Aspergillus fumigatus is a medically important opportunistic pathogen and a major cause of respiratory allergy. The species has long been considered an asexual organism. However, genome analysis has revealed the presence of genes associated with sexual reproduction, including a MAT-2 high-mobility group mating-type gene and genes for pheromone production and detection (Galagan et al., personal communication; Nierman et al., personal communication). We now demonstrate that A. fumigatus has other key characteristics of a sexual species. We reveal the existence of isolates containing a complementary MAT-1 alpha box mating-type gene and show that the MAT locus has an idiomorph structure characteristic of heterothallic (obligate sexual outbreeding) fungi. Analysis of 290 worldwide clinical and environmental isolates with a multiplex-PCR assay revealed the presence of MAT1-1 and MAT1-2 genotypes in similar proportions (43% and 57%, respectively). Further population genetic analyses provided evidence of recombination across a global sampling and within North American and European subpopulations. We also show that mating-type, pheromone-precursor, and pheromone-receptor genes are expressed during mycelial growth. These results indicate that A. fumigatus has a recent evolutionary history of sexual recombination and might have the potential for sexual reproduction. The possible presence of a sexual cycle is highly significant for the population biology and disease management of the species.

Fruit body formation in filamentous fungi is a complex and yet hardly understood process. We show here that protein turnover control is crucial for Aspergillus nidulans development. Deletion of genes encoding COP9 signalosome (CSN) subunits 1, 2, 4, or 5 resulted in identical blocks in fruit body formation. The CSN multiprotein complex controls ubiquitin-dependent protein degradation in eukaryotes. Six CSN subunits interacted in a yeast two-hybrid analysis, and the complete eight-subunit CSN was recruited by a functional tandem affinity purification tag fusion of subunit 5 (CsnE). The tagged CsnE was unable to recruit any CSN subunit in a strain deleted for subunit 1 or subunit 4. Mutations in the JAMM metalloprotease core of CsnE resulted in mutant phenotypes identical to those of csn deletion strains. We propose that a correctly assembled CSN including a functional JAMM links protein turnover to fungal sexual development.development ͉ filamentous fungi F ungal fruit bodies are sexual reproduction structures that generate meiotic spores. The model mold Aspergillus nidulans develops a closed spherical fruit body (cleistothecium) including different tissue types: Hülle cells surround and nurse the growing cleistothecium, pericarp cells develop the protecting wall, and inner ascogenous cells mature into sexual spores (1, 2). Massive reconstruction of vegetative hyphae is required to build the complex three-dimensional fruit body. The regulation of this development is hardly understood in any fungus (3). A genetic screen recently identified csnD and csnE resembling genes for subunits of the COP9 signalosome (CSN) of animals and plants to be essential for fruit body formation of A. nidulans (4).CSN is a multiprotein complex composed of proteins containing PCI and MPN interaction domains (5, 6). Csn5/Jab1 is the only subunit conserved in all eukaryotes, and it carries an MPNϩ domain containing the JAMM motif conferring metalloprotease (deneddylation) activity (6, 7). CSN controls by its MPNϩ domain the activity of cullin-RING E3 ligases by cleaving the ubiquitin-like protein Nedd8/Rub1 from the cullin (8, 9). Neddylated E3 ubiquitin ligases are key mediators of posttranslational labeling of proteins for the proteasome (10). The CSN thus controls eukaryotic ubiquitin-dependent protein degradation.The complete eight-subunit CSN, composed of six PCI and two MPN domain proteins, was described for eukaryotes as humans (11), mice (12), plants (13), flies (14), and Dictyostelium (15). In fungi, definitive evidence for an eight-subunit CSN is lacking so far. CSN complex purification from Neurospora crassa revealed subunits 1-7, but subunit 8 was identified neither in the purification experiment nor in the genome sequence by bioinformatics means (16). In fission yeast subunits 6 and 8 have not been identified yet (17), and in the CSN-related complex of Saccharomyces cerevisiae only subunit Csn5 (yeast Rri1p) is well conserved (18).The fungal CSN complexes known to date are not essential for viability but are involved in cellu...

SummaryThe COP9 signalosome complex (CSN) is a crucial regulator of ubiquitin ligases. Defects in CSN result in embryonic impairment and death in higher eukaryotes, whereas the filamentous fungus Aspergillus nidulans survives without CSN, but is unable to complete sexual development. We investigated overall impact of CSN activity on A. nidulans cells by combined transcriptome, proteome and metabolome analysis. Absence of csn5/csnE affects transcription of at least 15% of genes during development, including numerous oxidoreductases. csnE deletion leads to changes in the fungal proteome indicating impaired redox regulation and hypersensitivity to oxidative stress. CSN promotes the formation of asexual spores by regulating developmental hormones produced by PpoA and PpoC dioxygenases. We identify more than 100 metabolites, including orsellinic acid derivatives, accumulating preferentially in the csnE mutant. We also show that CSN is required to activate glucanases and other cell wall recycling enzymes during development. These findings suggest a dual role for CSN during development: it is required early for protection against oxidative stress and hormone regulation and is later essential for control of the secondary metabolism and cell wall rearrangement.

Cand1 can be separated into two functional polypeptides. C-terminal Cand1 binds first at the cullin adaptor site. N-terminal Cand1 blocks the neddylation site subsequently. Defects in the split fungal Cand1 impair development more than defects in CSN.

The signalosome (CSN) is a conserved multiprotein complex involved in regulation of eukaryotic development and is also required to activate ribonucleotide reductase for DNA synthesis. In Aspergillus nidulans, csnD/csnE are key regulators of sexual development. Here, we investigated whether the csnD/csnE genes are involved in the DNA damage response in this fungus. The growth of the csnD/csnE deletion mutants was reduced by subinhibitory concentrations of hydroxyurea, camptothecin, 4-nitroquinoline oxide, and methyl methanesulfonate. A. nidulans increases csnD/csnE mRNA levels when it is challenged by different DNAdamaging agents. There is no significant transcriptional induction of the csnE promoter fused with lacZ gene in the presence of DNA-damaging agents, suggesting that increased mRNA accumulation is due to increased mRNA stability. Septation was not inhibited in the csnD/csnE deletion mutants while DuvsB DcsnE presented an increase in septation upon DNA damage caused by methyl methanesulfonate, suggesting that uvsB ATR and csnE genetically interact during checkpoint-dependent inhibition of septum formation. The double DcsnD/ DcsnE DnpkA mutants were more sensitive to DNA-damaging agents than were the respective single mutants. Our results suggest that csnD/csnE genes are involved in the DNA damage response and that NpkA and UvsB ATR genetically interact with the signalosome.