Browsing by Subject "Neurospora crassa"
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Item Assessing conserved function of conidiation regulators in two distantly related ascomycetes, Aspergillus nidulans and Neurospora crassa(2012-07-16) Chung, Da WoonConidiation is a common and critical asexual reproductive mode in fungi. The ascomycetes, the largest group in the kingdom Fungi undergo conidiation. The wide array of morphological difference in a conidiophore and conidial size, shape, and cellular organization demonstrates the importance of evolution in driving the morphological and functional diversity. An important unanswered question is how these conidiation processes evolve. We hypothesized that a conidiation regulatory pathway was present in the ancestral species, and became specialized in the extant species to lead to morphological and functional diversity. To address this hypothesis we assessed the conserved function of conidiation regulators in two distantly related ascomycetes, Aspergillus nidulans and Neurospora crassa. Using sequence similarity analysis, N. crassa orthologs were characterized to seven main conidiation regulatory genes in A. nidulans (fluG, flbC, flbD, abaA, wetA, medA, and stuA). Expression of the N. crassa orthologs complemented defective conidiation in the A. nidulans fluG, flbD, wetA, medA, and stuA mutants. In contrast, abaA and flbC and the N. crassa orthologs did not share conserved biochemical function. Taken in context of other recent studies of conidiation regulators, there are four distinct evolutionary patterns: (i) Non-homologous genes with analogous roles in conidiation (?brlA? and ?fl?), (ii) Orthologs with retained biochemical function that lack analogous role in conidiation (?fluG?, ?flbD?, and ?wetA?), (iii) Orthologs with retained biochemical function and analogous roles in conidiation (?medA? and ?stuA?), and (iv) Orthologs with biochemical function not conserved but with analogous roles in conidiation (?abaA? and ?flbC?). These studies set the stage for long-term studies of how evolution proceeded during the evolution of conidiation at different levels of phylogenetic diversity. An understanding of how evolutionary mechanisms shape the dynamics of developmental pathways will be significant for our understanding of fungal evolution of other novel adaptations such as pathogenesis.Item The Biogenesis of Small Interfering RNA in Neurospora Crassa(2013-01-16) Chang, Shwu-Shin; Yi Liu, Ph.D.RNA interference is a well-conserved gene silencing mechanism in eukaryotes. It regulates various biological processes including development, genome defense and heterochromatin formation. RNAi is initiated by the production of dsRNA, which is processed by Dicer to produce small interfering RNA (siRNA). In the filamentous fungus, Neurospora crassa, two types of siRNA have been characterized. One is involved in transgene-induced silencing, termed quelling; the other type is induced by DNA damage and functions to slow down protein translation after DNA damage. Both of these siRNAs originate from repetitive sequences in the Neurospora genome. We show that the components of the homologous recombination (HR) machinery are required to generate these types of small RNA specifically at repetitive regions. Furthermore, chromatin remodeling and DNA replication enzymes are required for efficient HR activity and small RNA production. Lastly, we show that the two small RNA pathways are mechanistically similar by demonstrating that quelling-induced siRNA can also be induced upon DNA damage. Our results suggest that the small RNA biogenesis machinery is recruited specifically to the repetitive loci after homologous recombination, which may result in the formation of aberrant DNA structures. dsRNA not only triggers the RNAi pathway, but also initiates a signaling cascade that results in activating the transcription of ~60 genes, including the RNAi components, in Neurospora. The function of the dsRNA activated genes suggests that RNAi is part of a broad ancient host defense response against viral and transposon infection. A genetic screen has been designed to identify the components involved in this dsRNA triggered transcriptional response; several mutants have been identified and characterized.Item Evolution of Genes and Gene Networks in Filamentous Fungi(2011-10-21) Greenwald, Charles JoaquinThe Pezizomycotina, commonly known as the filamentous fungi, are a diverse group of organisms that have a major impact on human life. The filamentous fungi diverged from a common ancestor approximately 200 ? 700 million years ago. Because of the diversity and the wealth of biological and genomic tools for the filamentous fungi it is possible to track the evolutionary history of genes and gene networks in these organisms. In this dissertation I focus on the evolution of two genes (lolC and lolD) in the LOL secondary metabolite gene cluster in Epichlo? and Neotyphodium genera, the evolution of the MAP kinase-signaling cascade in the filamentous fungi, the regulation of the gene networks involved in asexual development in Neurospora crassa, and the identification of two genes in the N. crassa asexual development gene network, acon-2 and acon-3. I find that lolC and lolD originated as an ancient duplication in the ancestor of the filamentous fungi, which were later recruited in the LOL gene cluster in the fungal endophyte lineage. In the MAP kinase-signaling cascade, I find that the MAPK component is the most central gene in the gene network. I also find that the MAPK signaling cascade originated as three copies in the ancestor to eukaryotes, an arrangement that is maintained in filamentous fungi. My observations of gene expression profiling during N. crassa asexual development show tissue specific expression of genes. Both the vegetative mycelium and the aerial hyphae contribute to the formation of macroconidiophores. Also, with the help of genomic tools recently developed by researchers in the filamentous fungal community, I identified NCU00478 and NCU07617 as the genes with mutations responsible for two aconidial strains of N. crassa, acon-2 and acon-3 respectively.Item Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase with group I intron RNAs(2002) Myers, Christopher Allan; Lambowitz, AlanItem Interaction of the Neurospora crassa mitochondrial tyrosyl-tRNA synthetase with group I introns(2002-05) Chen, Xin; Lambowitz, AlanThe Neurospora crassa mitochondrial tyrosyl-tRNA synthetase, or CYT-18 protein, functions in splicing by binding specifically to the group I intron catalytic core. To investigate how the CYT-18 promotes group I intron splicing, I used an Escherichia coli genetic assay based on the phage T4 td intron to test the ability of CYT-18 to suppress mutations that cause structural defects around its binding site in the P4-P6 domain of the group I intron catalytic core. My results show that most mutations at either P4 bp-1 or P6 bp-1 inhibit self-splicing, but can be suppressed by CYT-18. CYT-18 can compensate efficiently for mutations that disrupt base-pairing at either P4 bp-1 or P6 bp-1, for mutations at P6 bp-1 that disrupt the base-triple interaction with J3/4-3, and for nucleotide substitutions at either position that are predicted to be suboptimal for base stacking. Mutations at the junction of the P4-P6 stacked helices lead to grossly impaired tertiary-structure formation centered in the P4-P6 domain. CYT-18-suppressiable mutants bind the protein with increased Kd up to 79-fold while the koff values are within twofold. My results indicate that the P4-P6 junction is a linchpin region in group I intron and that CTY-18 binding induces the formation of the correct structure in this region, leading to folding of the group I intron catalytic core. Using a similar genetic system, I also show that the tertiary interactions of P5- L9 and P8-L2 are important for group I intron splicing. CYT-18 C-terminal domain is required for suppression of the mutations that disrupt these tertiary interactions. Together with previous RNA footprinting and modeling data, my results suggest that P8 in group I intron RNA might contact with regions in the C-terminal domain of the CYT-18 protein and that the interactions between CYT-18 and both P4-P6 and P3-P9 domains help stabilize the tertiary interactions in group I intron and promote the splicing of group I introns.Item Mapping Circadian Output Pathways in Neurospora crassa(2013-12-09) Bennett, Lindsay DanielleCircadian clocks are ubiquitous in eukaryotic organisms, providing the ability to anticipate regularly occurring stressful environmental changes. The molecular clock leads to a change in physiology of the organism such that it is prepared for predictable changes. While the external signals detected by the clock, as well as the molecular mechanism of clock components have been extensively characterized, less is known about how the clock manifests time of day information to the organism as a whole. Our lab has focused on identifying output pathways from the clock, using the model organism Neurospora crassa. We have previously demonstrated the circadian regulation of the conserved Mitogen Activated Protein Kinase (MAPK) OS-2 pathway, a homolog of the mammalian p38 pathway, and necessary for maintaining osmotic homeostasis in Neurospora. I present data indicating the circadian regulation of the 2 other MAPK pathways in Neurospora, the mammalian ERK1 and ERK2 like MAPKs, MAK-1 and MAK-2, and show that they are outputs of the clock. Furthermore, I identified around 500 genes that are mis-regulated when MAK-1 is deleted; greater than 25% of those genes are predicted to be clock-controlled. I demonstrated that the clock is signaling through the MAK-1 pathway to regulate 3 clock-controlled genes (ccgs) that encode proteins involved in several different biological processes including, stress response, cell wall formation, and mitochondrial phosphate transport. I established the circadian regulation of the transcript levels of 2 of the MAK-1 cascade components, mek-1 and mak-1. Additionally, I found that the accumulation of MEK-1 protein is clock-controlled, suggesting this is one mechanism by which the clock regulates the activity of MAK-1. Additional studies were carried out to elucidate the proteins that directly regulate the expression of mek-1 and mak-1; however, the mechanisms of direct clock control remain unclear and require further investigation. The finding that the circadian clock regulates all MAPK pathways in Neurospora, combined with the conservation of both the circadian clock and MAPK pathways in mammals provide compelling evidence that mammalian MAPK pathways are also regulated as clock output pathways to control circadian physiology. There is a strong link between aberrations in mammalian clocks, MAPKs, and disease, and therefore, an urgent need to further characterize the circadian regulation of the MAPK families, which will reveal new avenues for therapeutic treatments.Item The Mechanism of Double-Stranded RNA Response in Neurospora(2009-01-09) Choudhary, Swati; Liu, YiIn eukaryotic cells, recognition of double-stranded RNA (dsRNA) by the enzyme Dicer initiates the RNA interference (RNAi) pathway, resulting in post-transcriptional gene silencing. Argonaute proteins play a critical role in this conserved pathway, which is present in protists, fungi, plants and animals. In addition, dsRNA can trigger the interferon response as part of the immune response in vertebrates. In this study, we show that the production of dsRNA triggers the transcriptional induction of qde-2 (an Argonaute gene) and dcl-2 (a Dicer gene), two central components of the RNAi pathway in the filamentous fungus Neurospora crassa. The induction of QDE-2 by dsRNA is required for efficient gene silencing, indicating that this is a regulatory mechanism that allows the optimal function of the RNAi pathway. In addition, we demonstrate that Dicer proteins (DCLs) regulate QDE-2 post-transcriptionally, suggesting a role for DCLs or siRNA in QDE-2 accumulation. A genome-wide search revealed that additional RNAi components and homologs of antiviral and interferon-stimulated genes are also dsRNA-activated genes (DRAGs) in Neurospora. Our results suggest that the activation of the RNAi components is part of a broad ancient host defense response against viral and transposon infections. In order to understand the signaling mechanisms underlying this dsRNA response, we undertook a study of the dsRNA response elements (dsREs) in the promoter regions of qde-2 and other DRAGs. We demonstrate that different regions of the qde-2 promoter orchestrate early and late transcriptional induction in response to dsRNA. In the qde-2 promoter, a GC-rich element and downstream CAAT repeats were found to be important for the early response. In addition, the GC-rich dsRE was found in the promoters of other DRAGs, and was sufficient for dsRNA-induced transcriptional response. These results suggest that these DRAGs share the transcriptional induction pathway triggered by dsRNA. Finally, we demonstrate that QDE-2 contains an additional 10KDa N-terminal RGG domain, which is important for binding small interfering RNAs (siRNAs) and therefore required for its stability as well as efficient RNAi.Item The Mechanism of RNA Interference in Neurospora(2007-08-08) Maiti, Mekhala; Liu, YiIn the canonical RNA interference (RNAi) pathway, small-interfering RNA (siRNA) duplexes generated by Dicer are incorporated into the RNA-induced-silencing complex (RISC), and subsequently converted to single-stranded siRNA. Generation of single stranded siRNA is a pre-requisite for recognition and cleavage of the target mRNA by Argonaute. In biochemical experiments, Argonaute generates single-stranded siRNA by cleaving the passenger strand of the siRNA duplex. Mutational analysis of Neurospora homologue of Argonaute-2, known as Quelling Deficient -2 (QDE-2), revealed that the endonuclease activity of QDE-2 is required for the generation of singlestranded siRNA in vivo. Further biochemical studies to understand the mechanism for removal of the nicked passenger strand from siRNA duplex, led to the identification of a novel QDE-2 interacting protein (QIP) with a putative exonuclease domain. Disruption of qip led to the impairment of RNAi and most of the siRNAs were accumulated in nickedduplex form. Furthermore, QIP functions as an exonuclease to remove the cleaved passenger strand in a QDE-2 dependent manner. Thus, the cleavage of the passenger strand by QDE2 and its subsequent removal by QIP are critical biochemical steps in Neurospora RNAi pathway. Quelling, an RNAi related phenomenon in Neurospora, is induced by multiple copies of transgene. It was proposed that QDE-1 (a RNA dependent RNA polymerase, RdRp) and QDE-3 (a RecQ helicase) functions in quelling pathway by generating double-stranded RNA (dsRNA) from transgenes. To further understand the importance of QDE-1 and QDE-3, quelling assays were performed in the qde-1ko and qde-3ko strains. In contrast to previous results, the requirement of QDE-1 and QDE-3 was bypassed when the transgene copy number was high. Moreover, gene silencing analyses using strains lacking all potential RdRps suggested that unlike in C.elegans and Arabidopsis, the amplification of secondary dsRNA or siRNA is largely absent in Neurospora. The search for potential regulatory mechanisms of RNAi components in Neurospora led to the identification of a dsRNA response pathway. Two key components of the Neurospora RNAi pathway, qde2 and dicer like protein-2 (dcl-2), are induced by dsRNA at transcriptional and posttranscriptional level. The induction of QDE-2 is required for efficient gene silencing, indicating the importance of this regulatory mechanism in RNAi pathway.Item Sexual Development and Meiotic Silencing in Neurospora crassa(2014-07-29) Suesc?n Torres, Ana VictoriaMeiotic silencing refers to the mechanism of silencing genes or chromosomes without a homologous counterpart (unpaired) during meiotic prophase I. Meiotic silencing has been described in several eukaryotes, including humans. Failure to complete meiotic silencing may be detrimental to the organism. There is a necessity for understanding the regulation of the process. Neurospora is a powerful model system to study gene silencing phenomena. Numerous genes have been determined to be involved in meiotic silencing in Neurospora; however, very little is known about the molecular mechanisms underlying the process. To understand the regulation of meiotic silencing, it is required to combine different approaches such as genetics, proteomics and biochemical analyses. There is a need for introducing biochemical approaches to the study of meiotic silencing and other processes occurring during sexual development in N. crassa. However, protein extraction from sexual tissue is challenging due to the mechanical difficulties associated with disruption of sexual structure. I standardized a strategy that optimizes protein extraction from sexual tissue. Using this strategy, I studied protein-protein interactions among components of the meiotic silencing machinery and determine the proteome of sexual development. I identified new protein interactions during meiotic silencing in N. crassa, and established protein-binding partners for the suppressor of meiotic silencing SMS-5. These interacting partners, PAF400 and Pianissimo represent new molecular components involved in the nuclear initial stage of the meiotic silencing mechanism. Interactions between SMS-5, PAF400, and Pianissimo may represent the connection between chromatin remodeling, DNA repair, signaling transduction pathways and meiotic silencing. I describe the experiments and data analyses used to develop a comprehensive proteomics data set and a functional catalogue for N. crassa sexual development. I used a global proteomics approach and comparative protein functional analysis to investigate the potential molecular differences between two stages of sexual development in filamentous fungi. The data show that secondary metabolites biosynthesis and cellulase activity are required in fruiting body maturation. N. crassa functional catalogue of sexual development proteins will serve as a reference tool for further studies related to sexual development not only in N. crassa, but also in other filamentous ascomycetes.Item Sugar sensing and regulation of conidiation in Neurospora crassa(Texas A&M University, 2004-11-15) Xie, XinThe orange bread mold Neurospora crassa is a useful model for the study of filamentous fungi. One of the asexual reproduction cycles in N. crassa, macroconidiation, can be induced by several environmental cues, including glucose starvation. The rco-3 gene is a regulator of sugar transport and macroconidiation in N. crassa and was proposed to encode a sugar sensor (Madi et al., 1997). To identify genes that are functionally related to RCO-3, three distinct suppressors of the sorbose resistance phenotype of rco-3 were isolated and characterized. The dgr-1 mutant phenotypically resembles rco-3 and may be part of the rco-3 signaling pathway. Epistatic relationship among rco-3, dgr-1 and the suppressors were carried out by analyzing rco-3; dgr-1 and sup; dgr-1 double mutants. These analyses indicate that rco-3 is epistatic to dgr-1. A cDNA microarray containing 1363 N. crassa genes was generated to examine the transcriptional response of wild type cells grown in the presence of glucose or starved for glucose for two hours. Comparing N. crassa profiling data with the published diauxic shift data from S. cerevisiae (DeRisi et al., 1997) revealed that S. cerevisiae and N. crassa share a similar, but not identical, transcriptional response pattern for genes belonging to central carbon metabolism. The microarray results indicate that N. crassa utilizes glucose through fermentation and respiration simultaneously in aerobic culture, a finding that is consistent with previous measurements of ethanol production and enzyme activities in N. crassa. The same microarray was used to examine the transcriptional response to glucose status in rco-3 and dgr-1 mutants. The two mutants display similar expression patterns for most of the genes on the microarray supporting a close functional relationship between them. In addition, I identified a high affinity glucose transport gene in N. crassa, whose transcription is under the control of glucose, rco-3 and dgr-1.Item Translational Control Mechanisms Analyzed in Neurospora crassa(2013-07-11) Wei, JiajieThe Neurospora crassa arg-2 gene encodes the small subunit of carbamoyl phosphate synthetase, the first enzyme in fungal arginine (Arg) biosynthesis. The arginine attenuator peptide (AAP), specified by an upstream open reading frame (uORF), stalls ribosomes at its termination codon in response Arg to control the translation of arg-2. In project 1, the effect of AAP and Arg on ribosome peptidyl transferase center (PTC) activity was analyzed in N. crassa and wheat germ cell-free translation extracts using the transfer of nascent AAP to puromycin as an assay. The results show that inhibition of PTC activity by the AAP and Arg is the basis for the AAP?s function. The mode of PTC inhibition appears unusual because neither a specific amino acid nor a specific nascent peptide chain length was required for AAP to function. In eukaryotic translation initiation, the stringency of start codon selection impacts initiation efficiencies at AUG codons in different contexts and at near-cognate codons (NCCs) that differ from AUG by a single nucleotide. In project 2, a codon-optimized firefly luciferase reporter was used to examine the stringency of start codon selection in N. crassa. In vivo and in vitro results indicated that the hierarchy of initiation in N. crassa is similar to that in human cells. The preferred context was more important for efficient initiation from NCCs than from AUG. In project 3, the use of NCCs was also specifically examined for the N. crassa cpc-1 gene. cpc-1 and Saccharomyces cerevisiae GCN4 are homologs specifying a transcription activator, which drives the primary transcriptional response to amino acid starvation. In vitro studies showed that uORF1 and uORF2 in cpc-1 are functionally analogous to uORF1 and uORF4 in GCN4. uORF1 promotes reinitiation at downstream start codons. uORF2 inhibits translation from the main cpc-1 start codon. Four NCCs in the CPC1 reading frame and upstream of uORF2 can also be used for translation initiation. In summary, we explored uORF-mediated translational regulation and the use of NCCs as initiation codons. Taken together, these studies establish N. crassa as a model system to examine mechanisms contributing to translational control including initiation and termination.