Project description:To determine the genes directly and indirectly under the control of the Grainy-head homolog (GHH) transcription factor in Neurospora crassa Three different sample types (Aerial Hyphae & Conidia; Mycelia; or Whole Colonies) of both wild-type (FGSC #2489) and grainy-head homolog (FGSC #13563) strains of Neurospora crassa were subjected to transcriptome analyses to determine the genes differentially expressed in the ghh background compared to wild type.
Project description:Many fungi form complex three-dimensional fruiting bodies, within which the meiotic machinery for sexual spore production has been considered to be largely conserved over evolutionary time. Indeed, much of what we know about meiosis in plant and animal taxa has been deeply informed by studies of meiosis in Saccharomyces and Neurospora. Nevertheless, the genetic basis of fruiting body development and its regulation in relation to meiosis in fungi is barely known, even within the best studied multicellular fungal model Neurospora crassa. We characterized morphological development and genome-wide transcriptomics in the closely related species Neurospora crassa, Neurospora tetrasperma, and Neurospora discreta, across eight stages of sexual development. Despite diverse life histories within the genus, all three species produce vase-shaped perithecia. Transcriptome sequencing provided gene expression levels of 2479 orthologous genes among all three species. Expression of key meiosis genes and sporulation genes, corresponded to developmental differences among these Neurospora species during sexual development. Screening N. crassa knockout crosses of genes selected for their expression differences across species, eight genes, whose functions were previously unknown, are found to be critical for the successful formation of perithecia. The absence of these genes in mutant crosses resulted in either no perithecium formation or in arrested development at an early stage. Our results provide insight into the genetic basis of Neurospora sexual reproduction, which is also of great importance with regard to other multicelluar ascomycetes, including fungal pathogens closely related to Neurospora in the Sordariomycetes, such as Fusarium spp, Magnaporthe oryzae, and Nectria haematococca mRNA were sampled and compared from eight time points across sexual reproduction in three Neurospora species
Project description:RNA-seq from Neurospora crassa at 5 time points of light induction, with 2 replicates for each, totalling 10 samples RNA-seq from Neurospora crassa at 5 time points of light induction, with 2 replicates for each, totalling 10 samples
Project description:Facultative heterochromatin in the filamentous fungus Neurospora crassa is identified by the repressive histone mark H3K27me3 and is primarily subtelomeric, while constitutive heterochromatin, marked by the DIM-5-catalzyed H3K9me3, is found at centromeres, telomeres, and smaller dispersed regions. In strains lacking constitutive heterochromatin (e.g., Δdim-5), H3K27me2/3 relocalizes to the regions formerly marked by H3K9me3. H3K27me3 is catalyzed by the SET-7 histone methyltransferase subunit of the Polycomb Repressive Complex 2 (PRC2); another PRC2 member, Neurospora p55 (NPF) regulates subtelomeric H3K27me2/3. Despite the de-repression of >70 genes, a Δset-7 strain has no distinguishable phenotype. To investigate the facultative heterochromatin contribution to genome organization, we performed high-throughput “chromosome conformation capture” (Hi-C) on mutants with impacted H3K27me2/3 deposition. A Δset-7 strain has decreased inter-/intra-subtelomeric contacts among others; this pattern is mirrored in a Δnpf strain, which lacks subtelomeric H3K27me3. In a Δset-7 strain, telomere bundles were often uncoupled from the nuclear membrane and de-repressed genes were subtelomeric. The chromosome conformation of a Δset-7;Δdim-5 double mutant was similar to Δset-7, suggesting that facultative heterochromatin relocalization does not compensate for H3K9me3 loss and rescue the Neurospora genome organization in strains with defective constitutive heterochromatin. Overall design: Hi-C: We analyzed a total of three strains of Neurospora crassa by chromatin conformation capture followed by high throughput sequencing (Hi-C). Each strain has two replicates. Strains were grown from conidia ~4 hours, crosslinked, and conidia were made into spheroplasts. Genomic DNA from spheroplasts was digested with HindIII, ends were filled in with dNTPs (including biotin-conjugated dCTP), blunt ends were ligated, crosslinks were reversed, DNA was soniciated, ligation products were purified with streptavidin beads, and libraries were prepared for sequencing. The datasets of wild type strain, NMF39 - deposited in GEO sebmission GSE71024, serves as the reference for the mutants deficient in facultative heterochromatin formation (N4718 delta set-7, N4721 delta npf, and N5542 delta set-7;delta dim-5). ChIP-seq: We analyzed two strains of Neurospora crassa (control strain: WT N3752, test strain delta npf N4721) by Chromatin Immunoprecipitation for the histone mark H3K27me3. Strains were grown, crosslinked, lysed, the histone mark H3K27me3 was immunopurified, and associated DNA was sequenced). RNA-seq: We analyzed a total of five strains (four different genetic backgrounds) by poly-A+ mRNA-sequencing for gene expression changes by monitoring poly-adenylated messenger RNA levels. A wild type strain (N3752) serves as the reference strain, and the strains N4718 and N4730 (delta set-7), N4721 (delta npf), and N5916 (delta set-7;delta dim-5) served as the test strains. Please note that all python scripts (".py.txt" files) are provided as series supplementary file.
Project description:The protein kinase Ime2 is known to have an important role in meiosis in the yeast Saccharomyces cerevisiae. However, the Neurospora crassa IME2 homolog functions in self/nonself recognition as well as in the development of fungal sexual structures called protoperithecia. In N. crassa, protoperithecia are induced upon nitrogen starvation. We were interested in gene expression differences between an ime-2 deletion strain and a wild type strain, and due to the role of ime-2 during sexual development, carried out these arrays on media that was lacking in nitrogen. There is one condition and three samples in this experiment (wild type and Dime-2 strains grown on minimal media lacking nitrogen), and dye swaps were performed
Project description:Multi-targeting priming (MTP) for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences. We demonstrated superior performance of two MTPs compared to oligo-dT microarray profling and RNA tag sequencing the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development. Priming with MTPs in addition to oligo-dT resulted in higher sensitivity, a greater number of well-measured genes, more genes significantly differentially expressed, and a greater power to detect meager differences. Neurospora crassa mat A FGSC#2489 2 developmental stages and oligo(dT) primers.
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:Multi-targeting priming (MTP) for genome-wide gene expression assays provides selective targeting of multiple sequences and counter-selection against undesirable sequences. We demonstrated superior performance of two MTPs compared to oligo-dT microarray profling and RNA tag sequencing the response of Saccharomyces cerevisiae to nitrogen deficiency and profiling Neurospora crassa early sexual development. Priming with MTPs in addition to oligo-dT resulted in higher sensitivity, a greater number of well-measured genes, more genes significantly differentially expressed, and a greater power to detect meager differences. Overall design: Neurospora crassa mat A FGSC#2489 2 developmental stages and oligo(dT) primers.
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:Purpose: We explore gene expression changes when Neurospora crassa wild type responds to different carbon sources in Vogel's medium. Method: We obtained mRNA samples of Neurospora crassa WT in Vogel's minimal medium (VMM) with different carbon source and used RNA-seq technique to measure the trancriptome changes. Results: We identified many genes of transcription factors and enzymes that were up regulated or down regulated in response to the different carbon stimulation. Conclusion: Our data represents a systematic transcriptome profiling of filamentous fungi on different carbon source and identify COL-26 as a critical regulator in degradation of starch components. Overall design: mRNA profiles of cultures of WT after 4 hours of exposure to media of interest were generated by deep sequencing, in triplicate, using Illumina HiSeq2000. Soluble carbon sources are 2 mM and insoluble carbon source are 2% (w/v).