Project description:Histones are not statically embedded, but are constantly exchanged outside of DNA replication. This study reports the characterization and validation of a histone turnover reporter strain of Neurospora crassa, and the method employed. This strain utilizes FLAG-tagged histone H3 under the control of a light-inducible promoter. This study also preliminarily explores histone turnover defects at constitutive heterochromatin with the loss of the heterochromatin proteins DIM-2, HDA-1, DIM-5, and HPO.
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: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 Three developmental stages and two different primers used for reverse transcription: mycelium oligo(dT) M1 protoperithecia oligo(dT) PP1 perithecia oligo(dT) PT1 mycelium oligo(dT)+ Multi-Targeted Primer [MTP] (M2) protoperithecia oligo(dT)+ MTP (PP2) perithecia oligo(dT)+ MTP (PT2)
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.
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: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: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.
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