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The Cys2His2 zinc finger protein gene c2h2 of Schizophyllum commune is involved in mushroom formation. Its inactivation results in a strain that is arrested at the stage of aggregate formation. In this study, the c2h2 orthologue of Agaricus bisporus was over-expressed in this white button mushroom forming basidiomycete using Agrobacterium-mediated transformation. Morphology, cap expansion rate, and total number and biomass of mushrooms were not affected by over-expression of c2h2. However, yield per day of the c2h2 over-expression strains peaked 1 day earlier. These data and expression analysis indicate that C2H2 impacts timing of mushroom formation at an early stage of development, making its encoding gene a target for breeding of commercial mushroom strains. Electronic supplementary material The online version of this article (doi:10.1007/s00253-016-7574-9) contains supplementary material, which is available to authorized users.

Recent genome-wide studies have demonstrated that fungi possess the machinery to alternatively splice pre-mRNA. However, there has not been a systematic categorization of the functional impact of alternative splicing in a fungus. We investigate alternative splicing and its functional consequences in the model mushroom forming fungus Schizophyllum commune. Alternative splicing was demonstrated for 2,285 out of 12,988 expressed genes, resulting in 20% additional transcripts. Intron retentions were the most common alternative splicing events, accounting for 33% of all splicing events, and 43% of the events in coding regions. On the other hand, exon skipping events were rare in coding regions (1%) but enriched in UTRs where they accounted for 57% of the events. Specific functional groups, including transcription factors, contained alternatively spliced genes. Alternatively spliced transcripts were regulated differently throughout development in 19% of the 2,285 alternatively spliced genes. Notably, 69% of alternatively spliced genes have predicted alternative functionality by loss or gain of functional domains, or by acquiring alternative subcellular locations. S. commune exhibits more alternative splicing than any other studied fungus. Taken together, alternative splicing increases the complexity of the S. commune proteome considerably and provides it with a rich repertoire of alternative functionality that is exploited dynamically.

Mushrooms are of great value as a food source. The Netherlands has a large share of the European white button mushroom (Agaricus bisporus) production. This market will increase in the future due to larger demand for high quality food with a growing world population. Environmental factors play a role in the initiation of mushroom formation. Mushroom formation by A. bisporus is repressed by 1-octen-3-ol and high CO2, but stimulated by low temperature. Fruiting is responsive to blue light in Schizophyllum commune and repressed by high CO2 levels. Signaling of environmental conditions leads to transcription factor activity. A set of S. commune transcription factors and a blue light sensor involved in fruiting body development have been identified. This research showed that transcription factors BriI and Hom1 stimulate vegetative growth, while biomass formation is repressed by Wc-2, Hom2, and Fst4. The transcription factor tea1 was also down-regulated in the Δwc-2Δwc-2, Δhom2Δhom2 and Δfst4Δfst4 strains. The Δtea1Δtea1 strain produced more biomass than the wild-type and was severely affected in mushroom formation. Together, these data show that transcription factors Wc-2, Hom2, Fst4, and Tea1 link mushroom initiation and repression of vegetative growth. CO2 has profound effects on functioning of organisms. Therefore, it is sensed throughout the tree of life, in most cases via the cAMP-Pka signaling pathway. Here, a clear link was shown between high CO2 and cAMP levels and repression of fruiting in S. commune. Addition of cAMP in the medium mimicked high CO2 conditions by repressing fruiting body development. Overexpression of pde2, a gene encoding phosphodiesterase that degrades cAMP, resulted in fruiting body development even at elevated CO2 levels. Furthermore, it was investigated whether the homeodomain protein Hom2 is a target for Pka. The Δhom2Δhom2 dikaryon formed more biomass than the wild-type at ambient CO2 levels, while biomass of both strains was similar at 5% CO2. Hom2 contains 4 predicted Pka RRXS phosphorylation motifs. The serine codons of these motifs were replaced by alanine. This resulted in transformant strains that showed growth inhibition and prompt fructification at low CO2. Together, it is proposed that Pka phosphorylates Hom2, thereby maintaining the mycelium in the vegetative phase. Dephosphorylation of Hom2 at low CO2 switches the dikaryon from the vegetative into the generative phase. All hom2 orthologues of Agaricomycetes contain 2-5 RRXS motifs indicating that the role of Hom2 in fruiting is conserved. The S. commune transcription factor c2h2 is involved in mushroom formation. Its inactivation results in arrest at aggregate formation. In this study, the A. bisporus c2h2 orthologue was overexpressed in this basidiomycete. Morphology, cap expansion rate, total numbers and biomass of mushrooms harvested during culturing were not affected by over-expression of c2h2. However, more class II mushrooms were formed compared to the wild-type and the yield-per-day peaked one day earlier. These data and expression analysis indicate that C2H2 functions early in fruiting body development, while it also seems to have a role in selective tissues of young mushrooms. Data indicate that c2h2 is a target for breeding of commercial mushroom strains.

Establishment of the dikaryotic mycelium and formation of fruiting bodies are highly complex developmental programmes that are activated by a combination of environmental cues. A wide variety of proteins are expected to regulate and coordinate these programmes or to fulfil enzymatic conversions or structural roles. With the identification of the first genes involved in mushroom development, we are only at the beginning of understanding fruiting body formation. The process of identification of genes will be accelerated by whole genome expression studies and increased availability of molecular tools to assign functions to genes. Establishment of the dikaryon and emergence of fruiting bodies in basidiomycetes are regulated by the mating-type genes. These genes encode DNA-binding proteins and pheromones and their receptors. Regulation of fruiting by the mating-type genes is mediated by downstream transcription factors. Several genes encoding such regulatory proteins have now been identified. Regulatory circuits ultimately activate genes encoding structural proteins or enzymes that are involved in fruiting body formation. The role of hydrophobins is well established. They enable hyphae to escape the aqueous environment to allow fruiting body development. Moreover, they coat aerial structures and line air channels in mushrooms. The hydrophobic coating irreversibly directs growth of hyphae into the air, allows dispersal of spores and ensures gas exchange in fruiting bodies under humid conditions. Apart from hydrophobins, phenolics polymerised by the action of laccases may contribute to surface hydrophobicity of fruiting bodies. These enzymes have also been proposed to cross-link cell walls of hyphae in the fruiting bodies but this still has to be established. Experimental evidence indicates that cytochrome P450 enzymes, lectins, haemolysins and expansins also function in mushroom development. Lectins may be involved in aggregation of hyphae, haemolysins in signalling particularly to induce apoptosis of selected hyphae in the fruiting body, while expansins may be involved in cell wall modification and extension.