ABSTRACT: In order to study changes in gene expression during mushroom development in Schizophyllum commune, genome wide gene expression was analysed in 4 developmental stages: vegetative mycelium, stage I aggregates, stage II primordia and mature mushrooms. In order to study the effects of absence of expression of sc3 and sc4, genome-wide expression was analysed in a dikaryon in which those genes have been inactivated by targeted gene deletion. 5 samples: - vegetative mycelium - stage I aggregates - stage II primordia - mature mushrooms - deltaSC3 deltaSC4 dikaryon RNA was obtained from 3 biological replicates and pooled
Project description:In order to study the effect of light on gene expression in Schizophyllum commune, genome wide gene expression was analysed in 4-day-old monokaryotic and dikaryotic wild type colonies, grown either in light or in darkness. 4 samples: - monokaryon, grown for 4 days in the light - monokaryon, grown for 4 days in the dark - dikaryon, grown for 4 days in the light - dikaryon, grown for 4 days in the dark RNA was obtained from 3 biological replicates and pooled
Project description:In order to study changes in gene expression during mushroom development in Schizophyllum commune, genome wide gene expression was analysed in 4 developmental stages: vegetative mycelium, stage I aggregates, stage II primordia and mature mushrooms 4 samples: - vegetative mycelium - stage I aggregates - stage II primordia - mature mushrooms RNA was obtained from 3 biological replicates and pooled
Project description:Large scale assessment of the transcriptomes of the vegetative mycelium and primordium will facilitate the generation of a more comprehensive picture of the fruiting process in basidiomycetes. We coupled 5'-Serial Analysis of Gene Expression (5'-SAGE) to high-throughput pyrosequencing from 454 Life Sciences to analyze the transcriptomes and identify up-regulated genes (URGs) among vegetative mycelium (Myc) and stage 1 primordium (S1-Pri) of Coprinopsis cinerea during fruiting body development. We evaluated the expression of >3,000 genes in the two respective growth stages and demonstrated that almost one-third of these genes were preferentially expressed in either stage, which implicated a significant transcriptomic switch during fruiting body initiation. We annotated >34,000 and >45,000 transcription start sites (TSSs) in the transcriptomes of Myc and S1-Pri respectively. We identified a wealth of potential URGs related to early fruiting events, although their functions and roles are not exactly known. This study serves to advance our understanding of the molecular mechanisms of fruiting body development in the model mushroom C. cinerea. 5'-SAGE analysis of the transcriptomes of vegetative mycelium and primordium of C. cinerea by 454 GS20 high-throughput pyrosequencer
Project description:The M-bM-^@M-^Xdevelopmental hourglassM-bM-^@M-^Y concept suggests that intermediate developmental stages are most resistant to evolutionary changes and that differences between species arise through divergence later in development. This high conservation during mid-development is illustrated by the M-bM-^@M-^XwaistM-bM-^@M-^Y of the hourglass and it represents a low probability of evolutionary change. Earlier molecular surveys both on animals and plants have shown that the genes expressed at the waist stage are more ancient and more conserved in their expression. The existence of such a developmental hourglass has not been explored in fungi, another eukaryotic kingdom. In this study, we generated a series of transcriptomic data covering the entire lifecycle of a model mushroom-forming fungus, Coprinopsis cinerea, and we observed a molecular hourglass over its development. The M-bM-^@M-^Xyoung fruiting bodyM-bM-^@M-^Y (YFB) is the stage that expresses the evolutionarily oldest (lowest transcriptome age index, TAI) transcriptome and gives the strongest signal of purifying selection (lowest transcriptome divergence index, TDI). We also demonstrated that all three kingdoms M-bM-^@M-^S animals, plants and fungi M-bM-^@M-^S display high expression levels of genes in M-bM-^@M-^Xinformation storage and processingM-bM-^@M-^Y at the waist stages, whereas the genes in M-bM-^@M-^XmetabolismM-bM-^@M-^Y become more highly expressed later. Besides, the three kingdoms all show underrepresented M-bM-^@M-^Xsignal transductionM-bM-^@M-^Y at the waist stages. The synchronic existence of a molecular M-bM-^@M-^XhourglassM-bM-^@M-^Y across the three kingdoms reveals a mutual strategy for eukaryotes to incorporate evolutionary innovations. NimbleGen custom microarray with total RNAs extracted from two biological replicates for each stage of mycelium, fruiting initials (~2mm tall), stage 2 primordium (~1cm tall), young fruiting body (~2cm tall) and the fully-expanded cap of the mature fruiting body (~4cm tall). Mycelia from 5 agar plates were collected and pooled to form one replicate. For the other stages, 4-5 independent structures were isolated and their RNA extracted as one replicate sample.
Project description:Mycelia of filamentous fungi explore new substrates by means of hyphae that extend from the periphery of the colony. Previously, it has been shown by immuno-labelling, reporter studies and in situ hybridization that these exploring hyphae are heterogenic with respect to protein secretion and transcription. We performed single hyphal tip RNA profiling using microarrays to assess the differences in RNA accumulation in neighboring exploring hyphae Single tips from 5 neighboring exploring hyphae from a single colony were selected for RNA extraction, amplification and hybridization on Affymetrix microarrays
Project description:The analysis of fungi secreted proteins has been increasingly employed as a powerful strategy in the prospecting of new catalysts with potential biotechnological application. Since enzyme production is strongly modulated by several factors such as pH, available nutrients, water content, oxygen levels and temperature, the evaluation of growth conditions is of utmost importance to achieve optimal enzyme production. Here, a non-sequenced wood-rotting fungus, L. crinitus, was selected as a model organism for secretome analysis by means of in vitro enzymatic assays and in-depth characterization via proteomics. For enzyme production, the fungus was cultured with several types and concentrations of carbon and nitrogen sources and variable water content. Five different carbon substrates (glucose, maltose, starch, sucrose, carboxymethylcellulose (CMC), glycerol and fructose) and three nitrogen containing compounds (urea, sodium nitrate and ammonium chloride) were used as substitutes of the original carbon and nitrogen sources in culture media. Interestingly, the biomass yield as well as the array of secreted proteins differed drastically under different growing conditions. A mixture of soluble secreted extracts derived from different culture conditions was analyzed both by shotgun mass spectrometry and through protein separation by two-dimensional gel electrophoresis (2DE) prior to identification via LC-MS/MS. Proteins were identified by sequence homology searches using mass spectrometry (MS)-driven BLAST. The spectrum of secreted enzymes comprised various types of CAZymes (carbohydrate-active enzymes), oxidase/reductases, proteases, lipase/esterases, proteins with non-related functions thereby classified as miscellany proteins and hypothetical or unknown predicted proteins. Although pre-separation by 2DE improved the number of identifications (protein map of 150 spots corresponding to 171 identifications) compared to the shotgun approach (98 identifications) both strategies revealed similar distribution of proteins within the functional categories described above. Culture media with reduced water content stimulated the expression of oxidases/reductases such as Lac, MnP, GMC oxidoreductases and glyoxal oxidase while hydrolases were induced during submerged fermentation. The diversity of proteins observed within both the CAZyme and oxidoreductase groups revealed in this fungus a powerful arsenal of enzymes dedicated to the breakdown and consumption of lignocellulose. Moreover, further secretome characterization after sequencing and analysis of the L. crinitus genome can potentially lead to the discovery of novel enzymes of industrial and biotechnological interest. Importantly, information on sequencing data could also reveal novel enzymes which production is strongly stimulated only in specific growing conditions.
Project description:Lipid droplets from 6 week old Arabidopsis leaves were purified on sucrose gradient. The abundance of the proteins in the lipid droplet fraction was established by label free and compared to soluble, membrane and plastoglobule fractions, and total leaf extract in order to identify the proteins specifically enriched in lipid droplets. Three independent biological replicates, from three independent cultures were performed. For each biological replicates, three preparations of lipid droplets (and other fractions) within each of the three independent cultures were obtained.
Project description:Plant-beneficial fungi from the genus Trichoderma (Hypocreales, Ascomycota) can control oomyceteous plant-pathogenic Pythium myriotylum (Peronosporales, Oomycota) and thus serve as bioeffectors for the eco-friendly products of crop protection. However, the underlying mechanisms of microbe-microbe interactions have yet to be fully understood. In this study, we focused on the role of the Trichoderma secretome induced by P. myriotylum mycelia. For this purpose, we selected strains showing strong (T. asperellum, T. atroviride, T. virens), moderate (T. cf. guizhouense, T. reesei), and weak (T. parepimyces) activities, respectively, and cultured with the sterilized P. myriotylum mycelia. Secreted proteins were analyzed using label-free LC-MS/MS, bioinformatic localization prediction, gene ontology (GO) annotation, and ortholog analysis. The exoproteomic analysis quantified proteins in the six Trichoderma spp., suggesting unequal antagonistic mechanisms among the strong and weak strains, respectively, with different proportions of putative cellulases, proteases, redox enzymes, and extracellular proteins of unknown function. Notably, proteolysis-related proteins were abundant, while the abundant proteases tended not to be conserved across the species (i.e., non-orthologous). Putative cellobiohydrolases were detected abundantly in all Trichoderma species except for the weak antagonist T. parepimyces, even though its genome encodes for these proteins. Notably, secretomes of the most potent anti-Pythium bioeffectors tended to have higher endo-cellulase activity. Cellulose and other glucans are major components of the oomycete cell wall, which was partly reflected in the cellulases produced by the Trichoderma species. The varying abundances of orthologous proteins suggested the evolution of differing transcription regulation mechanisms across the Trichoderma genus in response to the ubiquitous presence of Oomycota.