Regulation of mushroom formation in Schizophyllum commune

Mushrooms are fungal adaptations for the aerial dispersal of sexual spores. Their main economic value is their use as food. Despite their economical interest, relatively little is known about how mushrooms are formed. Schizophyllum commune is the only mushroom-forming fungus in which genes can be inactivated by homologous recombination and therefore it serves as a model system for mushroom development. The process of mushroom formation in S. commune starts with the aggregation of hyphae into stage I aggregates. These aggregates develop into stage II primordia and further differentiate into mature fruiting bodies. In order to improve the process of targeted deletion of genes by homologous recombination, a dedicated deletion vector (pDelCas) was developed that allows efficient screening of the transformants using two antibiotics. In addition, gene ku80 was inactivated, resulting in an increased relative incidence of homologous recombination. The genome of S. commune is 38.5 MBp in size and contains 13.210 predicted genes. A comparison with the genomes of other species showed that hydrophobins and several classes of transcription factors are over-represented in S. commune and other mushroom-forming species. Genome-wide expression analysis showed that hydrophobins, signal transduction genes and transciptional regulation genes are differentially expressed during mushroom development. Also, anti-sense gene expression was found to occur widely during development, especially in stage II aggregates. Of the 471 predicted transcription factor genes of S. commune, 311 were expressed during mushroom development. Deletion strains were produced for 10 of these transcription factor genes. In the light, wild type dikaryons form asymmetrical colonies that have produced mushrooms after 10 days of growth. Homozygous dikaryons in which bri1, hom2 or wc-2 were deleted, formed symmetrical colonies and no mushrooms developed. Homozygous dikaryons in which fst4 was deleted had an irregular shape similar to the wild type but they formed no stage I aggregates. Colonies of strains in which c2h2 was deleted were asymmetrical when grown in the light and they did form aggregates, but these did not develop into primordia. Strains in which fst3, hom1 or gat1 were inactivated formed more but smaller mushrooms than the wild type. Expression analyses on deletion strains of hom2, wc-2 and fst4 showed that hydrophobins and the regulators c2h2 and hom1 were downregulated. Furthermore, mushroom formation in the strain in which wc-2 was deleted could be induced by a heat shock by introducing a construct encompassing the wc-2 gene under control of the inducible hsp3 promoter. A strain in which the hydrophobin genes sc3 and sc4 are deleted does not form mushrooms. Genes encoding structural mushroom proteins are lowly expressed in this strain. In contrast, genes encoding regulators involved in mushroom development are normally expressed, suggesting that a signal from aerial hyphae is required to activate these regulators. The findings presented in the Thesis may be instrumental to explain why certain mushrooms can not be cultivated commercially and also represents an excellent lead to improve production of commercial mushrooms