conditions.

Light microscopy, live-cell image acquisition, and analysis:

Cells were grown in glass-bottom dishes (World Precision Instruments, Berlin) in 2 ml of minimal medium (MM) without glucose + 2% glycerol + pyridoxine and/or arginine or MM + 2% ethanol (or threonine) + pyridoxine and/or arginine medium. Cells were incubated at 37° for 10 hr and images were captured at RT with an Axiophot microscope (Zeiss, Jena), a Planapochromatic x63 oil immersion objective lens, and a 50 W Hg lamp. Images were collected and analyzed with a Hamamatsu Orca ER II camera system, and the Wasabi software (version 1.2).

Electron microscopy:

For scanning electron microscopy (SEM), colonies grown on ethanol plates were transferred with a piece of agar into 5% glutaraldehyde for fixation. After several washings with water, the pieces were transferred to glycol-monoethyl ether and incubated overnight at room temperature. They were then transferred to water-free acetone and critical point dried. The samples were then sputter coated with gold and observed with a Hitachi S-530 SEM.

Molecular cloning of the rosA gene:

We used the protein sequence of S. macrospora Pro1 to screen the A. nidulans DNA sequence database at the Whitehead Institute (Cambridge, MA). We found two sequences with similarity to Pro1 and used them to isolate a corresponding cosmid (PUI library kindly provided by B. Miller, University of Idaho, Moscow, ID). The characterization of one of these genes, rosA, is presented here, while the results obtained for the second gene will be published elsewhere. After subcloning of a 6-kb XbaI/KpnI restriction fragment, the full rosA genomic locus was sequenced on both strands, which confirmed the sequence obtained in the genome project. RT-PCR was used to generate a corresponding cDNA, and two introns of 82 and 45 bp were detected at the 5'-end of the gene. The border sequences of the introns were in agreement with the consensus sequences described for fungi (GURR et al. 1987). The position of the larger intron is conserved in orthologs of S. macrospora, Sordaria brevicollis, and Neurospora crassa (Figure 1). The gene from A. nidulans was named repressor of sexual development (rosA) because of the phenotype of the corresponding deletion strain (see below).

View larger version (84K): In this window In a new window Download PPT slide   FIGURE 1.— (A) Scheme of the rosA gene locus. (B) Alignment of RosA with Pro1 from S. macrospora (accession no. Q9UVG3) and the homolog from N. crassa (accession no. Q9P326). Shaded background indicates amino acids that were identical in two sequences and solid background indicates amino acids that matched in all three proteins. The alignment was done with DNAStar using Megalign (Clustal) with a gap penalty and a gap-length penalty of 10. The cysteine residues involved in the coordination of the Zn atoms are highlighted with an asterisk above the sequences. The position of the conserved intron is indicated by an arrowhead. The predicted bipartite NLS is indicated by a dashed line above the sequence. The A. nidulans sequence is available under accession no. CAD58393

 
The deduced RosA protein has a molecular mass of 79.6 kD and a calculated isoelectric point of 7.6. At the N terminus of the protein, we detected the highly conserved Zn(II)₂Cys₆ motif and a putative bipartite nuclear localization signal, which overlaps with the predicted DNA-binding domain (Figure 1). In addition, we found a threonine-rich region (aa 178–236) and two putative PEST sequences (PESTfind analysis program at http://www.expasy.org; amino acids 133–253 and 646–659). The identity of the amino acid sequence of the Zn(II)₂Cys₆ region to the same motif in other transcription factors was between 55 and 70%. In the case of Pro1 from S. macrospora and orthologs from other fungi, long stretches of identical amino acid sequences were found in the C terminus of the proteins in addition to the identity of the DNA-binding motifs (Figure 1). The region from amino acid 80 to 352 displayed weak similarity (20% identity) to the corresponding regions of S. macrospora or N. crassa. The protein sequence identity between full-length RosA and Pro1 of S. macrospora or of the Pro1 orthologs from N. crassa and S. brevicollis was 38%. In contrast, sequence identities among the latter three Pro1 proteins are 90%, and the corresponding genes complement the pro1 mutation in S. macrospora. In addition to Pro1, MASLOFF et al. (2002) detected another similar protein, Pro1A, in the database of N. crassa. This transcription factor displayed only 40% identical amino acids to Pro1 and could not complement the pro1 mutant phenotype of S. macrospora. The identity between A. nidulans RosA and Pro1A was only 21%. In two other Aspergilli, A. fumigatus and A. oryzae, we found proteins with identities to RosA of 42.5 and 36.0%, respectively. We found no significant hits in the genomes of S. cerevisiae, Schizosaccharomyces pombe, the filamentous ascomycete Ashbya gossypii, or the basidiomycetous fungi Ustilago maydis and Coprinus cinereus.

Deletion of rosA:

To assess the role of RosA, we deleted the gene from the genome. In the 6-kb KpnI/XbaI restriction fragment, a 2.5-kb BamHI/EcoRI of the rosA open reading frame was replaced by the nutritional marker gene argB. With this cloning strategy, we removed a DNA fragment containing 400 bp upstream of the ATG and the open reading frame, except the last 100 bp to the stop codon. The construct (pKV18) was introduced into the argB auxotrophic strain (SRF200) and transformants were screened by Southern blot for a homologous recombination event. We found one colony, among 80 transformants, with the expected banding pattern (Figure 2). The gene replacement event was confirmed by PCR and other restriction digests. To make sure that the strain did not contain any other mutations, we crossed it to an argB auxotrophic strain (RMS011) and selected strains from the progeny with the rosA-deletion event. Phenotypic inspection of independent rosA progeny strains revealed no significant differences to the wild-type strain regarding timing of asexual or sexual development, morphology of the conidiophores, number of conidia, or number of cleistothecia or ascospores, when grown under standard laboratory conditions on agar plates. Because common laboratory strains, such as SRF200 or RMS011, harbor a mutation in the developmental regulator veA, we wanted to prove whether the rosA deletion causes a phenotype in a veA⁺ background. We crossed SKV8 with WIM126 and selected a rosA, veA⁺ strain (SKVw3) with no other mutations and compared it to the rosA⁺, veA⁺ strain FGSC4. The presence of the veA⁺ allele was confirmed by sequence analysis of the corresponding part of the gene. We incubated the strains in the presence and absence of light and found that the number of cleistothecia was increased in the rosA-deletion strain in the dark. This result suggests that rosA partially represses sexual development in A. nidulans and that this function depends on veA. The phenotypic difference between wild type and rosA strains became more drastic when we changed the concentration of the carbon source or grew them in the presence of 0.6 M KCl. When the glucose concentration was reduced from 100 to 10 mM, cleistothecia formation was dramatically reduced in wild type, whereas in rosA-deletion strains mature cleistothecia with fertile ascospores were still produced (Figure 3). Interestingly, cleistothecia were located in the border region of the rosA colony rather than in the center as in wild type. We quantified this effect by counting the Hülle cell nests in a circle of 0.5 cm diameter at a distance of 0.5 cm from the colony border and found 26 (±12) for rosA and 0.7 (±2) for wild type. Similarly, addition of 0.6 M KCl still allowed the production of mature cleistothecia with fertile ascospores in rosA-deletion strains, while in wild-type strains cleisthothecia production was much more strongly inhibited.

View larger version (41K): In this window In a new window Download PPT slide   FIGURE 2.— Deletion of the rosA gene. (A) Scheme of the knockout construct pKV18. (B) PCR analysis for the demonstration of the rosA-deletion event using the three oligonucleotides indicated in A. PCR fragments were separated on a 1% agarose gel and stained with ethidium bromide. (C) Southern blot analysis of a rosA-deletion strain. Genomic DNA of a wild-type (SRF200) and a rosA-deletion strain was isolated, restricted with EcoRI (left blot) and XhoI (right blot), separated on a 1% agarose gel, blotted, and hybridized with the 32P-labeled probe indicated in A.

 

View larger version (62K): In this window In a new window Download PPT slide   FIGURE 3.— Phenotype of a rosA-deletion strain. Point-inoculated rosA strain pKVw3 and wild-type FGSC4, both veA+, were grown for 4 days at 37° on solid minimal medium. (A) No difference between rosA and wild type is observed on agar plates containing 100 mM glucose and incubated in the light. (B) On agar plates incubated in the dark, the number of cleistothecia is increased in the rosA strain. (C and D) Comparison of wild-type and rosA strains on low-glucose medium (10 mM). Neither wild type nor the rosA strain produce cleistothecia in the light. In the dark, the rosA strain undergoes sexual development and produces cleistothecia. (E) Enlarged view of the region boxed in D. (F) Further enlargement of the boxed area in E shows a cleistothecium embedded in yellow Hülle cells. (G) Crushed cleistothecium, which released mature asci and red ascospores. Bar, 1.5 cm in A–D; 2 mm in E; 400 µm in F; and 200 µm in G.

 
Since it is known that development is repressed in submersed culture, we asked whether a rosA strain would be able to initiate the sexual cycle under these conditions. Indeed, we found Hülle cell formation after 48 hr of cultivation of the rosA strain in liquid culture (Figure 4). Although the number of Hülle cells increased after prolonged incubation, further developmental stages, such as cleistothecia were not observed. This phenotype was independent of the veA gene and fully complemented with a genomic copy of rosA (pKV15; results not shown).

View larger version (88K): In this window In a new window Download PPT slide   FIGURE 4.— Phenotype of a rosA-deletion strain in liquid minimal medium. Medium was inoculated with 106 spores/ml and incubated on a horizontal shaker (200 rpm) for 72 hr. (A) Mycelia of wild type (SKV29) and (B) mycelia of the rosA-deletion strain SKV12 as seen in the scanning electron microscope. (C) Phase-contrast picture of an individual Hülle cell. (D) A bulk of Hülle cells of the MutA(p)-GFP-expressing strain SKV30 generated in liquid culture. The picture was taken with a combination of phase-contrast and fluorescence illumination. The Hülle cell in the middle shows green fluorescence in the cytoplasm. (E) Northern blot analysis of three developmental regulators during growth in submersed culture. Cultures of wild type (wt) and mutant (rosA) were harvested after 24, 48, and 72 hr, RNA isolated, and subjected to Northern blotting. The membranes were hybridized with gene-specific DNA fragments labeled with 32P. Prior to hybridization, rRNA was stained with methylene blue as loading control.

 
The question of whether the Hülle cells produced in submersed culture were physiologically active was addressed by analysis of the expression of a Hülle-cell-specific gene, the -1,3-glucanase mutA (WEI et al. 2001). A corresponding GFP fusion construct (Mut(p)-gfp) was transformed into a rosA-deletion strain. We analyzed several independent transformants for GFP fluorescence, because the plasmid integrates ectopically into the genome of A. nidulans, and expression of the construct might vary depending on the integration site and the copy number. Hülle cells appeared in liquid medium after 48 hr. About 10% of the cells showed GFP fluorescence. This small number might reflect different physiological states of the Hülle cells, but nevertheless suggests that the cells are viable and active (Figure 4; WEI et al. 2001).

Interaction of rosA with other developmental regulators:

The phenotype of Hülle cell production in submersed culture was already described for strains overexpressing either nsdD or veA (HAN et al. 2001; KIM et al. 2002). To test whether deletion of rosA causes an induction of a nsdD or a veA transcript, we performed Northern blot analyses. Mycelium (SKVw3) was grown for 24, 48, and 72 hr in liquid culture and then processed for RNA analysis. In the wild-type strain (FGSC4), nsdD was expressed at a low level at all time points while it was upregulated in the rosA strain (two independent experiments). Similarly, the veA gene was slightly induced in the absence of rosA. StuA, which induces Hülle-cell-specific genes (SCHERER et al. 2002), was also upregulated in the rosA strain in comparison to wild type after 48 and 72 hr (Figure 4).

Overexpression of rosA:

To overexpress rosA, we replaced the endogenous promoter by the highly inducible alcohol dehydrogenase (alcA) promoter from A. nidulans. We introduced the resulting construct (pKV12) as a single copy into A. nidulans wild type (RMS011). Under repressing conditions (growth on 2% glucose) no significant difference with respect to hyphal growth or asexual or sexual development was observed. However, induction of the gene with 2% ethanol (or threonine) as carbon source resulted in a drastic developmental phenotype, which was also observed when the induction level was decreased by using 2% glycerol instead of ethanol (results not shown). Transferred colonies grew only with a reduced extension rate in comparison to the wild type and further development was completely inhibited. Instead, massive production of aerial hyphae occurred and led to a cotton-like appearance of the colonies (Figure 5). Of 20 transformed strains, 16 showed this phenotype, suggesting that the phenotype was not due to disruption of a developmental gene upon ectopic integration of the overexpression construct. A similar phenotype is well known for a class of mutants called fluffy. This class is composed of, e.g., transcription factors and other genes encoding proteins involved in signal transduction. One candidate of the latter group is fadA, which codes for the -subunit of a heterotrimeric G-protein (YU et al. 1996). When a constitutively active allele of fadA is introduced into an A. nidulans wild-type strain, the colony adopts a fluffy appearance. To analyze whether RosA acts as a transcription factor downstream of the activated G-protein, we introduced a constitutively active allele of fadA into a rosA-deletion strain (SKV8) by cotransformation of pYU8 with the pyr4-containing plasmid pRG1. Among the obtained transformants 40% of the clones displayed the fluffy phenotype. The same percentage of fluffy colonies was obtained after cotransformation of pYU8 into the wild-type strain RMS011. This suggests that FadA signaling is independent of RosA.

View larger version (99K): In this window In a new window Download PPT slide   FIGURE 5.— Overexpression of the rosA gene. Comparison of a wild type (RMS011) and SKV6 (alcA(p)::rosA) grown on glucose (left) and ethanol medium (right). Plates were incubated for 4 days at 37°. (Bottom, left and right) Scanning electron microscopic pictures of a wild type (left) and the overexpression strain (right). Bar represents 1.5 cm (colonies) and 100 µm (bottom, left and right).

 

Transcription of rosA is upregulated during asexual development and upon carbon starvation:

The regulation of rosA expression was studied by Northern blot and RT-PCR analyses of mycelium harvested at different developmental stages. To synchronize development, hyphae were grown in liquid culture for 20 hr and then filtered through a miracloth membrane and transferred to the surface of an agar plate. Under those conditions, asexual development is initiated immediately after transfer and stalks of conidiophores are visible after 6 hr; metulae, phialides, and some spores after 12 hr; and after 20 hr asexual development is completed. Sexual development is then initiated after 48 hr. The rosA transcript was detectable only in the hyphal stage using nested PCR with cDNA as template, which was generated in a first reaction by RT-PCR (data not shown). The rosA transcript became detectable by Northern blot analysis only in early developmental stages and peaked in the 12-hr sample. These results suggest that the expression is tightly developmentally regulated (Figure 6). Since the carbon supply in developing cultures may be limiting, we tested whether rosA expression is affected by the nutritional status of the mycelium. We grew A. nidulans for 20 hr in liquid culture, filtered the mycelium, washed it with medium without glucose, and incubated the material for 3 hr in liquid medium lacking any carbon source. The rosA gene was transiently induced after 3 hr of carbon starvation (Figure 6). As a control for the rosA expression pattern, we probed the same Northern blot with the rodA gene. This gene encodes a hydrophobin, which forms the rodlet layers of the conidiospores and is thus expressed late during asexual development (STRINGER et al. 1991). Surprisingly, the rodA transcript was also induced upon prolonged starvation in liquid culture. The rosA transcript was not detectable during sexual development either in Northern blot analysis or by nested RT-PCR (data not shown).

View larger version (75K): In this window In a new window Download PPT slide   FIGURE 6.— Transient expression of rosA during development and upon starvation. A. nidulans was induced for differentiation and mycelium harvested at the time points indicated. In addition, mycelia grown in liquid culture were harvested after 20 hr, washed, and transferred in medium lacking any carbon source. After 3 hr and after 22 hr of incubation at 37° mycelium was harvested and processed for transcript analysis. RNA was isolated from all samples and 15 µg processed for Northern blots using a 1-kb 32P-labeled rosA-specific probe of the open reading frame generated by PCR. As a control we have hybridized the RNA of the asexual stage and after starvation with the rodA-specific probe generated by PCR. As a further loading control we stained the ribosomal RNA after transfer to the membrane with methylene blue.

 

RosA localization:

The putative transcriptional regulator RosA contains a potential nuclear localization signal (NLS) overlapping with the Zn binuclear cluster DNA-binding domain at the N terminus of the protein (Figure 1). To check whether the NLS is functional, we fused RosA with GFP. Expression under the endogenous rosA promoter did not give any GFP signal, which is in agreement with the low expression level observed in the Northern blot analysis. Therefore, we placed rosA under the control of the inducible A. nidulans alcA promoter and fused GFP to the 3'-end. Although transformants were grown in the presence of ethanol to induce the alcA promoter, we could not detect any GFP signal. When we changed the GFP molecule from the C to the N terminus of the protein, we obtained a GFP signal (Figure 7). Interestingly, the full-length RosA protein did not accumulate efficiently in nuclei, but nuclei were not excluded. This was observed after induction of the construct with ethanol and also after low expression in the presence of glycerol. Thus it seems that only some of the GFP-RosA proteins were able to enter the nucleus. When we successively truncated the C terminus of the protein, we achieved efficient nuclear translocation of versions shorter than 293 amino acids, suggesting that the region from amino acid 294 to 449 negatively affects nuclear import. Nuclear residence was proven through colocalization with a nuclear-targeted dsRedT4 protein (Figure 7). The RosA localization pattern was identical in a veA1 mutant and in a veA wild type. To test whether the RosA^294-449 was sufficient to reduce the efficiency of nuclear import of another nuclear protein, we fused this polypeptide to the C terminus of the transcriptional regulator StuA. We are using a GFP fusion of this C-terminal StuA fragment routinely for nuclear labeling (SUELMANN et al. 1997). Interestingly, the GFP-StuA protein was not efficiently translocated into the nuclei when the RosA fragment was added (Figure 7).

View larger version (22K): In this window In a new window Download PPT slide   FIGURE 7.— Subcellular localization of RosA as GFP-fusion protein. (A) Expression of full-length RosA and a truncated version as indicated with the amino acid numbers in parentheses. Nuclei were visualized with nuclear-targeted dsRedT4. (B, left) Scheme of different RosA versions fused to GFP at the N terminus. (B, right) Localization of the corresponding constructs shown on the left. The sizes of the proteins are indicated in the pictures. All RosA fusion proteins were expressed under the control of the alcA promoter and grown for 10 hr at 37° in medium with 2% ethanol as carbon source. (C) GFP-StuA(NLS) was fused to RosA294-449 and expressed under the control of the alcA promoter (pKV72) in strain SRF200, yielding SKV48. As a control GFP-StuA(NLS) (pKV71) used (SKV47). Germlings were grown on ethanol medium as in B.

 

Six-cysteine binuclear cluster DNA-binding domains are a characteristic class of fungal transcription factors (TODD and ANDRIANOPOULOS 1997). In the complete S. cerevisiae genome 56 and in N. crassa at least 27 (http://www.broad.mit.edu/annotation/fungi/neurospora/gene_by_hmmer.html) different putative Zn(II)₂Cys₆ proteins have been identified (SCHJERLING and HOLMBERG 1996; MASLOFF et al. 2002). In comparison, 123 potential Zn(II)₂Cys₆ binuclear cluster proteins have been annotated in the A. nidulans genome database (http://www.broad.mit.edu/annotation/fungi/aspergillus/genes_by_hmmer.html). The DNA-binding motif was first described in the S. cerevisiae Gal4 regulator and has been found since in >80 characterized proteins, mainly from ascomycetes (PAN and COLEMAN 1990). Many of these transcription factors have been described as regulators of different metabolic pathways, ranging from carbon metabolism to the synthesis of secondary metabolites (TODD and ANDRIANOPOULOS 1997). In addition, some Zn(II)₂Cys₆ proteins have been shown to regulate developmental processes in fungi. Thus, in S. cerevisiae one factor, Ume6, is involved in meiosis and has activating and repressing properties (RUBIN-BEJERANO et al. 1996). Two transcription factors from this family that regulate fungal morphogenesis have been described in N. crassa (fl) and in S. macrospora (pro1). When the fl gene in N. crassa was mutated, the strain failed to form asexual spores, while mutation of pro1 in S. macrospora blocked the formation of mature perithecia (BAILEY and EBBOLE 1998; MASLOFF et al. 1999). In both cases the loss-of-function phenotype suggested a gene-activating function. This was confirmed in the case of Pro1 by a functional analysis in S. cerevisiae, where a transactivation domain was found in the N-terminal half of the protein (MASLOFF et al. 2002).

Here, we describe RosA, a protein of A. nidulans with sequence similarity to the Pro1 transcription factor from S. macrospora, but serving a distinctly different role from that of Pro1. Whereas a loss-of-function mutation in S. macrospora leads to a block in late sexual development, with the formation of aberrant perithecia, we present evidence that rosA acts as a repressor of sexual development under low-carbon conditions and in liquid medium in A. nidulans. These observed different functions in Sordaria and Aspergillus contrast with the degree of sequence similarity and conservation of one of the two introns, which argues for a common ancestor of these genes. In agreement with the hypothesis of different functions for Pro1 and RosA is a series of experiments in which we tested whether pro1 and rosA could substitute each other. Overexpression of Sordaria pro1 in A. nidulans did not produce the expected fluffy phenotype, and, similarly, transformation of rosA into the S. macrospora pro1 mutant did not rescue the mutant phenotype (S. PöGGELER, personal communication; data not shown).

The rosA mutant, in contrast to the wild-type strain, produced mature cleistothecia under low-glucose (10 mM) and high-salt (0.6 M KCl) conditions and initiated the sexual cycle—reflected by the production of Hülle cells—in submersed culture. Whereas the induction of sexual development in rosA on agar plates was dependent on veA, the production of Hülle cells in liquid medium was veA independent. These phenotypic differences suggest that RosA represses sexual development under certain environmental conditions. In agreement with the role of RosA as a repressor of sexual development is the finding that three transcription factors with known roles in sexual development—nsdD, veA, and stuA—were transcriptionally induced in liquid culture in the rosA strain (Figure 4). This result, together with the strong induction of rosA 12 hr after the initiation of asexual development, correlates nicely with the findings from HAN et al. (2001), who observed downregulation of nsdD 10 hr after the induction of asexual development. It remains to be studied whether nsdD, veA, and stuA are direct targets of RosA.

Interestingly, we observed that rosA also undergoes a transient upregulation 3 hr after removal of glucose. Since sexual development occurs after the depletion of the carbon source and therefore depends on reserve materials (WEI et al. 2001), it could be that RosA inhibits the onset of sexual development after short-term starvation periods. This starvation-mediated induction of rosA could explain the upregulation 12 hr after the onset of asexual development, since conidiophores grow into the air and may be carbon limited. However, we still do not know whether the increase of the rosA transcript is restricted to some cell types such as the conidiophores or the substrate mycelium.

The sexual-development-repressing properties of RosA resemble those of the LsdA protein recently characterized (LEE et al. 2001). Deletion of lsdA led to the induction of sexual development under high-osmotic conditions, where sexual development of wild type is blocked. This suggests that LsdA mediates the repression of the sexual cycle under these environmental conditions.

If RosA represses sexual development, the question is, How can we explain the drastic hyphal growth reduction and fluffy phenotype after overexpression? A possible answer is that this phenotype is caused by misregulation of different morphogenetic and metabolic pathways. On the other hand, it could be that growth and development reduction is a normal feature of RosA when induced by carbon depletion. Thus, a transient upregulation of rosA would prevent the immediate induction of the sexual cycle when inappropriate. During this short time, the metabolism could adapt to the starving situation, and the cell would start inducing, e.g., enzymes for the mobilization of reserve material. The drastic overexpression phenotype does not allow any speculation about a role of RosA during late sexual development.

In addition to the transcriptional regulation of rosA, it appears that the protein activity also could be regulated post-transcriptionally. We found that RosA localized mainly in the cytoplasm and only some fraction of the protein accumulated in nuclei. In contrast, truncated versions of the protein with <294 aa were transported efficiently into the nuclei. This demonstrates that the predicted NLS is functional and suggests that nuclear import could be post-transcriptionally regulated, for instance, by changes in protein conformation to expose the NLS.

In summary, we show in this work that the induction of the sexual cycle in A. nidulans requires the integration of several environmental signals that might be transduced through different cascades and specific transcription factors. RosA could be involved in the transmission of signals, which integrate sexual development with growth under submersed, high-salt, or carbon-limited conditions. To further unravel the functions and the interplays of the transcription factors, it will be the challenge of future research to identify target genes for VeA, NsdD, and RosA.

We thank A. Hassel and G. Kost (Philipps-University of Marburg) for assistance in taking SEM pictures and U. Ugalde (University of San Sebastian, San Sebastian, Spain) and N. Requena (University of Tübingen, Tübingen, Germany) for critically reviewing the manuscript. We are also grateful to S. Pöggeler (University of Bochum, Bochum, Germany) for the S. macrospora complementation experiment and J. Yu (University of Wisconsin, Madison, WI) for various fluffy strains and genes. We wish to thank K. S. Chae, Chonbuk National University, Chonju, Chonbuk, Korea, for sending us several A. nidulans strains. This work was supported by the German Science Foundation Priority Programme (DFG) SFB 395, the Fonds der Chemischen Industrie, and the Max-Planck-Institute for Terrestrial Microbiology, Marburg, Germany.

Sequence data from this article have been deposited with the EMBL/GenBank Data Libraries under accession no. CAD58393.

ADAMS, T. H., J. K. WIESER and J.-H. YU, 1998 Asexual sporulation in Aspergillus nidulans. Microbiol. Mol. Biol. Rev. 62: 35–54.[Abstract/Free Full Text]

ARAMAYO, R., T. H. ADAMS and W. E. TIMBERLAKE, 1989 A large cluster of highly expressed genes is dispensable for growth and development in Aspergillus nidulans. Genetics 122: 65–71.[Abstract/Free Full Text]

BAILEY, L. A., and D. J. EBBOLE, 1998 The fluffy gene of Neurospora crassa encodes a Gal4p-type C₆ zinc cluster protein required for conidial development. Genetics 148: 1813–1820.[Abstract/Free Full Text]

BRAUS, G. H., S. KRAPPMANN and S. E. ECKERT, 2002 Sexual development in ascomycetes—fruit body formation of Aspergillus nidulans, pp. 215–244 in Molecular Biology of Fungal Development, edited by H. D. OSIEWACZ. Marcel Decker, New York.

BUSBY, T. M., K. Y. MILLER and B. L. MILLER, 1996 Suppression and enhancement of the Aspergillus nidulans medusa mutation by altered dosage of the bristle and stunted genes. Genetics 143: 155–163.[Abstract]

BUSCH, S., S. E. ECKERT, S. KRAPPMANN and G. H. BRAUS, 2003 The COP9 signalosome is an essential regulator of development in the filamentous fungus Aspergillus nidulans. Mol. Microbiol. 49: 717–730.[CrossRef][Medline]

CALVO, A. M., R. A. WILSON, J. W. BOK and N. P. KELLER, 2002 Relationship between secondary metabolism and fungal development. Microbiol. Mol. Biol. Rev. 66: 447–459.[Abstract/Free Full Text]

CHAE, K.-S., J. H. KIM, Y. CHOI, D. M. HAN and K.-Y. JAHNG, 1995 Isolation and characterization of a genomic DNA fragment complementing an nsdD mutation of Aspergillus nidulans. Mol. Cell 5: 146–150.

CHAMPE, S. P., and L. D. SIMON, 1992 Cellular differentiation and tissue formation, pp. 63–91 in Morphogenesis: An Analysis of the Development of Biological Form, edited by E. F. ROSSOMANDO and S. ALEXANDER. Marcel Dekker, New York.

DUTTON, J. R., S. JOHNS and B. L. MILLER, 1997 StuAp is a sequence-specific transcription factor that regulates developmental complexity in Aspergillus nidulans. EMBO J. 16: 5710–5721.[CrossRef][Medline]

DYER, P. S., M. PAOLETTI and D. B. ARCHER, 2003 Genomics reveals sexual secrets of Aspergillus. Microbiology 149: 2301–2303.[Free Full Text]

ECKERT, S. E., B. HOFFMANN, C. WANKE and G. H. BRAUS, 1999 Sexual development of Aspergillus nidulans in tryptophan auxotrophic strains. Arch. Microbiol. 172: 157–166.[CrossRef][Medline]

FISCHER, R., 2002 Conidiation in Aspergillus nidulans, pp. 59–86 in Molecular Biology of Fungal Development, edited by H. D. OSIEWACZ. Marcel Decker, New York.

GURR, S. J., S. E. UNKLES and J. R. KINGHORN, 1987 The structure and organization of nuclear genes of filamentous fungi, pp. 93–138 in Gene Structure in Eukaryotic Microbes, edited by J. R. KINGHORN. IRL Press, Oxford/Washington, DC.

HAN, K. H., and R. A. PRADE, 2002 Osmotic stress-coupled maintenance of polar growth in Aspergillus nidulans. Mol. Microbiol. 43: 1065–1078.[CrossRef][Medline]

HAN, K. H., K. Y. HAN, J. H. YU, K. S. CHAE, K. Y. JAHNG et al., 2001 The nsdD gene encodes a putative GATA-type transcription factor necessary for sexual development of Aspergillus nidulans. Mol. Microbiol. 41: 299–309.[CrossRef][Medline]

HAN, K.-H., J.-A. SEO and J.-H. YU, 2004 A putative G protein-coupled receptor negatively controls sexual development in Aspergillus nidulans. Mol. Microbiol. 51: 1333–1345.[CrossRef][Medline]

HILL, T. W., and E. KäFER, 2001 Improved protocols for Aspergillus minimal medium: trace element and minimal medium salt stock solutions. Fungal Genet. Newsl. 48: 20–21.

KäFER, E., 1965 Origins of translocations in Aspergillus nidulans. Genetics 52: 217–232.[Free Full Text]

KAROS, M., and R. FISCHER, 1999 Molecular characterization of HymA, an evolutionarily highly conserved and highly expressed protein of Aspergillus nidulans. Mol. Genet. Genomics 260: 510–521.

KAWASAKI, L., O. SANCHEZ, K. SHIOZAKI and J. AGUIRRE, 2002 SakA MAP kinase is involved in stress signal transduction, sexual development and spore viability in Aspergillus nidulans. Mol. Microbiol. 45: 1153–1163.[CrossRef][Medline]

KIM, H.-S., K.-Y. HAN, K.-J. KIM, D.-M. HAN, K.-Y. JAHNG et al., 2002 The veA gene activates sexual development in Aspergillus nidulans. Fungal Genet. Biol. 37: 72–80.[CrossRef][Medline]

LARA-ORTIZ, T., H. RIVEROS-ROSAS and J. AGUIRRE, 2003 Reactive oxygen species generated by microbial NADPH oxidase NoxA regulate sexual development in Aspergillus nidulans. Mol. Microbiol. 50: 1241–1255.[CrossRef][Medline]

LEE, D. W., S. KIM, S.-J. KIM, D. M. HAN, K.-Y. JAHNG et al., 2001 The lsdA gene is necessary for sexual development inhibition by salt in Aspergillus nidulans. Curr. Genet. 39: 237–243.[CrossRef][Medline]

MASLOFF, S., S. PöGGELER and U. KüCK, 1999 The pro1(+) gene from Sordaria macrospora encodes a C₆ zinc finger transcription factor required for fruiting body development. Genetics 152: 191–199.[Abstract/Free Full Text]

MASLOFF, S., S. JACOBSEN, S. PöGGELER and U. KüCK, 2002 Functional analysis of the C₆ zinc finger gene pro1 involved in fungal sexual development. Fungal Genet. Biol. 36: 107–116.[CrossRef][Medline]

MILLER, K. Y., J. WU and B. L. MILLER, 1992 StuA is required for cell pattern formation in Aspergillus. Genes Dev. 6: 1770–1782.[Abstract/Free Full Text]

MOONEY, J. L., and L. N. YAGER, 1990 Light is required for conidiation in Aspergillus nidulans. Genes Dev. 4: 1473–1482.[Abstract/Free Full Text]

PAN, T., and J. E. COLEMAN, 1990 GAL4 transcription factor is not a "zinc finger" but forms a Zn(II)₂Cys₆ binuclear cluster. Proc. Natl. Acad. Sci. USA 87: 2077–2081.[Abstract/Free Full Text]

PASCON, R. C., and B. L. MILLER, 2000 Morphogenesis in Aspergillus nidulans requires Dopey (DopA), a member of a novel family of leucine zipper-like proteins conserved from yeast to humans. Mol. Microbiol. 36: 1250–1264.[CrossRef][Medline]

RUBIN-BEJERANO, I., S. MANDEL, K. ROBZYK and Y. KASSIR, 1996 Induction of meiosis in Saccharomyces cerevisiae depends on conversion of the transcriptional repressor Ume6 to a positive regulator by its regulated association with the transcriptional activator Ime1. Mol. Cell. Biol. 16: 2518–2526.[Abstract]

SAMBROOK, J., and D. W. RUSSEL, 1999 Molecular Cloning: A Laboratory Manual. Cold Spring Harbor Laboratory Press, Cold Spring Harbor, NY.

SCHERER, M., H. WEI, R. LIESE and R. FISCHER, 2002 Aspergillus nidulans catalase-peoxidase gene (CpeA) is transcriptionally induced during sexual development through the APSES-transcription factor StuA. Eukaryot. Cell 1: 725–735.[Abstract/Free Full Text]

SCHJERLING, P., and S. HOLMBERG, 1996 Comparative amino acid sequence analysis of the C₆ zinc cluster family of transcriptional regulators. Nucleic Acids Res. 24: 4599–4607.[Abstract/Free Full Text]

SOHN, K. T., and K. S. YOON, 2002 Ultrastructural study on the cleistothecium development in Aspergillus nidulans. Mycobiology 30: 117–127.

STRINGER, M. A., R. A. DEAN, T. C. SEWALL and W. E. TIMBERLAKE, 1991 Rodletless, a new Aspergillus developmental mutant induced by directed gene inactivation. Genes Dev. 5: 1161–1171.[Abstract/Free Full Text]

SUELMANN, R., N. SIEVERS and R. FISCHER, 1997 Nuclear traffic in fungal hyphae: in vivo study of nuclear migration and positioning in Aspergillus nidulans. Mol. Microbiol. 25: 757–769.[CrossRef][Medline]

TODD, R. B., and A. ANDRIANOPOULOS, 1997 Evolution of a fungal regulatory gene family: the Zn(II)2Cys6 binuclear cluster DNA binding motif. Fungal Genet. Biol. 21: 388–405.[CrossRef][Medline]

TOEWS, M. A., J. WARMBOLD, S. KONZACK, P. E. RISCHITOR, D. VEITH et al., 2004 Establishment of mRFP1 as fluorescent marker in Aspergillus nidulans and construction of expression vectors for high-throughput protein tagging using recombination in Escherichia coli (GATEWAY). Curr. Genet. 45: 383–389.[CrossRef][Medline]

TSITSIGIANNIS, D. I., R. ZARNOWSKI and N. P. KELLER, 2004 The lipid body protein, PpoA, coordinates sexual and asexual sporulation in Aspergillus nidulans. J. Biol. Chem. 279: 11344–11353.[Abstract/Free Full Text]

VALLIM, M. A., K. Y. MILLER and B. L. MILLER, 2000 Aspergillus SteA (Sterile12-like) is a homeodomain-C₂/H₂-Zn⁺² finger transcription factor required for sexual reproduction. Mol. Microbiol. 36: 290–301.[CrossRef][Medline]

WARING, R. B., G. S. MAY and N. R. MORRIS, 1989 Characterization of an inducible expression system in Aspergillus nidulans using alcA and tubulin coding genes. Gene 79: 119–130.[CrossRef][Medline]

WEI, H., M. SCHERER, A. SINGH, R. LIESE and R. FISCHER, 2001 Aspergillus nidulans alpha-1,3 glucanase (mutanase), mutA, is expressed during sexual development and mobilises mutan. Fungal Genet. Biol. 34: 217–227.[CrossRef][Medline]

WEI, H., N. REQUENA and R. FISCHER, 2003 The MAPKK-kinase SteC regulates conidiophore morphology and is essential for heterokaryon formation and sexual development in the homothallic fungus Aspergillus nidulans. Mol. Microbiol. 47: 1577–1589.[CrossRef][Medline]

YELTON, M. M., J. E. HAMER and W. E. TIMBERLAKE, 1984 Transformation of Aspergillus nidulans by using a trpC plasmid. Proc. Natl. Acad. Sci. USA 81: 1470–1474.[Abstract/Free Full Text]

YU, J.-H., J. WIESER and T. H. ADAMS, 1996 The Aspergillus FlbA RGS domain protein antagonizes G-protein signaling to block proliferation and allow development. EMBO J. 15: 5184–5190.[Medline]

ZONNEVELD, B. J. M., 1977 Biochemistry and ultrastructure of sexual development in Aspergillus, pp. 58–80 in Genetics and Physiology of Aspergillus, edited by J. E. SMITH and J. A. PATEMAN. Academic Press, London.

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