Metabolomics,Unknown,Transcriptomics,Genomics,Proteomics

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The role of photoreceptors in the light modulated cellulase gene expression in Neurospora crassa

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 enyzmes and the light response. The filamentous fungus, 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. Genome wide 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. Three deletion strains (delta-wc-1 (FGSC 11712), delta-wc-2 (FGSC 11124) and delta-vvd (FGSC 11556)) and the wild type strain (FGSC 2489) at two different timepoints (28h or 40h) were analyzed. Cy3 and Cy5 dye swaps were performed.

ORGANISM(S): Neurospora crassa

SUBMITTER: Doris Tisch

PROVIDER: E-GEOD-32871 | biostudies-arrayexpress |

REPOSITORIES: biostudies-arrayexpress

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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

Project description:To better understand the roles of WC-1, WC-2 and other putative photoreceptors in mediation of light signals and to identify new components in light sensing cascades, we used microarrays with genome-wide coverage to characterize light-inducible transcriptional changes in Neurospora crassa. After unsupervised hierarchical clustering of 90 microarrays, 316 genes, representing about 3.21% of the total genome, showed robust and consistent light responses. As expected, WC-1 and WC-2 knockout strains both exhibited severe impairments in response to a continuous white light stimulus, confirming their dominant roles in light sensing cascades. Knockout strains of 5 newly-identified light-inducible transcription factors were further characterized for their potential function in mediating light signals. Interestingly, one early light-inducible transcription factor, Submerged Protoperithecia-1 (SUB-1), appeared to be essential in mediating a large number of late light response, providing a direct molecular connection between early and late light responses. Furthermore, a novel motif, which is overrepresented among late light responsive genes, was demonstrated to be necessary for full induction of late light responses in vivo. Taken together, these results reveal a simple hierarchical light-sensing cascade in Neurospora, which is initially invoked by light-activated White Collar Complex (WCC) binding to Light Response Elements (LREs) of the primary target genes, including SUB-1. Subsequently, the induction of SUB-1 and other activators activate downstream targets. Thus, light responses of well-defined timing and amplitude can be orchestrated to meet the differential needs of underlying biological processes. Keywords: time course, light response Two-color microarray, totally 135 arrays were used to characterize light response in different photoreceptor knockout strains. Alexa Fluor 555 was consistently used to label cDNA synthesized from reference RNA, which is a mixture containing equal amounts of RNA samples harvested from different circadian time points and light treatment durations. The same batch of pooled RNA was used as a reference for each array experiment. Alexa Fluor 647 was used exclusively to label cDNA representing sample RNA.

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The role of photoreceptors in the light modulated cellulase gene expression in Neurospora crassa

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