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Filamentous fungi that thrive on plant biomass are the major producers of hydrolytic enzymes used to decompose lignocellulose for biofuel production. Although induction of cellulases is regulated at the transcriptional level, how filamentous fungi sense and signal carbon-limited conditions to coordinate cell metabolism and regulate cellulolytic enzyme production is not well characterized. By screening a transcription factor deletion set in the filamentous fungus Neurospora crassa for mutants unable to grow on cellulosic materials, we identified a role for the transcription factor, VIB1, as essential for cellulose utilization. VIB1 does not directly regulate hydrolytic enzyme gene expression or function in cellulosic inducer signaling/processing, but affects the expression level of an essential regulator of hydrolytic enzyme genes, CLR2. Transcriptional profiling of a Δvib-1 mutant suggests that it has an improper expression of genes functioning in metabolism and energy and a deregulation of carbon catabolite repression (CCR). By characterizing new genes, we demonstrate that the transcription factor, COL26, is critical for intracellular glucose sensing/metabolism and plays a role in CCR by negatively regulating cre-1 expression. Deletion of the major player in CCR, cre-1, or a deletion of col-26, did not rescue the growth of Δvib-1 on cellulose. However, the synergistic effect of the Δcre-1; Δcol-26 mutations circumvented the requirement of VIB1 for cellulase gene expression, enzyme secretion and cellulose deconstruction. Our findings support a function of VIB1 in repressing both glucose signaling and CCR under carbon-limited conditions, thus enabling a proper cellular response for plant biomass deconstruction and utilization.

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.

Background: 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., Results: 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., Conclusions: 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.

Filamentous fungi secrete a wide range of enzymes, including cellulases and hemicellulases, with potential applications in the production of lignocellulosic biofuels. Of the cellulolytic fungi, Hypocrea jecorina (anamorph Trichoderma reesei) is the best characterized in terms of cellulose degradation, but other cellulolytic fungi, such as the model filamentous fungus Neurospora crassa, can serve a crucial role in building our knowledge about the fungal response to biomass due to the many molecular and genetic tools available for this organism. Here we cloned and expressed GH5-1 (NCU00762), a secreted endoglucanase in N. crassa. The protein was produced using a ccg-1 promoter under conditions in which no other cellulases are present. Native GH5-1 (nGH5-1) and this recombinant GH5-1 (rGH5-1) were purified to gauge differences in glycosylation and activity; both rGH5-1 and nGH5-1 were similarly glycosylated, with an estimated molecular weight of 52kDa. On azo-carboxymethylcellulose, rGH5-1 activity was equal to that of nGH5-1, and on cellulose (Avicel) rGH5-1 was 20% more active. The activity of a GH5-1-GFP fusion protein (rGH5-1-GFP-6xHis) was similar to rGH5-1 and nGH5-1. To determine the binding pattern of catalytically active rGH5-1-GFP-6xHis to plant cell walls, Arabidopsis seedlings were incubated with rGH5-1-GFP-6xHis or Pontamine Fast Scarlet 4B (S4B), a cellulose-specific dye. Confocal microscopy showed that rGH5-1-GFP-6xHis bound in linear, longitudinal patterns on seedling roots, similar to S4B. The functional expression and characterization of rGH5-1 and its GFP fusion derivative set important precedents for further investigation of biomass degradation by filamentous fungi, especially N. crassa, with applications for characterization and manipulation of novel enzymes., (Copyright © 2010 Elsevier Inc. All rights reserved.)

Background: In filamentous ascomycete fungi, the utilization of alternate carbon sources is influenced by the zinc finger transcription factor CreA/CRE-1, which encodes a carbon catabolite repressor protein homologous to Mig1 from Saccharomyces cerevisiae. In Neurospora crassa, deletion of cre-1 results in increased secretion of amylase and β-galactosidase., Methodology/principal Findings: Here we show that a strain carrying a deletion of cre-1 has increased cellulolytic activity and increased expression of cellulolytic genes during growth on crystalline cellulose (Avicel). Constitutive expression of cre-1 complements the phenotype of a N. crassa Δcre-1 strain grown on Avicel, and also results in stronger repression of cellulolytic protein secretion and enzyme activity. We determined the CRE-1 regulon by investigating the secretome and transcriptome of a Δcre-1 strain as compared to wild type when grown on Avicel versus minimal medium. Chromatin immunoprecipitation-PCR of putative target genes showed that CRE-1 binds to only some adjacent 5'-SYGGRG-3' motifs, consistent with previous findings in other fungi, and suggests that unidentified additional regulatory factors affect CRE-1 binding to promoter regions. Characterization of 30 mutants containing deletions in genes whose expression level increased in a Δcre-1 strain under cellulolytic conditions identified novel genes that affect cellulase activity and protein secretion., Conclusions/significance: Our data provide comprehensive information on the CRE-1 regulon in N. crassa and contribute to deciphering the global role of carbon catabolite repression in filamentous ascomycete fungi during plant cell wall deconstruction.

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.

The phytopathogen Pseudomonas syringae competes with other epiphytic organisms, such as filamentous fungi, for resources. Here we characterize a gene in P. syringae pv. syringae B728a and P. syringae pv. tomato DC3000, termed phcA, that has homology to a filamentous fungal gene called het-c. phcA is conserved in many P. syringae strains, but is absent in one of the major clades, which includes the P. syringae pathovar phaseolicola. In the filamentous fungus Neurospora crassa, HET-C regulates a conserved programmed cell death pathway called heterokaryon incompatibility (HI). Ectopic expression of phcA in N. crassa induced HI and cell death that was dependent on the presence of a functional het-c pin-c haplotype. Further, by co-immunoprecipitation experiments, a heterocomplex between N. crassa HET-C1 and PhcA was associated with phcA-induced HI. P. syringae was able to attach and extensively colonize N. crassa hyphae, while an Escherichia coli control showed no association with the fungus. We further show that the P. syringae is able to use N. crassa as a sole nutrient source. Our results suggest that P. syringae has the potential to utilize phcA to acquire nutrients from fungi in nutrient-limited environments like the phyllosphere by the novel mechanism of HI induction.

Filamentous fungi that thrive on plant biomass are the major producers of hydrolytic enzymes used to decompose lignocellulose for biofuel production. Although induction of cellulases is regulated at the transcriptional level, how filamentous fungi sense and signal carbon-limited conditions to coordinate cell metabolism and regulate cellulolytic enzyme production is not well characterized. By screening a transcription factor deletion set in the filamentous fungus Neurospora crassa for mutants unable to grow on cellulosic materials, we identified a role for the transcription factor, VIB1, as essential for cellulose utilization. VIB1 does not directly regulate hydrolytic enzyme gene expression or function in cellulosic inducer signaling/processing, but affects the expression level of an essential regulator of hydrolytic enzyme genes, CLR2. Transcriptional profiling of a Δvib-1 mutant suggests that it has an improper expression of genes functioning in metabolism and energy and a deregulation of carbon catabolite repression (CCR). By characterizing new genes, we demonstrate that the transcription factor, COL26, is critical for intracellular glucose sensing/metabolism and plays a role in CCR by negatively regulating cre-1 expression. Deletion of the major player in CCR, cre-1, or a deletion of col-26, did not rescue the growth of Δvib-1 on cellulose. However, the synergistic effect of the Δcre-1; Δcol-26 mutations circumvented the requirement of VIB1 for cellulase gene expression, enzyme secretion and cellulose deconstruction. Our findings support a function of VIB1 in repressing both glucose signaling and CCR under carbon-limited conditions, thus enabling a proper cellular response for plant biomass deconstruction and utilization. [ABSTRACT FROM AUTHOR]

Filamentous fungi that thrive on plant biomass are the major producers of hydrolytic enzymes used to decompose lignocellulose for biofuel production. Although induction of cellulases is regulated at the transcriptional level, how filamentous fungi sense and signal carbon-limited conditions to coordinate cell metabolism and regulate cellulolytic enzyme production is not well characterized. By screening a transcription factor deletion set in the filamentous fungus Neurospora crassa for mutants unable to grow on cellulosic materials, we identified a role for the transcription factor, VIB1, as essential for cellulose utilization. VIB1 does not directly regulate hydrolytic enzyme gene expression or function in cellulosic inducer signaling/processing, but affects the expression level of an essential regulator of hydrolytic enzyme genes, CLR2. Transcriptional profiling of a Δvib-1 mutant suggests that it has an improper expression of genes functioning in metabolism and energy and a deregulation of carbon catabolite repression (CCR). By characterizing new genes, we demonstrate that the transcription factor, COL26, is critical for intracellular glucose sensing/metabolism and plays a role in CCR by negatively regulating cre-1 expression. Deletion of the major player in CCR, cre-1, or a deletion of col-26, did not rescue the growth of Δvib-1 on cellulose. However, the synergistic effect of the Δcre-1; Δcol-26 mutations circumvented the requirement of VIB1 for cellulase gene expression, enzyme secretion and cellulose deconstruction. Our findings support a function of VIB1 in repressing both glucose signaling and CCR under carbon-limited conditions, thus enabling a proper cellular response for plant biomass deconstruction and utilization. [ABSTRACT FROM AUTHOR]