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ABSTRACT: Light represents an important environmental cue, which exerts considerable influence on the metabolism of fungi. Studies with the biotechnological fungal workhorse Trichoderma reesei (Hypocrea jecorina) have revealed an interconnection between transcriptional regulation of cellulolytic enzymes and the light response. Neurospora crassa has been used as a model organism to study light and circadian rhythm biology. We therefore investigated whether light also regulates transcriptional regulation of cellulolytic enzymes in N. crassa.
We show that the N. crassa photoreceptor genes wc-1, wc-2 and vvd are involved in regulation of cellulase gene expression, indicating that this phenomenon is conserved among filamentous fungi. The negative effect of VVD on production of cellulolytic enzymes is thereby accomplished by its role in photoadaptation and hence its function in White collar complex (WCC) formation. In contrast, the induction of vvd expression by the WCC does not seem to be crucial in this process. Additionally, we found that WC-1 and WC-2 not only act as a complex, but also have individual functions upon growth on cellulose.
Genome wide transcriptome analysis of photoreceptor mutants and evaluation of results by analysis of mutant strains identified several candidate genes likely to play a role in light modulated cellulase gene expression. Genes with functions in amino acid metabolism, glycogen metabolism, energy supply and protein folding are enriched among genes with decreased expression levels in the wc-1 and wc-2 mutants. The ability to properly respond to amino acid starvation, i. e. up-regulation of the cross pathway control protein cpc-1, was found to be beneficial for cellulase gene expression. Our results further suggest a contribution of oxidative depolymerization of cellulose to plant cell wall degradation in N. crassa.
BMC Genomics 03/2012; 13:127. · 4.07 Impact Factor
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ABSTRACT: Hemicellulose, the second most abundant plant biomass fraction after cellulose, is widely viewed as a potential substrate 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, a model filamentous fungus, expresses and secretes enzymes required for plant cell wall deconstruction. To better understand genes specifically associated with degradation of hemicellulose, we applied secretome and transcriptome analysis to N. crassa grown on beechwood xylan. We identified 34 secreted proteins and 353 genes with elevated transcription on xylan. The xylanolytic phenotype of strains with deletions in genes identified from the secretome and transcriptome analysis of the wild type was assessed, revealing functions for known and unknown proteins associated with hemicellulose degradation. By evaluating phenotypes of strains containing deletions of predicted TF genes in N. crassa, we identified a TF (XLR-1; xylan degradation regulator 1) essential for hemicellulose degradation that is an ortholog to XlnR/XYR1 in Aspergillus and Trichoderma species, respectively, a major transcriptional regulator of genes encoding both cellulases and hemicellulases. Deletion of xlr-1 in N. crassa abolished growth on xylan and xylose, but growth on cellulose and cellulolytic activity were only slightly affected. To determine the regulatory mechanisms for hemicellulose degradation, we explored the transcriptional regulon of XLR-1 under xylose, xylanolytic, and 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. This systematic analysis illustrates the similarities and differences in regulation of hemicellulose degradation among filamentous fungi.
Eukaryotic Cell 02/2012; 11(4):482-93. · 3.60 Impact Factor
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ABSTRACT: Transcription factors (TFs) are key nodes of regulatory networks in eukaryotic organisms, including filamentous fungi such as Neurospora crassa. The 178 predicted DNA-binding TFs in N. crassa are distributed primarily among six gene families, which represent an ancient expansion in filamentous ascomycete genomes; 98 TF genes show detectable expression levels during vegetative growth of N. crassa, including 35 that show a significant difference in expression level between hyphae at the periphery versus hyphae in the interior of a colony. Regulatory networks within a species genome include paralogous TFs and their respective target genes (TF regulon). To investigate TF network evolution in N. crassa, we focused on the basic leucine zipper (bZIP) TF family, which contains nine members. We performed baseline transcriptional profiling during vegetative growth of the wild-type and seven isogenic, viable bZIP deletion mutants. We further characterized the regulatory network of one member of the bZIP family, NCU03905. NCU03905 encodes an Ap1-like protein (NcAp-1), which is involved in resistance to multiple stress responses, including oxidative and heavy metal stress. Relocalization of NcAp-1 from the cytoplasm to the nucleus was associated with exposure to stress. A comparison of the NcAp-1 regulon with Ap1-like regulons in Saccharomyces cerevisiae, Schizosaccharomyces pombe, Candida albicans and Aspergillus fumigatus showed both conservation and divergence. These data indicate how N. crassa responds to stress and provide information on pathway evolution.
Microbiology 11/2010; 157(Pt 3):747-59. · 3.06 Impact Factor
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ABSTRACT: Fungal degradation of plant biomass may provide insights for improving cellulosic biofuel production. We show that the model cellulolytic fungus Neurospora crassa relies on a high-affinity cellodextrin transport system for rapid growth on cellulose. Reconstitution of the N. crassa cellodextrin transport system in Saccharomyces cerevisiae promotes efficient growth of this yeast on cellodextrins. In simultaneous saccharification and fermentation experiments, the engineered yeast strains more rapidly convert cellulose to ethanol when compared with yeast lacking this system.
Science 10/2010; 330(6000):84-6. · 31.20 Impact Factor
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ABSTRACT: The filamentous fungus Neurospora crassa is a model laboratory organism, but in nature is commonly found growing on dead plant material, particularly grasses. Using functional genomics resources available for N. crassa, which include a near-full genome deletion strain set and whole genome microarrays, we undertook a system-wide analysis of plant cell wall and cellulose degradation. We identified approximately 770 genes that showed expression differences when N. crassa was cultured on ground Miscanthus stems as a sole carbon source. An overlap set of 114 genes was identified from expression analysis of N. crassa grown on pure cellulose. Functional annotation of up-regulated genes showed enrichment for proteins predicted to be involved in plant cell wall degradation, but also many genes encoding proteins of unknown function. As a complement to expression data, the secretome associated with N. crassa growth on Miscanthus and cellulose was determined using a shotgun proteomics approach. Over 50 proteins were identified, including 10 of the 23 predicted N. crassa cellulases. Strains containing deletions in genes encoding 16 proteins detected in both the microarray and mass spectrometry experiments were analyzed for phenotypic changes during growth on crystalline cellulose and for cellulase activity. While growth of some of the deletion strains on cellulose was severely diminished, other deletion strains produced higher levels of extracellular proteins that showed increased cellulase activity. These results show that the powerful tools available in N. crassa allow for a comprehensive system level understanding of plant cell wall degradation mechanisms used by a ubiquitous filamentous fungus.
Proceedings of the National Academy of Sciences 12/2009; 106(52):22157-62. · 9.68 Impact Factor
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ABSTRACT: Heterokaryon incompatibility (HI) is a nonself recognition phenomenon occurring in filamentous fungi that is important for limiting resource plundering and restricting viral transfer between strains. Nonself recognition and HI occurs during hyphal fusion between strains that differ at het loci. If two strains undergo hyphal fusion, but differ in allelic specificity at a het locus, the fusion cell is compartmentalized and undergoes a rapid programmed cell death (PCD). Incompatible heterokaryons show a macroscopic phenotype of slow growth and diminished conidiation, and a microscopic phenotype of hyphal compartmentation and cell death. To understand processes associated with HI and PCD, we used whole-genome microarrays for Neurospora crassa to assess transcriptional differences associated with induction of HI mediated by differences in het-c pin-c haplotype. Our data show that HI is a dynamic and transcriptionally active process. The production of reactive oxygen species is implicated in the execution of HI and PCD in N. crassa, as are several genes involved in phosphatidylinositol and calcium signalling pathways. However, genes encoding mammalian homologues of caspases or apoptosis-inducing factor (AIF) are not required for HI or programmed cell death. These data indicate that PCD during HI occurs via a novel and possibly fungal-specific mechanism, making this pathway an attractive drug target for control of fungal infections.
Microbiology 09/2009; 155(Pt 12):3957-70. · 3.06 Impact Factor
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ABSTRACT: Treatment of Neurospora crassa cells with phytosphingosine (PHS) induces programmed cell death (PCD) by an unknown mechanism. To determine the relationship between PHS treatment and PCD, we determined changes in global gene expression levels in N. crassa during a time-course of PHS treatment. Most genes having differential expression levels compared to untreated samples showed an increase in relative expression level upon PHS exposure. However, genes encoding mitochondrial proteins were highly enriched among approximately 100 genes that showed a relative decrease in expression levels after PHS treatment, suggesting that repression of these genes might be related to the death-inducing effects of PHS. Since mutants in respiratory chain complex I are more resistant to both PHS and hydrogen peroxide (H(2)O(2)) than the wild-type strain, possibly related to the production of reactive oxygen species, we also compared gene expression profiles of a complex I mutant (nuo14) and wild-type in response to H(2)O(2). Genes with higher expression levels in the mutant, in the presence of H(2)O(2), are also significantly enriched in genes encoding mitochondrial proteins. These data suggest that complex I mutants cope better with drug-induced decrease in expression of genes encoding mitochondrial proteins and may explain their increased resistance to both PHS and H(2)O(2). As a way of identifying new components required for PHS-induced death, we analysed the PHS sensitivity of 24 strains carrying deletions in genes that showed a significant alteration in expression pattern when the wild-type was exposed to the sphingolipid. Two additional mutants showing increased resistance to PHS were identified and both encode predicted mitochondrial proteins, further supporting the role of the mitochondria in PHS-induced PCD.
Microbiology 07/2009; 155(Pt 9):3134-41. · 3.06 Impact Factor
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ABSTRACT: Identifying and characterizing transcriptional regulatory networks is important for guiding experimental tests on gene function. The characterization of regulatory networks allows comparisons among both closely and distantly related species, providing insight into network evolution, which is predicted to correlate with the adaptation of different species to particular environmental niches. One of the most intensely studied regulatory factors in the yeast Saccharomyces cerevisiae is the bZIP transcription factor Gcn4p. Gcn4p is essential for a global transcriptional response when S. cerevisiae experiences amino acid starvation. In the filamentous ascomycete Neurospora crassa, the ortholog of GCN4 is called the cross pathway control-1 (cpc-1) gene; it is required for the ability of N. crassa to induce a number of amino acid biosynthetic genes in response to amino acid starvation. Here, we deciphered the CPC1 regulon by profiling transcription in wild-type and cpc-1 mutant strains with full-genome N. crassa 70-mer oligonucleotide microarrays. We observed that at least 443 genes were direct or indirect CPC1 targets; these included 67 amino acid biosynthetic genes, 16 tRNA synthetase genes, and 13 vitamin-related genes. Comparison among the N. crassa CPC1 transcriptional profiling data set and the Gcn4/CaGcn4 data sets from S. cerevisiae and Candida albicans revealed a conserved regulon of 32 genes, 10 of which are predicted to be directly regulated by Gcn4p/CPC1. The 32-gene conserved regulon comprises mostly amino acid biosynthetic genes. The comparison of regulatory networks in species with clear orthology among genes sheds light on how gene interaction networks evolve.
Eukaryotic Cell 07/2007; 6(6):1018-29. · 3.60 Impact Factor
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Jay C Dunlap,
Katherine A Borkovich,
Matthew R Henn,
Gloria E Turner,
Matthew S Sachs,
N Louise Glass,
Kevin McCluskey,
Michael Plamann,
James E Galagan,
Bruce W Birren, [......],
Matthew Crawford,
Michael Koerhsen,
Phil Montgomery,
Lisa Larson,
Matthew Pearson,
Takao Kasuga, Chaoguang Tian,
Meray Baştürkmen,
Lorena Altamirano,
Junhuan Xu
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ABSTRACT: A consortium of investigators is engaged in a functional genomics project centered on the filamentous fungus Neurospora, with an eye to opening up the functional genomic analysis of all the filamentous fungi. The overall goal of the four interdependent projects in this effort is to accomplish functional genomics, annotation, and expression analyses of Neurospora crassa, a filamentous fungus that is an established model for the assemblage of over 250,000 species of non yeast fungi. Building from the completely sequenced 43-Mb Neurospora genome, Project 1 is pursuing the systematic disruption of genes through targeted gene replacements, phenotypic analysis of mutant strains, and their distribution to the scientific community at large. Project 2, through a primary focus in Annotation and Bioinformatics, has developed a platform for electronically capturing community feedback and data about the existing annotation, while building and maintaining a database to capture and display information about phenotypes. Oligonucleotide-based microarrays created in Project 3 are being used to collect baseline expression data for the nearly 11,000 distinguishable transcripts in Neurospora under various conditions of growth and development, and eventually to begin to analyze the global effects of loss of novel genes in strains created by Project 1. cDNA libraries generated in Project 4 document the overall complexity of expressed sequences in Neurospora, including alternative splicing alternative promoters and antisense transcripts. In addition, these studies have driven the assembly of an SNP map presently populated by nearly 300 markers that will greatly accelerate the positional cloning of genes.
Advances in genetics 02/2007; 57:49-96. · 3.39 Impact Factor
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ABSTRACT: To test the inferences of spotted microarray technology against a biochemically well-studied process, we performed transcriptional profiling of conidial germination in the filamentous fungus, Neurospora crassa. We first constructed a 70 base oligomer microarray that assays 3366 predicted genes. To estimate the relative gene expression levels and changes in gene expression during conidial germination, we analyzed a circuit design of competitive hybridizations throughout a time course using a Bayesian analysis of gene expression level. Remarkable consistency of mRNA profiles with previously published northern data was observed. Genes were hierarchically clustered into groups with respect to their expression profiles over the time course of conidial germination. A functional classification database was employed to characterize the global picture of gene expression. Consensus motif searches identified a putative regulatory component associated with genes involved in ribosomal biogenesis. Our transcriptional profiling data correlate well with biochemical and physiological processes associated with conidial germination and will facilitate functional predictions of novel genes in N.crassa and other filamentous ascomycete species. Furthermore, our dataset on conidial germination allowed comparisons to transcriptional mechanisms associated with germination processes of diverse propagules, such as teliospores of the phytopathogenic fungus Ustilago maydis and spores of the social amoeba Dictyostelium discoideum.
Nucleic Acids Research 02/2005; 33(20):6469-85. · 8.03 Impact Factor
