In this study, we undertook a functional characterization and transcriptome analysis that enabled a comprehensive study of the mating type loci of the mushroom Schizophyllum commune. Induced expression of both the bar2 receptor and the bap2(2) pheromone gene within 6 to 12 h after mates' contact was demonstrated by quantitative real-time PCR. Similar temporal expression patterns were confirmed for the allelic bbr1 receptor and bbp1 pheromone-encoding genes by Northern hybridization. Interestingly, the fusion of clamp connections to the subterminal cell was delayed in mating interactions in which one of the compatible partners expressed the bar2 receptor with a truncated C terminus. This developmental delay allowed the visualization of a green fluorescent protein (Gfp)-labeled truncated receptor at the cell periphery, consistent with a localization in the plasma membrane of unfused pseudoclamps. This finding does not support hypotheses envisioning a receptor localization to the nuclear membrane facilitating recognition between the two different nuclei present in each dikaryotic cell. Rather, Gfp fluorescence observed in such pseudoclamps indicated a role of receptor-pheromone interaction in clamp fusion. Transcriptome changes associated with mating interactions were analyzed in order to identify a role for pheromone-receptor interactions. We detected a total of 89 genes that were transcriptionally regulated in a mating type locus A-dependent manner, employing a cutoff of 5-fold changes in transcript abundance. Upregulation in cell cycle-related genes and downregulation of genes involved in metabolism were seen with this set of experiments. In contrast, mating type locus B-dependent transcriptome changes were observed in 208 genes, with a specific impact on genes related to cell wall and membrane metabolism, stress response, and the redox status of the cell.T he filamentous fungus Schizophyllum commune is a widely distributed mushroom that lives saprophytically and can cause white rot on wood. It has been extensively used to study mating interactions for almost 100 years (47). In addition to studies involving the mushroom Coprinopsis cinerea and the corn smut Ustilago maydis, the mating type genes have been extensively analyzed for this tetrapolar basidiomycete (4, 29). The recent publication of the S. commune genome sequence enables global expression analyses (41). During the life cycle of this fungus, compatible haploid monokaryons can mate to produce a fertile dikaryon with two nuclei per cell, one derived from each mating partner. Under appropriate environmental conditions, the dikaryon is able to form the sexual reproductive fruiting bodies (40). While the fusion of two monokaryotic hyphae (plasmogamy) is independent of mating types, specific steps of sexual development are regulated by the mating type genes encoded in the loci A and B (25, 47). For each of these loci, two linked, multiallelic subloci (termed ␣ and ␤) have been determined by recombination analyses (27,49,51,65). The A loci encode homeodomain ...

cFungi have been used as model systems to define general processes in eukaryotes, for example, the one gene-one enzyme hypothesis, as well as to study polar growth or pathogenesis. Here, we show a central role for the regulator protein Ras in a mushroomforming, filamentous basidiomycete linking growth, pheromone signaling, sexual development, and meiosis to different signal transduction pathways. ras1 and Ras-specific gap1 mutants were generated and used to modify the intracellular activation state of the Ras module. Transformants containing constitutive ras1 alleles (ras1 G12V and ras1 Q61L ), as well as their compatible mating interactions, did show strong phenotypes for growth (associated with Cdc42 signaling) and mating (associated with mitogenactivated protein kinase signaling). Normal fruiting bodies with abnormal spores exhibiting a reduced germination rate were produced by outcrossing of these mutant strains. Homozygous ⌬gap1 primordia, expected to experience increased Ras signaling, showed overlapping phenotypes with a block in basidium development and meiosis. Investigation of cyclic AMP (cAMP)-dependent protein kinase A indicated that constitutively active ras1, as well as ⌬gap1 mutant strains, exhibit a strong increase in Tpk activity. Ras1-dependent, cAMP-mediated signal transduction is, in addition to the known signaling pathways, involved in fruiting body formation in Schizophyllum commune. To integrate these analyses of Ras signaling, microarray studies were performed. Mutant strains containing constitutively active Ras1, deletion of RasGap1, or constitutively active Cdc42 were characterized and compared. At the transcriptome level, specific regulation highlighting the phenotypic differences of the mutants is clearly visible.

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The B mating-type locus of the tetrapolar basidiomycete Schizophyllum commune encodes pheromones and pheromone receptors in multiple allelic specificities. This work adds substantial new evidence into the organization of the B mating-type loci of distantly related S. commune strains showing a high level of synteny in gene order and neighboring genes. Four pheromone receptor-like genes were found in the genome of S. commune with brl1, brl2 and brl3 located at the B mating-type locus, whereas brl4 is located separately. Expression analysis of brl genes in different developmental stages indicates a function in filamentous growth and mating. Based on the extensive sequence analysis and functional characterization of brl-overexpression mutants, a function of Brl1 in mating is proposed, while Brl3, Brl4 and Brl2 (to a lower extent) have a role in vegetative growth, possible determination of growth direction. The brl3 and brl4 overexpression mutants had a dikaryon-like, irregular and feathery phenotype, and they avoided the formation of same-clone colonies on solid medium, which points towards enhanced detection of self-signals. These data are supported by localization of Brl fusion proteins in tips, at septa and in not-yet-fused clamps of a dikaryon, confirming their importance for growth and development in S. commune.

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