Much remains to be learned about the biology of mushroom-forming fungi, which are an important source of food, secondary metabolites and industrial enzymes. The wood-degrading fungus Schizophyllum commune is both a genetically tractable model for studying mushroom development and a likely source of enzymes capable of efficient degradation of lignocellulosic biomass. Comparative analyses of its 38.5-megabase genome, which encodes 13,210 predicted genes, reveal the species's unique wood-degrading machinery. One-third of the 471 genes predicted to encode transcription factors are differentially expressed during sexual development of S. commune. Whereas inactivation of one of these, fst4, prevented mushroom formation, inactivation of another, fst3, resulted in more, albeit smaller, mushrooms than in the wild-type fungus. Antisense transcripts may also have a role in the formation of fruiting bodies. Better insight into the mechanisms underlying mushroom formation should affect commercial production of mushrooms and their industrial use for producing enzymes and pharmaceuticals.

Fungi are well known to the casual observer for producing water-repelling aerial moulds and elaborate fruiting bodies such as mushrooms and polypores. Filamentous fungi colonize moist substrates (such as wood) and have to breach the water-air interface to grow into the air. Animals and plants breach this interface by mechanical force. Here, we show that a filamentous fungus such as Schizophyllum commune first has to reduce the water surface tension before its hyphae can escape the aqueous phase to form aerial structures such as aerial hyphae or fruiting bodies. The large drop in surface tension (from 72 to 24 mJ m -2 ) results from self-assembly of a secreted hydrophobin (SC3) into a stable amphipathic protein film at the water-air interface. Other, but not all, surface-active molecules (that is, other class I hydrophobins and streptofactin from Streptomyces tendae) can substitute for SC3 in the medium. This demonstrates that hydrophobins not only have a function at the hyphal surface but also at the medium-air interface, which explains why fungi secrete large amounts of hydrophobin into their aqueous surroundings.

SummaryMushrooms represent the most conspicuous structures of fungi. Their development is being studied in the model basidiomycete Schizophyllum commune. The genome of S. commune contains 472 genes encoding predicted transcription factors. Of these, fst3 and fst4 were shown to inhibit and induce mushroom development respectively. Here, we inactivated five additional transcription factor genes. This resulted in absence of mushroom development (in the case of deletion of bri1 and hom2), in arrested development at the stage of aggregate formation (in the case of c2h2) and in the formation of more but smaller mushrooms (in the case of hom1 and gat1). Moreover, strains in which hom2 and bri1 were inactivated formed symmetrical colonies instead of irregular colonies like the wild type. A genome-wide expression analysis identified several gene classes that were differentially expressed in the strains in which either hom2 or fst4 was inactivated. Among the genes that were downregulated in these strains were c2h2 and hom1. Based on these results, a regulatory model of mushroom development in S. commune is proposed. This model most likely also applies to other mushroom-forming fungi and will serve as a basis to understand mushroom formation in nature and to enable and improve commercial mushroom production.

The SDS-insoluble protein fraction of Agaricus bisporus fruiting bodies was solubilized with trifluoroacetic acid. On SDS-PAGE this fraction was found to contain one abundant protein with an apparent M, of 16 kDa. The N-terminal amino acid sequence of this protein was determined and RT-PCR used to isolate a cDNA clone which upon sequencing identified the protein as a typical class I hydrophobin (ABHI). The gene (ABHI) was isolated and sequenced, and a second hydrophobin gene (ABHZ) was found about 2.5 kbp downstream of ABHI. Purified ABHI self-assembled at hydrophobic-hydrophilic interfaces, producing the typical rodlet layer known from other hydrophobins. Similar rodlets were observed on the surface of the fruiting body, while immunological localization showed the hydrophobin to be particularly abundant at the outer surface of fruiting bodies, in the veil and in the core tissue of the stipe. Transcripts of ABHI were found only in fruiting-body hyphae. The ABHI hydrophobin is probably solely responsible for the hydrophobicity of the fruiting-body surface but may also line air channels within fruiting bodies.

Aerial mycelium and hyphal strands of Agaricus bisporus, strain U1, exhibited a rodlet pattern at their surfaces characteristic for assembled class I hydrophobins. An SDS-insolubleltrif Iuoroacetic-acid-soluble f r a d o n from strands was found to contain one abundant protein with an apparent molecular mass on gel of 19 kDa. Two sequences for this protein (ABH3), typical of class I hydrophobins, could be deduced by sequencing cDNA clones obtained by RT-PCR. The two forms of the protein could be assigned to different alleles present in the two homokaryons that constitute the heterokaryotic U1 strain. ABH3 displays all the in wit-properties of a typical class I hydrophobin such as SC3 from Schizophy//um commune but is not glycosylated or otherwise post-translationally modified because the molecular mass values deduced from the amino acid sequence (9228 and 9271 Da) and derived from mass spectrometry were in good agreement. The ABH3 transcript was found to be present in the vegetative mycelium of both primary and secondary mycelium but not in the fruiting bodies, whereas the reverse was found for the ABHl hydrophobin. Using an S. commune mutant with a disrupted SC3 gene it was found that ABH3 can substitute for SC3 in inducing formation of aerial hyphae, suggesting a role of ABH3 in the emergence of aerial hyphae and strands in A. bisporus.

Phleomycin is mutagenic by introducing double-strand breaks in DNA. The ble gene of Streptoalloteychus hindustanus, which confers resistance to this substance, is widely used as a selection marker for transformation. Schizophyllum commune grows on 25 g of phleomycin ml؊1 after introduction of a resistance cassette based on the ble gene. However, we here report that growth of resistant colonies on this concentration of phleomycin resulted in aberrant colony morphologies. Apparently, phleomycin was mutagenic despite acquired resistance. Therefore, a new selection system was developed based on resistance to the antibiotic nourseothricin. However, the transformation efficiency was tenfold lower than that obtained with phleomycin as a selection agent. This low transformation efficiency could be rescued by addition of a nonselective concentration of phleomycin during protoplast regeneration. This was accompanied by a higher incidence of single-copy integrations and with an increase of expression of key genes involved in double-strand break repair. Taken together, we conclude that the effect of a nonselective concentration of phleomycin strongly resembles the effect of restriction enzyme-mediated integration (REMI) but, unlike REMI, it does not depend on the presence of a target restriction site.Phleomycin and other bleomycins are widely used as selection agents for the transformation of algae (6, 9), protista (36), animals (4, 24), and fungi (2,3,15,17,35). They introduce double-strand breaks in the DNA when activated by metal ions (mainly iron) and oxygen (34). In addition, bleomycins damage RNA and attack cell walls (5). Resistance to phleomycin is conferred by the ble gene of Streptoalloteychus hindustanus. This gene encodes a 14-kDa protein that is capable of sequestering bleomycin-like molecules in a reversible way (12). The basidiomycete Schizophyllum commune can be efficiently transformed by using a phleomycin resistance cassette, in which the ble gene of S. hindustanus is placed under the control of the regulatory sequences of the S. commune glyceraldehyde-3-phosphate dehydrogenase gene (GPD) (30). However, we here show that phleomycin-resistant strains of S. commune are mutated upon exposure to phleomycin. Therefore, a cassette was constructed that confers resistance to a new selection marker, nourseothricin. Addition of a nonselective concentration of phleomycin during protoplast regeneration promoted singlecopy integration of the construct and resulted in an increased transformation frequency independent of the selection marker used. MATERIALS AND METHODSStrains and growth conditions. The co-isogenic S. commune strains 4-39 (CBS 341.81) and 4-40 (CBS 340.81) were used, as well as the uracil auxotroph 12-42 and the 4-39 derivative 4-39P. The latter strain has been transformed to phleomycin resistance using plasmid pHYM1.2. This plasmid contains the S. hindustanus ble gene under the control of the upstream and downstream regulatory sequences of the S. commune GPD gene (27). Strains were grown in minimal mediu...

The cDNA coding sequence of the Agaricus bisporus hydrophobin gene ABH1 under the regulation sequences of the Schizophyllum commune SC3 hydrophobin gene gave no expression in S. commune. In contrast, the genomic coding sequence (containing three introns) produced high levels of ABH1 mRNA when transformed to S. commune in the same configuration. Apparently, introns were needed for the accumulation of mRNAs from the ABH1 gene. When the effect of intron deletion on expression of the homologous genes SC3 and SC6 was examined, it was observed that only the genomic coding sequences were expressed in S. commune. Run-on analysis with nuclei harbouring intron-containing and intronless SC6 showed that this effect did not occur at the level of transcription initiation: genomic and cDNA sequences were equally active in this respect. When a 50 bp artificial intron containing the consensus splice and branch sites of S. commune introns, in addition to random-generated sequences, was introduced in the right orientation into the intronless SC3 transcriptional unit, accumulation of SC3 mRNA was restored. By polymerase chain reaction amplification, no unspliced SC3 mRNA species could be detected. Furthermore, the addition of an intron into the transcriptional unit of the gene for green fluorescent protein (GFP) effected clear fluorescence of the transgenic hyphae. Apparently, splicing is required for the normal processing of primary transcripts in S. commune.

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