RESUMO
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.
Assuntos
Perfilação da Expressão Gênica , Regulação Fúngica da Expressão Gênica , Genes Fúngicos Tipo Acasalamento , Schizophyllum/genética , Alelos , Northern Blotting , Ciclo Celular , Núcleo Celular/genética , Núcleo Celular/metabolismo , Parede Celular/metabolismo , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Carpóforos/metabolismo , Loci Gênicos , Proteínas de Fluorescência Verde/metabolismo , Membrana Nuclear/metabolismo , Receptores de Feromônios/genética , Receptores de Feromônios/metabolismo , Schizophyllum/crescimento & desenvolvimento , Schizophyllum/metabolismo , Transdução de Sinais , Fatores de Tempo , TranscriptomaRESUMO
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.
Assuntos
Sequência de Bases , Genoma Fúngico/genética , Modelos Biológicos , Schizophyllum/genética , Carpóforos/genética , Carpóforos/crescimento & desenvolvimento , Proteínas Fúngicas/genética , Proteínas Fúngicas/metabolismo , Perfilação da Expressão Gênica , Regulação da Expressão Gênica no Desenvolvimento , Regulação da Expressão Gênica de Plantas , Genes Fúngicos Tipo Acasalamento , Loci Gênicos/genética , Schizophyllum/crescimento & desenvolvimento , Fatores de Transcrição/genética , Fatores de Transcrição/metabolismo , Madeira/microbiologiaRESUMO
The cytochrome distribution in hyphal tip cells of Schizophyllum commune was visualized using resonance Raman mapping and CARS microscopy. For comparison, resonance Raman mapping and CARS imaging of cytochrome was also performed during branch formation and in completely developed central hyphae. Cytochrome, as an essential component of the electron transport chain in mitochondria, plays an important role in providing energy to actively growing mycelia. It could be shown that mitochondria at the growing hyphal tips and at branching regions are more active, i.e. contain more cytochrome, as compared to those in older parts of the hyphae. This finding is compatible with the idea of high energy consumption for biosynthesis and intracellular transport at the growing tip, while older hyphae have lower needs for ATP generated via the respiratory chain in mitochondria. To the best of our knowledge this is the first study reporting about the localization and distribution of cytochrome, as an indirect mitochondria localization within S. commune or other basidiomycetous fungi, by means of resonance Raman microspectroscopy and CARS microscopy. These Raman methods bear the potential of label-free in vivo mitochondria localization and investigation.