Identification of direct target genes of the Neurospora crassa essential plant biomass deconstruction transcription factors CLR-1, CLR-2 and XLR-1
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ABSTRACT:
ORGANISM(S): Neurospora crassa
SUBMITTER: James Craig
PROVIDER: E-GEOD-68517 | biostudies-arrayexpress |
REPOSITORIES: biostudies-arrayexpress
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Project description:abstract: The plant cell wall is composed of many complex polymers, and its deconstruction requires an equally complex orchestration of a wide array of enzymes. In Neurospora crassa, clr-1, clr-2 and xlr-1 have been identified as the key transcription factors involved in cell wall breakdown. In order to define their regulons, we performed ChIPseq upon these three transcription factors. CLR-1, CLR-2 and XLR-1 each bind to the most highly and differentially expressed gene populations, which include the cellulases for the CLRs and the hemicellulases for XLR-1. CLR-1 also bound to its regulon under non-inducing conditions; however, this did not translate into gene expression. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest yeast homolog, GAL4. Co-immunoprecipitation studies were able to show that CLR-1 and CLR-2 act as homodimers. Finally, we report on a conserved XLR-1 point mutation that is sufficient to drive hemicellulase expression under non-inducing conditions. Understanding how these transcription factors work in concert to break down plant biomass can inform decisions on how to best engineer future fungal strains for decreased enzyme costs. RNAseq and ChIPseq was performed upon knockout mutants and wild type strains growing on various carbon sources to determin the role of the transcription factors CLR-1, CLR-2, and XLR-1 in plant cell wall degradation
2015-09-01 | E-GEOD-68514 | biostudies-arrayexpress
Project description:abstract: The plant cell wall is composed of many complex polymers, and its deconstruction requires an equally complex orchestration of a wide array of enzymes. In Neurospora crassa, clr-1, clr-2 and xlr-1 have been identified as the key transcription factors involved in cell wall breakdown. In order to define their regulons, we performed ChIPseq upon these three transcription factors. CLR-1, CLR-2 and XLR-1 each bind to the most highly and differentially expressed gene populations, which include the cellulases for the CLRs and the hemicellulases for XLR-1. CLR-1 also bound to its regulon under non-inducing conditions; however, this did not translate into gene expression. Motif analysis of the bound genes revealed conserved DNA binding motifs, with the CLR-2 motif matching that of its closest yeast homolog, GAL4. Co-immunoprecipitation studies were able to show that CLR-1 and CLR-2 act as homodimers. Finally, we report on a conserved XLR-1 point mutation that is sufficient to drive hemicellulase expression under non-inducing conditions. Understanding how these transcription factors work in concert to break down plant biomass can inform decisions on how to best engineer future fungal strains for decreased enzyme costs. RNAseq and ChIPseq upon knockout mutants and sild type growing on various carbon sources to determin the role of the transcription factors: CLR-1, CLR-2, and XLR-1 in plant cell wall degradation
2015-09-01 | E-GEOD-68516 | biostudies-arrayexpress
Project description:Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential feedstock for the production of liquid fuels and other value-added materials. Degradation of hemicellulose by filamentous fungi requires production of many different enzymes, which are induced by biopolymers or its derivatives and regulated mainly at the transcriptional level through transcription factors (TFs). Neurospora crassa has been shown to express and secrete plant cell wall associated enzymes. To better understand genes specifically associated with degradation of hemicellulose, we identified 353 genes by transcriptome analysis of N. crassa wild type strain grown on beechwood xylan. Exposure to xylan induces 9 of the 19 predicted hemicellulase genes. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of wild type showed that none were essential for growth on beechwood xylan. The transcription factor XlnR/Xyr1 in Aspergillus and Trichoderma species is considered to be the major transcriptional regulator of genes encoding both cellulases and hemicellulases. We identified a xlnR/xyr1 homolog in N. crassa, NCU06971, termed xlr-1 (xylanase regulator 1). Deletion of xlr-1 in N. crassa abolishes the growth on xylan and xylose, but growth on cellulose was indistinguishable from wild type. To determine regulatory mechanisms associated with hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose and xylanolytic versus cellulolytic conditions. XLR-1 regulated only some predicted hemicellulase genes in N. crassa and was required for a full induction of several cellulase genes. Hemicellulase gene expression was induced by a combination of release from carbon catabolite repression (CCR) and induction. However, in N. crassa, xlr-1 is subject to non-CRE-1 mediated CCR. This systematic analysis provides the similarities and differences of hemicellulose degradation and regulation mechanisms used by N. crassa in comparison to other filamentous fungi. Four-condition experiments (minimal medium, xylan medium,xylose and Avicel medium) of mutant strain(xlr-1) compared to wild type strain; Cy3 and Cy5 dye swap
2012-03-26 | E-GEOD-34098 | biostudies-arrayexpress
Project description:The thermophilic filamentous fungi Myceliophthora thermophila (Sporotrichum thermophile) and Thielavia terrestris are proficient decomposers of cellulose, suggesting that they will be a rich source of thermostable industrial enzymes for lignocellulose degradation. To identify the genes and proteins involved in this process, we explored the transcriptomes of M. thermophila and T. terrestris growing at 45 ºC on either glucose, alfalfa, or barley straw by short-read sequencing of extracted mRNA. To better understand the adaptations that allow these fungi to grow at elevated temperatures, we compared their transcriptomes when growing at 34C to their transcritomes at 45C, and also to the transcriptome of the related fungus Chaetomium globosum, which does not grow at 45C. RNA was extracted from cultures in early growth stage growing with glucose, alfalfa, or barley straw as carbon source at 34C or 45C (M. thermophila and T. terrestris); duplicate cultures were sampled in some conditions.
2011-08-08 | E-GEOD-27323 | biostudies-arrayexpress
Project description:Fungi are an important source of enzymes for saccharification of plant polysaccharides and production of biofuels. Understanding of the regulation and induction of expression of genes encoding these enzymes is still incomplete. To explore the induction mechanism, we analysed the response of the industrially important fungus Aspergillus niger to wheat straw, with a focus on events occurring shortly after exposure to the substrate. RNA sequencing showed that over a third of the genes induced after 6 h of exposure to wheat straw were also induced during 6 h of carbon starvation, indicating that carbon starvation is probably an important factor in the early response to wheat straw. The up-regulation of the expression of a high number of genes encoding CAZymes that are active on plant-derived carbohydrates during early carbon starvation suggests that these enzymes could be involved in a scouting role during starvation, releasing inducing sugars from complex plant polysaccharides. Eight samples in total consisting of duplicate shake flask Aspergillus niger cultures from four conditions: 48h glucose, 6 h starvation, 6 h wheat straw, 24 h starvation
2014-08-22 | E-GEOD-57315 | biostudies-arrayexpress
Project description:The thermophilic fungus Malbranchea cinnamomea belongs to the order of Onygenales and is a promising source of thermostable, industrially relevant biocatalysts, as it can grow at temperatures of more than 50°C and is able to utilise many different types of plant biomass. Enzymes from M. cinnamomea that have been characterised so far include an α-amylase, an α-glucosidase, xylanases and an alkaline β-1,3-1,4-glucanase (lichenase), all of which have been reported to have temperature optima between 50°C and 80°C. With this study, we complement the knowledge of the enzymatic repertoire of M. cinnamomea with a transcriptomic analysis of strain FCH 10.5 to provide a more comprehensive view of its lignocellulolytic enzyme system. Genes differentially expressed during growth on two different polymeric substrates, beechwood xylan and wheat bran, point to differences in the fungal response to the deconstruction of a hardwood hemicellulose (beechwood xylan) and a cereal hemicellulose (wheat bran). The data presented here will form the basis for a systematic exploration of the full potential of this fungus as a source of thermostable enzymes. We sequenced the genome of M. cinnamomea FCH 10.5, which was isolated from the compost of a waste treatment plant in Hanoi, Vietnam (PMC5604768, https://www.ncbi.nlm.nih.gov/nuccore/FQSS02000000). For RNAseq, the fungus was grown on three different carbon sources (glucose, wheat bran, beechwood xylan) at 50°C. Mycelium was harvested after 4h and 48h and RNA was extracted. For RNAseq analysis, the RNA of 4h and 48h samples was mixed 1:1, to get information about both early- and late-response genes during growth on the different carbon sources. Two independent duplicate experiments were done for each substrate. Total RNA was extracted using TRIzol (Invitrogen) and chloroform, and further purified with the RNeasy Plant RNA Kit with on-column DNAse digestion (QIAGEN). The quality of the purified RNA was verified by agarose gel electrophoresis, Nanodrop (Thermo Scientific) and Qubit (Life Technologies). The NEBNext Ultra Directional RNA Library Prep Kit for Illumina (New England Biolabs) was used to process the samples according to the manufacturer’s instructions. Briefly, mRNA was isolated from total RNA using oligo-dT magnetic beads and used to synthesise cDNA. The cDNA was ligated with sequencing adapters and PCR amplified. The quality and yield after sample preparation were determined with the Fragment Analyzer (Advanced Analytical). The size of the resulting products was consistent with the expected size distribution (a broad peak between 300-500 bp). Standard Illumina primers for Illumina cBot and HiSeq 2500, and the HiSeq control software HCS v2.2.58 were used according to manufacturer’s protocols for clustering and DNA sequencing with a concentration of 16.0 pM. The Illumina data analysis pipelines RTA v1.18.64 and Bcl2fastq v2.17 were used for image analysis, base calling, and quality check. Sequencing was performed on an Illumina HiSeq 2500 sequencer. The assembled genome from the DNA sequencing was used as a reference to map the reads using the packages Tophat (v2.0.14. Linux_x86_64) and Bowtie (v2-2.1.0) with a default mismatch rate of 2%. The frequency with which a read was mapped on a transcript was determined based on the mapped locations from the alignment. To normalise for transcript length, fpkm (fragments per kilobase of transcript per million mapped reads) were calculated. For differential expression analysis, the read counts were loaded into the DESeq package v 1.10.1. Genes were considered differentially expressed if they showed a log2 fold change ≥ 1 and the adjusted p-value was < 0.05.
2017-10-27 | E-MTAB-6157 | biostudies-arrayexpress
Project description:Investigation of whole genome gene expression level changes in Streptomyces sp. SirexAA-E (ActE) when grown on different carbon sources. The results of this study demonstrate that ActE upregulates a small number of genes specific for the utilization of the avaliable carbon source. Cellulolytic Streptomyces sp. SirexAA-E (ActE), isolated from the pinewood-boring wasp Sirex noctilio, has a genome enriched for biomass utilization. The secreted proteomes obtained from growth on pure polysaccharides catalyzed hydrolysis of cellulose, mannan, and xylan with specific activities comparable to Spezyme CP, a commercial cellulase preparation. During reaction of an ActE secretome with cellulose, reducing sugar release was markedly stimulated in the presence of O2. ActE also expresses and secretes an expanded repertoire of enzymes during growth on natural and pre-treated biomass. These results indicate a new microbial contribution to biomass utilization that is widely distributed in natural environments by insects Streptomyces sp. ActE was grown in minimal medium supplimented with 0.5% carbon source (glucose, sigmacell-20, xylan, chitin, cellobiose, or AFEX). Cells were grown for 7 days and total RNA was extracted from the cell pellet. At least 3 biological replicates were performed for each carbon source (glucose, 3; sigmacell, 3; xylan, 5; chitin, 3; cellobiose 3; AFEX 3). Each biological replicate contained 3 technical replicates. The complete dataset were RMA Background Corrected, quantile normalized, the RMA algorithm was utilized by DNAStar ArrayStar.
2013-12-31 | E-GEOD-31748 | biostudies-arrayexpress
Project description:Various saprotrophic microorganisms, especially filamentous fungi, can efficiently degrade lignocellulose that is one of the most abundant natural material on earth. It consists of complex carbohydrates and aromatic polymers found in plant cell wall and thus in plant debris. Aspergillus fumigatus Z5 was isolated from compost heaps and showed highly efficient plant biomass-degradation capability.Genome analysis revealed an impressive array of genes encoding cellulases, hemicellulases, and pectinases involved in lignocellulosic biomass degradation. We sequenced the transcriptomes of Aspergillus fumigatus Z5 induced by sucrose, xylan, cellulose and rice straw, respectively. There were 444, 1711 and 1386 significantly differently (q-value ⤠0.0001 and |log2 of the ratio of the RPM values| ⥠2) expressed genes in xylan, cellulose and rice straw,respectively, relative to sucrose control. After incubation at 45 â, 145rpm for 20 hours with sucrose as the carbon source, mycelia were induced for 16 hours using xylan, cellulose and rice straw, respectively. Transcriptome induced by sucrose was used as the control when comparing the differences between other three transcriptomes (induced by xylan, cellulose and rice straw, respectively).
2015-11-22 | E-GEOD-55086 | biostudies-arrayexpress
Project description:Fungi are ubiquitous and are often confronted with the need to secure utilisable carbon from their external growth milieu through the use of extracellular proteins to scavenge for carbon from a vast array of complex polymeric carbon sources. This attribute is conserved across evolution in fungi. To understand how filamentous fungi extracellular proteins are modulated in response to the presence polymeric carbons in the environment, we have typed the array of the main extracellular proteins involved and their dynamics using a known hypercellulolytic fungus – Penicillium funiculosum (NCIM 1228), through multiplexed quantitative proteomics
2018-04-12 | PXD006445 | Pride
Project description:Transcriptional profiling of the A. niger WT (N402) strain treated with xylan (1%, w/v) for 6, 12 and 24 h. The main objective was to identify genes related to cellulases and hemicellulases after treatment with the polysaccharide xylan. The experiment was further validated by enzymatic assays. Three-condition experiment: WT-Xylan for 6, 12 and 24 h at 30oC in batch culture. Firstly, the WT (N402) strain was grown in minimal medium with fructose as carbon source (control), and then transferred to 1% (w/v) xylan as carbon source. 2 biological replicates per time point.
2010-10-21 | E-GEOD-24848 | biostudies-arrayexpress