Project description:Histone H1 variants, known as linker histones, are essential chromatin components in higher eukaryotes, yet compared to the core histones relatively little is known about their in vivo functions. The filamentous fungus Neurospora crassa encodes a single H1 protein that is not essential for viability. To investigate the role of N. crassa H1, we constructed a functional FLAG-tagged H1 fusion protein and performed genomic and molecular analyses. Cell fractionation experiments showed that H1-FLAG is a chromatin binding protein. Chromatin-immunoprecipitation combined with sequencing (ChIP-seq) revealed that H1-3XFLAG is globally enriched throughout the genome with a subtle preference for promoters of expressed genes. In mammals, the stochiometery of H1 impacts nucleosome repeat length. To determine if H1 impacts nucleosome occupancy or nucleosome positioning in N. crassa, we performed micrococcal nuclease digestion in wildtype and the ∆hH1 strain followed by sequencing (MNase-Seq). Deletion of hH1 did not significantly impact nucleosome positioning or nucleosome occupancy. Analysis of DNA methylation using methylC-sequencing (mC-Seq) revealed a modest but global increase in DNA methylation in the ∆hH1 mutant. Together, these data suggest that H1 acts as a non-specific chromatin binding protein that can limit accessibility of the DNA methylation machinery in N. crassa.
Project description:The aim of this experiment is to test the ability of the ortholog of Arabidopsis LFY gene from Leanworthia crassa (Lcr) to complement an Arabidopsis LFY mutant. Plants used are homozygous lfy6 mutants (EMS alleles) in Ler background which are transformed or not (for the lfy6 mutant) by genomic clones for Arabidopsis LFY (AthLFY) or Leanworthia crassa LFY (LcrLFY). Flowering was synchronized by growing plants in SD then shifting them to LD. 2 time points samples (wild type Ler) were taken at the end of the SD period as a reference for genes induced by shifting to LD, irrespective of the status at the LFY locus.
Project description:Heat-stable antifungal factor (HSAF) isolated from Lysobacter enzymogenes has shown a broad-spectrum of antifungal activities. However, little is known about its mode of action. In this study, we used the model filamentous fungus Neurospora crassa to investigate the antifungal mechanism of HSAF. We first used HSAF to treat N. crassa strain for different time points. Spore germination, growth phenotype and differential gene expression analysis were conducted by utilizing global transcriptional profiling combined with genetic and physiological analyses. Our data showed that HSAF could significantly inhibit the germination and aerial hyphae growth of N.crassa. RNA-seq analysis showed a group of genes associated with cell wall formation and remodeling were highly activated. Screening of N. crassa gene deletion mutants combined with scanning electron microscopic observation revealed 3 fungal cell wall integrity related genes played important role in the interaction between N. crassa and L. enzymogens. In addition, WGCNA analysis, accompany with confocal microscopy observation revealed that HSAF could trigger autophagy mediated degradation and eventually result in cell death in N. crassa. The findings of this work provided new insights into the interactions between the predatory Lysobacter and its fungal prey.
Project description:A colony of fungus is comprised of long, branching filamentous cells called hyphae. Genetic mechanisms underlying the development of hyphae are poorly understood. We sectioned hyphae of a model fungus, Neurospora crassa (pink bread mold), into six parts depending on the age of the cells; 1 hour, 3 hour, 9 hour, 15 hour, 21 hour and 27 hour old, respectively. This data submission reflects an RNAseq analysis of mRNA extracted from the 1 hour time-point. 1 mycelial mRNA profile of 1 hour growth (hyphal tip) from Neurospora crassa strain FGSC 2489, Data was generated using Illumina GAIIx.
Project description:Transcriptional profiling with next-generation sequencing methods demonstrated that a Neurospora crassa mutant with the three most highly expressed beta-glucosidase genes deleted had a transcriptional response to cellobiose similair to that of wild type N. crassa exposed to cellulose.
Project description:Purpose: To explore conservation of gene regulation by the transcription factor clr-2/clrB in Neurospora crassa and Aspergillus nidulans Methods: mRNA from wild type and clr-2/clrB mutants were collected after a culture shift from sucrose/glucose to Avicel (crystaline cellulose) or no carbon media Results: We show that N. crassa and A. nidulans have similair global transcriptional responses to Avicel, with several hundred genes showing specific induction, though the induced genes are more specifically targeted at cellulose for N. crassa and more targeted at hemicellulose and pectin for A. nidulans. clr-2/clrB has a conserved fundamental function in cellulose induction, though the mechanism has diverged. Misexpression of clr-2 is sufficeint for inducer free cellulase secretion in N. crassa, but neither clrB or heterologous clr-2 is sufficient for inducer free cellulase secretion in A. nidulans. Conclusions: Our study demonstrates a conserved and essential role in cellulose utilization for the transcription factor clr-2 in filamentous ascomycetes and demonstrates that manipulation of clr-2 expression can be used to control cellulase expression in some species. Biological triplicates of liquid culture N. crassa and A. nidulans were harvested at 4 hours and 6 hours, respectively, after a switch to media of interest. Global mRNA abundances from liquid cultures of N. crassa and A. nidulans were measured by sequencing on the Illumina Genome Analyzer IIx and HiSeq2000 platforms.
Project description:Quantitative acetyl-proteomics, a newly identified post-translational modification, is known to regulate transcriptional activity in different organisms. Neurospora crassa is a model ascomycete fungus maintained for biochemistry and molecular biology research; however, extensive studies of the functions of its acylation proteins have yet to be performed. In this study, using LC-MS/MS qualitative proteomics strategies, we identified 1909 modification sites on 940 proteins in N. crassa and analysed the functions of these proteins using GO enrichment, KEGG pathway, and subcellular location experiments. We classified the acetylation protein involvement in diverse pathways, and protein-protein interaction (PPI) network analysis further demonstrated that these proteins participate in diverse biological processes. In summary, our study comprehensively profiles the crosstalk of modified sites, and PPI among these proteins may form a complex network with both similar and distinct regulatory mechanisms, providing improved understanding of their biological functions in N. crassa.