Phanerochaete chrysosporium gene expression in different media
Ontology highlight
ABSTRACT:
ORGANISM(S): Phanerochaete chrysosporium
SUBMITTER: Dan Cullen
PROVIDER: E-GEOD-52922 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
Similar Datasets
Project description:Using whole genome microarrays based on the annotated genomes of Phanerochaete chrysosporium, we monitored the changes in its transcriptomes relevant to cell wall degradation during growth on three chemically distinct Populus trichocarpa (poplar) wood substrates. Results of this study are sumbitted for review in Biotechnology for Biofuels From a data set of 10004 unique gene models, each NimbleGen (Madison, WI) array targeted 9959 genes and featured 12 unique 60mers per gene, all in triplicate. RNA and protein were obtained from P. chrysosporium strain RP-78 (USDA Forest Mycology Center, Forest Products Laboratory, Madison WI) grown in malt extract agar for 10 days prior to inoculation with wood wafers. Three poplar wood substrates with distinct cell wall chemical properties were selected from several hundred 4-year old Populus trichocarpa trees grown in a common garden field trial at the University of British Columbia (Canada). We selected three poplar genotypes based on cell wall chemical traits. Substrate A corresponds to a genotype with a higher than average lignin content and lower that average glucose content; Substrate B, a lower than average lignin content and higher that average glucose content; Substrate C lignin and glucose contents are near the population average. Poplar wood stems were cut into 0.5 mm wafers on a microtome, sterilized for 20 min at 121°C, dried at 50°C overnight, and cooled to room temperature. The specimens were then inoculated in Petri dishes with actively growing mycelia. Approximately 5 g of wood wafers were placed in each Petri dish (exact weights were recorded), sealed and incubated at 22°C and 70 ± 5% relative humidity for 10, 20 or 30 days. For RNA, the degraded wafers were removed from the Petri dishes, immediately snap-frozen in liquid nitrogen and stored at -80°C for later use. Total RNA was converted to Cy3 labelled cDNA, hybridized to microarrays and scanned as previously described by Vanden Wymelenberg et al 2010 (Appl Enviro Microbiol 76:3599-3610).The 24 arrays per fungal species were scanned and data extracted using NimbleScan v.2.4. The raw data was loaded into GeneSpring, where the intensities were converted to log2 and quantile normalized, and all probes per gene averaged. This data was then exported and further analyzed in R. For substrates “A” and “B”, three replicates were used for each wood substrate/fungus and incubation period combination. For substrate “C” only 2 biological replicates were employed.
2015-05-19 | E-GEOD-69008 | biostudies-arrayexpress
Project description:Using whole genome microarrays based on the annotated genomes of Postia placenta, we monitored the changes in its transcriptomes relevant to cell wall degradation during growth on three chemically distinct Populus trichocarpa (poplar) wood substrates. The research goal is to identtify genes essential for cellulose depolymerization. From a data set of 12438 unique gene models, each NimbleGen (Madison, WI) array targeted 9959 genes and featured 10 unique 60mers per gene, all in triplicate. RNA and protein were obtained from P. placenta strain MAD-698-R (USDA Forest Mycology Center, Forest Products Laboratory, Madison WI) grown in malt extract agar for 10 days prior to inoculation with wood wafers. Three poplar wood substrates with distinct cell wall chemical properties were selected from several hundred 4-year old poplar (Populus trichocarpa) trees grown in a common garden field trial at the University of British Columbia (Canada). We selected three poplar genotypes based on cell wall chemical traits. Substrate A corresponds to a genotype with a higher than average lignin content and lower that average glucose content; Substrate B, a lower than average lignin content and higher that average glucose content; Substrate C lignin and glucose contents are near the population average. Poplar wood stems were cut into 0.5 mm wafers on a microtome, sterilized for 20 min at 121°C, dried at 50°C overnight, and cooled to room temperature. The specimens were then inoculated in Petri dishes with actively growing mycelia. Approximately 5 g of wood wafers were placed in each Petri dish (exact weights were recorded), sealed and incubated at 22°C and 70 ± 5% relative humidity for 10, 20 or 30 days. For RNA, the degraded wafers were removed from the Petri dishes, immediately snap-frozen in liquid nitrogen and stored at -80°C for later use. Total RNA was converted to Cy3 labelled cDNA, hybridized to microarrays and scanned as previously described by Vanden Wymelenberg et al 2010 (Appl Enviro Microbiol 76:3599-3610).The 24 arrays per fungal species were scanned and data extracted using NimbleScan v.2.4. The raw data was loaded into GeneSpring, where the intensities were converted to log2 and quantile normalized, and all probes per gene averaged. This data was then exported and further analyzed in R. For substrates “A” and “B”, three replicates were used for each wood substrate/fungus and incubation period combination. For substrate “C” only 2 biological replicates were employed.
2015-05-19 | E-GEOD-69004 | biostudies-arrayexpress
Project description:Transcript profiles of Phanerochaete chrysosporium grown on ball-milled aspen or ball-milled pine were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in lignocellulose degradation. From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from medium containing ball-milled aspen or ball-milled pine as the sole carbon source. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and M-BM-< volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20M-KM-^ZC. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturerM-bM-^@M-^Ys protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20M-KM-^ZC. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (6 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42M-KM-^ZC. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16M-KM-^ZC for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16M-KM-^ZC. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37M-KM-^ZC. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37M-KM-^ZC. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42M-KM-^ZC. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire dataset.
2011-06-02 | E-GEOD-27941 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE14734: Cellulose-induced regulation of Phanaerochaete chrysosporium genes GSE14735: Nutrient limitation-induced regulation of Phanaerochaete chrysosporium genes Refer to individual Series
2009-03-30 | E-GEOD-14736 | biostudies-arrayexpress
Project description:Culture filtrates from three wood decay fungi were analyzed after five- and 10-days growth in submerged cultures containing 0.75 % (w/v) finely ground lodgepole pine (Pinus contorta). Significant differences in proteome profiles were observed among the species.
2022-09-27 | PXD037036 | Pride
Project description:Transcript profiles of Phanerochaete chrysosporium grown on carbon-limited medium, nitrogen-limited medium and replete medium. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in lignin degradation. Keywords: gene expression under three different culture conditions From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from the five abovementioned media. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20˚C. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. Total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20˚C. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (15 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42˚C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16˚C for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16˚C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37˚C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37˚C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42˚C. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA; ) were applied to the entire dataset.
2009-03-30 | E-GEOD-14735 | biostudies-arrayexpress
Project description:Transcript profiles of Phanerochaete chrysosporium grown on different substrates were analyzed. Array design based on the DoE's Joint Genome Institute's v2.1 annotation. Goal is to define genes involved in cellulose degradation. Keywords: gene expression under different culture conditions From a data set of 10,004 unique alleles, each Roche NimbleGen (Madison, WI) array featured 12 unique 60mer probes per gene, all in triplicate. (Seven gene models composed mostly of repetitive DNA were represented by only 2 to 11 60mers.) Total RNA was purified from below mentioned media. In short, cultures were harvested by filtering through Miracloth (Calbiochem, EMD Biosciences, Gibbstown, NJ), squeeze dried and snap frozen in liquid nitrogen. Pellets were stored at -80 C until use. Extraction buffer was prepared by combining 10 ml 690 mM para-aminosalicylic acid (sodium salt) (Sigma-Aldrich, St. Louis, MO) with 10 ml 56 mM triisopropylnapthalene sulfonic acid (sodium salt) (Sigma-Aldrich), and placed on ice. To this was added 5 ml 5X RNB (1.0 M Tris, 1.25 M NaCl, 0.25 M EGTA). The pH of the 5X RNB was adjusted to 8.5 with NaOH. The mixture was kept on ice and shaken just before use. Frozen fungal pellets were ground to a fine powder with liquid nitrogen in an acid washed, pre-chilled mortar and pestle. The ground mycelia were transferred to Falcon 2059 tubes (VWR International, West Chester, PA), and extraction buffer was added to make a thick slurry. The samples were vortexed vigorously and placed on ice until all samples were processed. One half volume TE-saturated phenol (Sigma-Aldrich) and ¼ volume chloroform (Sigma-Aldrich) were added to each sample and vortexed vigorously. Samples were spun at 2940 x g in a fixed-angle rotor for 5 minutes. The aqueous layer was removed to a new tube, and phenol:chloroform extractions were repeated until the interface between the aqueous and organic layers was clear. The final aqueous extractions were placed in clean 2059 tubes, to which was added 0.1 volume 3M sodium acetate, pH 5.2, (DEPC-treated) and 2 volumes absolute ethanol. The tubes were shaken vigorously and stored overnight at -20˚C. The tubes were spun 1 hour at 2940 x g, the supernatants were decanted, and the pellets were resuspended in 4 ml RNase-free H2O. total RNA was purified using the RNeasy Maxi kit (Qiagen, Valencia, CA) according to the manufacturer’s protocol for RNA cleanup. RNAs were eluted from the RNeasy spin columns using two spins, for a final volume of 2 ml. The eluted RNAs were ethanol precipitated and stored overnight at -20˚C. The RNAs were spun 1 hour at 2940 x g, washed 1x with 70% ethanol, and resuspended in 50-100ul RNase-free H2O. Three biological replicates per medium were used (15 separate arrays). RNA was converted to double-strand cDNA and labeled with the Cy3 fluorophore sample for hybridization to the array by Roche NimbleGen (Iceland). In brief, 10ug of total RNA was incubated with 1X first strand buffer, 10 mM DTT, 0.5mM dNTPs, 100 pM oligo T7 d(T)24 primer, and 400U of SuperScript II (Invitrogen) for 60 min at 42˚C. Second strand cDNA was synthesized by incubation with 1X second strand buffer, 0.2mM dNTPs, 0.07U/ul DNA ligase (Invitrogen), 0.27U/ul DNA polymerase I (Invitrogen), 0.013U/ul RNase H (Invitrogen), at 16˚C for 2 hours. Immediately following, 10U T4 DNA polymerase (Invitrogen) was added for additional 5 minute incubation at 16˚C. Double-stranded cDNA was treated with 27ng/ul of RNase A (EpiCenter Technologies) for 10 minutes at 37˚C. Treated cDNA was purified using an equal volume of phenol:chloroform:isoamyl alcohol (Ambion), ethanol precipitated, washed with 80% ethanol, and resuspended in 20ul water. One ug of each cDNA sample was amplified and labeled with 1 unit per ul of Klenow Fragment (New England BioLabs) and 1 O.D. unit of Cy3 fluorophore (TriLink Biotechnologies, Inc.) for 2 hours at 37˚C. Array hybridization was carried out with 6ug of labeled cDNA suspended in NimbleGen hybridization solution for 17 hours at 42˚C. Arrays were scanned on the Axon4000B Scanner (Molecular Dynamics) and data was extracted from the scanned image using NimbleScan v2.4. DNASTAR ArrayStar v2.1 (Madison,WI) software was used to quantify and visualize data. Analyses were based on three biological replicates per culture medium. Quantile normalization and robust multi-array averaging (RMA) were applied to the entire dataset.
2009-03-30 | E-GEOD-14734 | biostudies-arrayexpress
Project description:The biodegradation of lignocellulose requires the disruption of its lignin, which shields the metabolically assimilable polysaccharides in this recalcitrant natural composite. Although a variety of microorganisms can attack lignocellulose, white rot basidiomycetes are uniquely efficient at this process, cleaving the recalcitrant intermonomer linkages of lignin via extracellular oxidative mechanisms and mineralizing many of the resulting fragments to carbon dioxide via intracellular processes. Considerable progress has been made in understanding this process in the model white rot fungus Phanerochaete chrysosporium, which expresses important components of its ligninolytic system in response to nutrient limitation, as part of its secondary metabolism. Biochemical and genetic evidence point to an important role in P. chrysosporium for secreted lignin peroxidases (LiPs), manganese peroxidases (MnPs), and H2O2-producing oxidases, which are thought to work together to cleave lignin into low molecular weight fragments. However, many aspects of ligninolysis by P. chrysosporium remain poorly understood. Although a definitive picture of the entire ligninolytic system in P. chrysosporium is not yet attainable, transcriptome analyses of the fungus grown on wood can provide useful clues. With the advent of the initial genome assembly and annotations (v1.0 and v2.1), microarray-based transcriptome analysis allowed examination of transcript levels of P. chrysosporium genes when grown in ball-milled wood and in defined growth media. This approach provided useful insights but was limited to 10048 v2.1 targets and complicated by the unpredictable manner in which the fungus responds to unnatural carbon sources in submerged basal salts media. A complete, fully coordinated ligninolytic system is likely not expressed by P. chrysosporium on ball-milled wood, because a potential route for regulatory feedback has been eliminated: the cellulose and hemicellulose in this substrate is readily accessible to enzymes, and thus ligninolysis is not essential for growth. An alternative approach is to compare levels of gene expression just before and after the onset of secondary metabolism and extracellular substrate oxidation by P. chrysosporium as it utilizes solid wood as its carbon source. If this can be done, and decay of the substrate is also confirmed, then the genes undergoing marked changes in expression during the metabolic transition can be identified with greater confidence. Although not all such genes are expected to have roles in biodegradation, this strategy may identify interesting candidates for future investigation. Here we report RNAseq-based transcriptomes to characterize changes in gene expression that occur during the transition to ligninolytic metabolism. Phanerochaete chrysosporium was inoculated onto thin sections of wood. RNA was purified from colonized material after 40 and 96 hours. Single read 100 bp Illumina runs were performed.
2015-09-16 | E-GEOD-69461 | biostudies-arrayexpress
Project description:This SuperSeries is composed of the following subset Series: GSE27941: Lignocellulose-induced regulation of Phanaerochaete chrysosporium genes GSE29656: Postia placenta MAD-698 gene expression in ball-milled aspen or ball-milled pine medium Refer to individual Series
2011-06-02 | E-GEOD-29659 | biostudies-arrayexpress
Project description:The biodegradation of lignocellulose requires the disruption of its lignin, which shields the metabolically assimilable polysaccharides in this recalcitrant natural composite. Although a variety of microorganisms can attack lignocellulose, white rot basidiomycetes are uniquely efficient at this process, cleaving the recalcitrant intermonomer linkages of lignin via extracellular oxidative mechanisms and mineralizing many of the resulting fragments to carbon dioxide via intracellular processes. Considerable progress has been made in understanding this process in the model white rot fungus Phanerochaete chrysosporium, which expresses important components of its ligninolytic system in response to nutrient limitation, as part of its secondary metabolism. Biochemical and genetic evidence point to an important role in P. chrysosporium for secreted lignin peroxidases (LiPs), manganese peroxidases (MnPs), and H2O2-producing oxidases, which are thought to work together to cleave lignin into low molecular weight fragments. However, many aspects of ligninolysis by P. chrysosporium remain poorly understood. Although a definitive picture of the entire ligninolytic system in P. chrysosporium is not yet attainable, transcriptome analyses of the fungus grown on wood can provide useful clues. With the advent of the initial genome assembly and annotations (v1.0 and v2.1), microarray-based transcriptome analysis allowed examination of transcript levels of P. chrysosporium genes when grown in ball-milled wood and in defined growth media. This approach provided useful insights but was limited to 10048 v2.1 targets and complicated by the unpredictable manner in which the fungus responds to unnatural carbon sources in submerged basal salts media. A complete, fully coordinated ligninolytic system is likely not expressed by P. chrysosporium on ball-milled wood, because a potential route for regulatory feedback has been eliminated: the cellulose and hemicellulose in this substrate is readily accessible to enzymes, and thus ligninolysis is not essential for growth. An alternative approach is to compare levels of gene expression just before and after the onset of secondary metabolism and extracellular substrate oxidation by P. chrysosporium as it utilizes solid wood as its carbon source. If this can be done, and decay of the substrate is also confirmed, then the genes undergoing marked changes in expression during the metabolic transition can be identified with greater confidence. Although not all such genes are expected to have roles in biodegradation, this strategy may identify interesting candidates for future investigation. Here we report RNAseq-based transcriptomes to characterize changes in gene expression that occur during the transition to ligninolytic metabolism.
2015-09-16 | GSE69461 | GEO