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new anti-inflammatory metabolites by microbial transformation of medrysone.microbial transformation of the anti-inflammatory steroid medrysone (1) was carried out for the first time with the filamentous fungi cunninghamella blakesleeana (atcc 8688a), neurospora crassa (atcc 18419), and rhizopus stolonifer (tsy 0471). the objective was to evaluate the anti-inflammatory potential of the substrate (1) and its metabolites. this yielded seven new metabolites, 14α-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (2), 6β-hydroxy-6α-methylpregn-4-ene-3,11,20-trione (3), 15β-hydroxy ...201627104348
molecular dissection of so (soft) protein in stress-induced aggregation and cell-to-cell interactive functions in filamentous fungal multicellularity.filamentous fungi grow by organizing multicellularity through hyphal compartmentalization and cell fusion. so (soft) protein, which was originally identified in neurospora crassa, plays distinct functional roles in cell-to-cell interactions, such as septal plugging and cell fusion. we previously reported that aoso, an aspergillus oryzae so homologue, forms aggregates at the septal pore in response to stress, as well as upon hyphal wounding. however, the functional regions that mediate the multic ...201627109373
experimental evolution reveals that high relatedness protects multicellular cooperation from cheaters.in multicellular organisms, there is a potential risk that cheating mutants gain access to the germline. development from a single-celled zygote resets relatedness among cells to its maximum value each generation, which should accomplish segregation of cheating mutants from non-cheaters and thereby protect multicellular cooperation. here we provide the crucial direct comparison between high- and low-relatedness conditions to test this hypothesis. we allow two variants of the fungus neurospora cr ...201627139112
backbone and side-chain (1)h, (13)c, and (15)n chemical shift assignments for the apo-form of the lytic polysaccharide monooxygenase nclpmo9c.the apo-form of the 23.3 kda catalytic domain of the aa9 family lytic polysaccharide monooxygenase nclpmo9c from neurospora crassa has been isotopically labeled and recombinantly expressed in pichia pastoris. in this paper, we report the (1)h, (13)c, and (15)n chemical shift assignments of this lpmo.201627147444
recombination-independent recognition of dna homology for repeat-induced point mutation (rip) is modulated by the underlying nucleotide sequence.haploid germline nuclei of many filamentous fungi have the capacity to detect homologous nucleotide sequences present on the same or different chromosomes. once recognized, such sequences can undergo cytosine methylation or cytosine-to-thymine mutation specifically over the extent of shared homology. in neurospora crassa this process is known as repeat-induced point mutation (rip). previously, we showed that rip did not require mei-3, the only reca homolog in neurospora, and that it could detect ...201627148882
hyphal chemotropism in fungal pathogenicity.the ability to grow as filamentous hyphae defines the lifestyle of fungi. hyphae are exposed to a variety of chemical stimuli such as nutrients or signal molecules from mating partners and host organisms. how fungi sense and process this chemical information to steer hyphal growth is poorly understood. saccharomyces cerevisiae and neurospora crassa have served as genetic models for the identification of cellular components functioning in chemotropism. a recent study in the pathogen fusarium oxys ...201627150623
histone h1 limits dna methylation in neurospora crassa.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 h1-3xflag ...201627172195
salt-responsive lytic polysaccharide monooxygenases from the mangrove fungus pestalotiopsis sp. nci6.lytic polysaccharide monooxygenases (lpmos) belong to the "auxiliary activities (aa)" enzyme class of the cazy database. they are known to strongly improve the saccharification process and boost soluble sugar yields from lignocellulosic biomass, which is a key step in the efficient production of sustainable economic biofuels. to date, most lpmos have been characterized from terrestrial fungi, but novel fungal lpmos isolated from more extreme environments such as an estuary mangrove ecosystem cou ...201627213015
gln3 is a main regulator of nitrogen assimilation in candida glabrata.after candida albicans, the yeast candida glabrata ranks second as an aetiological agent of candidaemia and is the most frequently encountered non-candida albicans species in patients with invasive candidiasis. transcriptome analysis in c. albicans, c. glabrata and cryptoccocus neoformans has revealed that, when engulfed by macrophages, these yeasts upregulate genes involved in nutrient acquisition, including nitrogen transporters such as the general amino acid permease gap1, the dicarboxylic am ...201627222014
neurospora crassa tox-1 gene encodes a ph- and temperature-tolerant mini-cellulase.cellulases that endure extreme conditions are essential in various industrial sectors. this study reports a mini-cellulase gene tox-1 from neurospora crassa. the gene tox-1 was cloned in escherichia coli after chimerization with the yebf gene and substitutions of certain isoleucine and valine with leucine residues. the yeast transformants could grow on rice straw-agar medium. the 44-amino acid peptide and its two mutant variants displayed potent cellulase activities in congo red assay and enzyma ...201627229865
pre-mrna splicing is modulated by antifungal drugs in the filamentous fungus neurospora crassa.for this study, we sought to identify pre-mrna processing events modulated by changes in extracellular ph, inorganic phosphate, and antifungal drugs. we examined genes with at least four putative introns whose transcriptional level responded to these effectors. we showed that the intron retention levels of genes encoding asparagine synthetase 2, c6-zinc finger regulator (fluffy), and a farnesyltransferase respond to amphotericin b, ketoconazole, and other effectors. in general, the assayed antif ...201627239448
cell wall composition plays a key role on sensitivity of filamentous fungi to chitosan.chitosan antifungal activity has been reported for both filamentous fungi and yeast. previous studies have shown fungal plasma membrane as main chitosan target. however, the role of the fungal cell wall (cw) in their response to chitosan is unknown. we show that cell wall regeneration in neurospora crassa (chitosan sensitive) protoplasts protects them from chitosan damage. caspofungin, a β-1,3-glucan synthase inhibitor, showed a synergistic antifungal effect with chitosan for n. crassa but not f ...201627259000
neurospora chromosomes are organized by blocks of importin alpha-dependent heterochromatin that are largely independent of h3k9me3.eukaryotic genomes are organized into chromatin domains with three-dimensional arrangements that presumably result from interactions between the chromatin constituents-proteins, dna, and rna-within the physical constraints of the nucleus. we used chromosome conformation capture (3c) followed by high-throughput sequencing (hi-c) with wild-type and mutant strains of neurospora crassa to gain insight into the role of heterochromatin in the organization and function of the genome. we tested the role ...201627260477
involvement of mak-1 and mak-2 map kinases in cell wall integrity in neurospora crassa.among three mapk disruptants of neurospora crassa, δmak-1 was sensitive and δmak-2 was hypersensitive to micafungin, a beta-1,3-glucan synthase inhibitor, than the wild-type or δos-2 strains. we identified six micafungin-inducible genes that are involved in cell wall integrity (cwi) and found that mak-1 regulated the transcription of non-anchored cell wall protein gene, ncw-1, and the beta-1,3-endoglucanase gene, bgt-2, whereas mak-2 controlled the expression of the glycosylhydrolase-like protei ...201627268441
an epichloë festucae homologue of mob3, a component of the stripak complex, is required for the establishment of a mutualistic symbiotic interaction with lolium perenne.in both sordaria macrospora and neurospora crassa, components of the conserved stripak (striatin-interacting phosphatase and kinase) complex regulate cell-cell fusion, hyphal network development and fruiting body formation. interestingly, a number of epichloë festucae genes that are required for hyphal cell-cell fusion, such as noxa, noxr, proa, mpka and mkka, are also required for the establishment of a mutualistic symbiotic interaction with lolium perenne. to determine whether mobc, a homologu ...201627277141
rbdb, a rhomboid protease critical for srebp activation and virulence in aspergillus fumigatus.srebp transcription factors play a critical role in fungal virulence; however, the mechanisms of sterol regulatory element binding protein (srebp) activation in pathogenic fungi remains ill-defined. screening of the neurospora crassa whole-genome deletion collection for genes involved in hypoxia responses identified a gene for an uncharacterized rhomboid protease homolog, rbdb, required for growth under hypoxic conditions. loss of rbdb in aspergillus fumigatus also inhibited growth under hypoxic ...201727303716
crosses heterozygous for hybrid neurospora translocation strains show transmission ratio distortion disfavoring homokaryotic ascospores made following alternate segregation.by introgressing neurospora crassa translocations into n. tetrasperma, we constructed heterokaryons bearing haploid nuclei of opposite mating types, and either the translocation and normal sequence chromosomes (i.e., [t + n]) or a duplication and its complementary deficiency (i.e., [dp + df]). the [t + n] heterokaryons result from alternate segregation of homologous centromeres, whereas adjacent-1 segregation generates [dp + df]. self-cross of either heterokaryon produces [t + n] and [dp + df] p ...201627317785
structure of the frequency-interacting rna helicase: a protein interaction hub for the circadian clock.in the neurospora crassa circadian clock, a protein complex of frequency (frq), casein kinase 1a (ck1a), and the frq-interacting rna helicase (frh) rhythmically represses gene expression by the white-collar complex (wcc). frh crystal structures in several conformations and bound to adp/rna reveal differences between frh and the yeast homolog mtr4 that clarify the distinct role of frh in the clock. the frq-interacting region at the frh n-terminus has variable structure in the absence of frq a kno ...201627340124
spiral-based microfluidic device for long-term time course imaging of neurospora crassa with single nucleus resolution.real-time imaging of fluorescent reporters plays a critical role in elucidating fundamental molecular mechanisms including circadian rhythms in the model filamentous fungus, neurospora crassa. however, monitoring n. crassa for an extended period of time with single nucleus resolution is a technically challenging task due to hyphal growth that rapidly moves beyond a region of interest during microscopy experiments. in this report, we have proposed a two-dimensional spiral-based microfluidic platf ...201627345439
light governs asexual differentiation in the grey mould fungus botrytis cinerea via the putative transcription factor bcltf2.botrytis cinerea is a plant pathogenic fungus known for its utilization of light as environmental cue to regulate asexual differentiation: conidia are formed in the light, while sclerotia are formed in the dark. as no orthologues of known regulators of conidiation (e.g., aspergillus nidulans brla, neurospora crassa fl) exist in the leotiomycetes, we initiated a de novo approach to identify the functional counterpart in b. cinerea. the search revealed the light-responsive c2h2 transcription facto ...201627347834
homology directed repair is unaffected by the absence of sirnas in drosophila melanogaster.small interfering rnas (sirnas) defend the organism against harmful transcripts from exogenous (e.g. viral) or endogenous (e.g. transposons) sources. recent publications describe the production of sirnas induced by dna double-strand breaks (dsb) in neurospora crassa, arabidopsis thaliana, drosophila melanogaster and human cells, which suggests a conserved function. a current hypothesis is that break-induced small rnas ensure efficient homologous recombination (hr). however, biogenesis of sirnas ...201627353331
regulation of neurospora crassa cell wall remodeling via the cot-1 pathway is mediated by gul-1.impairment of the neurospora crassa nuclear dbf2-related kinase-encoding gene cot-1 results in pleiotropic effects, including abnormally thick hyphal cell walls and septa. an increase in the transcript abundance of genes encoding chitin and glucan synthases and the chitinase gh18-5, but not the cell wall integrity pathway transcription factor rlm-1, accompany the phenotypic changes observed. deletion of chs-5 or chs-7 in a cot-1 background results in a reduction of hyperbranching frequency chara ...201727363849
a proteomic and genetic analysis of the neurospora crassa conidia cell wall proteins identifies two glycosyl hydrolases involved in cell wall remodeling.a proteomic analysis of the conidial cell wall identified 35 cell wall proteins. a comparison with the proteome of the vegetative hyphae showed that 16 cell wall proteins were shared, and that these shared cell wall proteins were cell wall biosynthetic proteins or cell wall structural proteins. deletion mutants for 34 of the genes were analyzed for phenotypes indicative of conidial cell wall defects. mutants for two cell wall glycosyl hydrolases, the cgl-1 β-1,3-glucanase (ncu07523) and the nag- ...201627381444
structure and function of the yeast listerin (ltn1) conserved n-terminal domain in binding to stalled 60s ribosomal subunits.the ltn1 e3 ligase (listerin in mammals) has emerged as a paradigm for understanding ribosome-associated ubiquitylation. ltn1 binds to 60s ribosomal subunits to ubiquitylate nascent polypeptides that become stalled during synthesis; among ltn1's substrates are aberrant products of mrna lacking stop codons [nonstop translation products (nsps)]. here, we report the reconstitution of nsp ubiquitylation in neurospora crassa cell extracts. upon translation in vitro, ribosome-stalled nsps were ubiquit ...201627385828
purification and some molecular properties of rice germ calmodulin.calmodulin (cam) from rice germ (oryza sativa l) was purified to homogeneity by hydrophobic interaction chromatography and gel filtration. the protein showed a single spot by sds-page. this purified protein had multiple absorption maxima at 276~279, 268, 265, 258, and 253 nm. like other plant cam, the protein contained one mole of tm3lys, cysteine, and tyrosine, and tryptophan was not detected. hydrophobic properties of rice germ, spinach, and neurospora crassa cam were directly tested by an hpl ...199827388650
identification and functional analysis of endogenous nitric oxide in a filamentous fungus.in spite of its prevalence in animals and plants, endogenous nitric oxide (no) has been rarely reported in fungi. we present here our observations on production of intracellular no and its possible roles during development of neurospora crassa, a model filamentous fungus. intracellular no was detected in hypha 8-16 hours after incubation in vogel's minimal liquid media and conidiophores during conidiation using a fluorescent indicator (daf-fm diacetate). treatment with cptio, an no scavenger, si ...201627425220
qirnapredictor: a novel computational program for the prediction of qirnas in neurospora crassa.recently, a new type of small interfering rnas (qirnas) of typically 20~21 nucleotides was found in neurospora crassa and rice and has been shown to regulate gene silencing in the dna damage response. identification of qirnas is fundamental for dissecting regulatory functions and molecular mechanisms. in contrast to other expensive and time-consuming experimental methods, the computational prediction of qirnas is a conveniently rapid method for gaining valuable information for a subsequent exper ...201627428111
transcriptional regulation using the q system in transgenic zebrafish.methods to label cell populations selectively or to modify their gene expression are critical tools in the study of developmental or physiological processes in vivo. a variety of approaches have been applied to the zebrafish model, capitalizing on tol2 transposition to generate transgenic lines with high efficiency. here we describe the adoption of the q system of neurospora crassa, which includes the qf transcription factor and the upstream activating sequence (quas) to which it binds. these co ...201627443927
modulation of circadian gene expression and metabolic compensation by the rco-1 corepressor of neurospora crassa.neurospora crassa is a model organism for the study of circadian clocks, molecular machineries that confer ∼24-hr rhythms to different processes at the cellular and organismal levels. the frequency (frq) protein is a central component of the neurospora core clock, a transcription/translation negative feedback loop that controls genome-wide rhythmic gene expression. a genetic screen aimed at determining new components involved in the latter process identified regulation of conidiation 1 (rco-1), ...201627449058
regulation of neurospora catalase-3 by global heterochromatin formation and its proximal heterochromatin region.catalase-3 (cat-3) constitutes the main catalase activity in growing hyphae of neurospora crassa, and its activity increases during exponential growth or is induced under different stress conditions. although extensive progress has been made to identify catalase regulators, the regulation mechanism of cat-3 at the chromatin level still remains unclear. here, we aim at investigating the molecular regulation mechanisms of cat-3 at the chromatin level. we found that cat-3 protein levels increased i ...201627458122
circadian clock regulation of mrna translation through eukaryotic elongation factor eef-2.the circadian clock has a profound effect on gene regulation, controlling rhythmic transcript accumulation for up to half of expressed genes in eukaryotes. evidence also exists for clock control of mrna translation, but the extent and mechanisms for this regulation are not known. in neurospora crassa, the circadian clock generates daily rhythms in the activation of conserved mitogen-activated protein kinase (mapk) pathways when cells are grown in constant conditions, including rhythmic activatio ...201627506798
suppressor of fusion, a fusarium oxysporum homolog of ndt80, is required for nutrient-dependent regulation of anastomosis.heterokaryon formation is an essential step in asexual recombination in fusarium oxysporum. filamentous fungi have an elaborate nonself recognition machinery to prevent formation and proliferation of heterokaryotic cells, called heterokaryon incompatibility (hi). in f. oxysporum the regulation of this machinery is not well understood. in neurospora crassa, vib-1, a putative transcription factor of the p53-like ndt80 family of transcription factors, has been identified as global regulator of hi. ...201627531696
the introduction of the fungal d-galacturonate pathway enables the consumption of d-galacturonic acid by saccharomyces cerevisiae.pectin-rich wastes, such as citrus pulp and sugar beet pulp, are produced in considerable amounts by the juice and sugar industry and could be used as raw materials for biorefineries. one possible process in such biorefineries is the hydrolysis of these wastes and the subsequent production of ethanol. however, the ethanol-producing organism of choice, saccharomyces cerevisiae, is not able to catabolize d-galacturonic acid, which represents a considerable amount of the sugars in the hydrolysate, ...201627538689
molecular components of the neurospora crassa ph signaling pathway and their regulation by ph and the pac-3 transcription factor.environmental ph induces a stress response triggering a signaling pathway whose components have been identified and characterized in several fungi. neurospora crassa shares all six components of the aspergillus nidulans ph signaling pathway, and we investigate here their regulation during an alkaline ph stress response. we show that the n. crassa pal mutant strains, with the exception of δpal-9, which is the a. nidulans pali homolog, exhibit low conidiation and are unable to grow at alkaline ph. ...201627557053
deep sequencing of mycovirus-derived small rnas from botrytis species.rna silencing is an ancient regulatory mechanism operating in all eukaryotic cells. in fungi, it was first discovered in neurospora crassa, although its potential as a defence mechanism against mycoviruses was first reported in cryphonectria parasitica and, later, in several fungal species. there is little evidence of the antiviral potential of rna silencing in the phytopathogenic species of the fungal genus botrytis. moreover, little is known about the rna silencing components in these fungi, a ...201627578449
a lytic polysaccharide monooxygenase with broad xyloglucan specificity from the brown-rot fungus gloeophyllum trabeum and its action on cellulose-xyloglucan complexes.fungi secrete a set of glycoside hydrolases and lytic polysaccharide monooxygenases (lpmos) to degrade plant polysaccharides. brown-rot fungi, such as gloeophyllum trabeum, tend to have few lpmos, and information on these enzymes is scarce. the genome of g. trabeum encodes four auxiliary activity 9 (aa9) lpmos (gtlpmo9s), whose coding sequences were amplified from cdna. due to alternative splicing, two variants of gtlpmo9a seem to be produced, a single-domain variant, gtlpmo9a-1, and a longer va ...201627590806
functional expression and characterization of a glucuronoyl esterase from the fungus neurospora crassa: identification of novel consensus sequences containing the catalytic triad.the complete hydrolysis of lignocellulose requires the actions of a variety of enzymes, including those that cleave the linkage between lignin and hemicellulose. the enzyme glucuronoyl esterase (ge) that constitutes a novel family of carbohydrate esterases, ce15, has been shown to display a unique ability to cleave the ester linkage between lignin alcohols and xylan-bound 4-o-methyl-d-glucuronic acid of hemicellulose. we herein report identification, expression, and functional characterization o ...201627600355
recombination-independent recognition of dna homology for repeat-induced point mutation.numerous cytogenetic observations have shown that homologous chromosomes (or individual chromosomal loci) can engage in specific pairing interactions in the apparent absence of dna breakage and recombination, suggesting that canonical recombination-mediated mechanisms may not be the only option for sensing dna/dna homology. one proposed mechanism for such recombination-independent homology recognition involves direct contacts between intact double-stranded dna molecules. the strongest in vivo ev ...201627628707
controlled growth of nanostructured biotemplates with cobalt and nitrogen codoping as a binderless lithium-ion battery anode.biomass can serve as a sustainable template for the synthesis of carbon materials but is limited by the intrinsic properties of the precursor organism. in this study we demonstrate that the properties of a fungal biotemplate can be tuned during cultivation, establishing a new electrode manufacturing process and ultimately improving the electrochemical performance of the biomass-derived electrode. more specifically, the carbon/nitrogen ratio of neurospora crassa mycelia mats was shifted by 5-fold ...201627636014
dual chromatin recognition by the histone deacetylase complex hchc is required for proper dna methylation in neurospora crassa.dna methylation, heterochromatin protein 1 (hp1), histone h3 lysine 9 (h3k9) methylation, histone deacetylation, and highly repeated sequences are prototypical heterochromatic features, but their interrelationships are not fully understood. prior work showed that h3k9 methylation directs dna methylation and histone deacetylation via hp1 in neurospora crassa and that the histone deacetylase complex hchc is required for proper dna methylation. the complex consists of the chromodomain proteins hp1 ...201627681634
epigenetic control of phenotypic plasticity in the filamentous fungus neurospora crassa.phenotypic plasticity is the ability of a genotype to produce different phenotypes under different environmental or developmental conditions. phenotypic plasticity is a ubiquitous feature of living organisms, and is typically based on variable patterns of gene expression. however, the mechanisms by which gene expression is influenced and regulated during plastic responses are poorly understood in most organisms. while modifications to dna and histone proteins have been implicated as likely candi ...201627694114
internalization of heterologous sugar transporters by endogenous α-arrestins in the yeast saccharomyces cerevisiae.when expressed in saccharomyces cerevisiae using either of two constitutive yeast promoters (pgk1prom and ccw12prom), the transporters cdt-1 and cdt-2 from the filamentous fungus neurospora crassa are able to catalyze, respectively, active transport and facilitated diffusion of cellobiose (and, for cdt-2, also xylan and its derivatives). in s. cerevisiae, endogenous permeases are removed from the plasma membrane by clathrin-mediated endocytosis and are marked for internalization through ubiquiti ...201627694235
accumulation of specific sterol precursors targets a map kinase cascade mediating cell-cell recognition and fusion.sterols are vital components of eukaryotic cell membranes. defects in sterol biosynthesis, which result in the accumulation of precursor molecules, are commonly associated with cellular disorders and disease. however, the effects of these sterol precursors on the metabolism, signaling, and behavior of cells are only poorly understood. in this study, we show that the accumulation of only ergosterol precursors with a conjugated double bond in their aliphatic side chain specifically disrupts cell-c ...201627708165
expression of two novel β-glucosidases from chaetomium atrobrunneum in trichoderma reesei and characterization of the heterologous protein products.two novel gh3 family thermostable β-glucosidases from the filamentous fungus chaetomium atrobrunneum (cel3a and cel3b) were expressed in trichoderma reesei, purified by two-step ion exchange chromatography, and characterized. both enzymes were active over a wide range of ph as compared to neurospora crassa β-glucosidase gh3-3, which was also expressed in t. reesei and purified. the optimum temperature of both c. atrobrunneum enzymes was around 60 °c at ph 5, and both enzymes had better thermal a ...201627714589
the q-system: a versatile expression system for drosophila.binary expression systems are flexible and versatile genetic tools in drosophila. the q-system is a recently developed repressible binary expression system that offers new possibilities for transgene expression and genetic manipulations. in this review chapter, we focus on current state-of-the-art q-system tools and reagents. we also discuss in vivo applications of the q-system, together with gal4/uas and lexa/lexaop systems, for simultaneous expression of multiple effectors, intersectional labe ...201627730575
the hsp90 co-chaperones sti1, aha1, and p23 regulate adaptive responses to antifungal azoles.heat shock protein 90 (hsp90) is essential for tumor progression in humans and drug resistance in fungi. however, the roles of its many co-chaperones in antifungal resistance are unknown. in this study, by susceptibility test of neurospora crassa mutants lacking each of 18 hsp90/calcineurin system member genes (including 8 hsp90 co-chaperone genes) to antifungal drugs and other stresses, we demonstrate that the hsp90 co-chaperones sti1 (hop1 in yeast), aha1, and p23 (sba1 in yeast) were required ...201627761133
fungal sex: the ascomycota.this article provides an overview of sexual reproduction in the ascomycetes, a phylum of fungi that is named after the specialized sacs or "asci" that hold the sexual spores. they have therefore also been referred to as the sac fungi due to these characteristic structures that typically contain four to eight ascospores. ascomycetes are morphologically diverse and include single-celled yeasts, filamentous fungi, and more complex cup fungi. the sexual cycles of many species, including those of the ...201627763253
synchronizing stochastic circadian oscillators in single cells of neurospora crassa.the synchronization of stochastic coupled oscillators is a central problem in physics and an emerging problem in biology, particularly in the context of circadian rhythms. most measurements on the biological clock are made at the macroscopic level of millions of cells. here measurements are made on the oscillators in single cells of the model fungal system, neurospora crassa, with droplet microfluidics and the use of a fluorescent recorder hooked up to a promoter on a clock controlled gene-2 (cc ...201627786253
identification of critical amino acid residues and functional conservation of the neurospora crassa and rattus norvegicus orthologues of neuronal calcium sensor-1.neuronal calcium sensor-1 (ncs-1) is a member of neuronal calcium sensor family of proteins consisting of an amino terminal myristoylation domain and four conserved calcium (ca(2+)) binding ef-hand domains. we performed site-directed mutational analysis of three key amino acid residues that are glycine in the conserved site for the n-terminal myristoylation, a conserved glutamic acid residue responsible for ca(2+) binding in the third ef-hand (ef3), and an unusual non-conserved amino acid argini ...201627796528
reorganization of plasma membrane lipid domains during conidial germination.neurospora crassa, a filamentous fungus, in the unicellular conidial stage has ideal features to study sphingolipid (sl)-enriched domains, which are implicated in fundamental cellular processes ranging from antifungal resistance to apoptosis. several changes in lipid metabolism and in the membrane composition of n. crassa occur during spore germination. however, the biophysical impact of those changes is unknown. thus, a biophysical study of n. crassa plasma membrane, particularly sl-enriched do ...201727815222
analysis of the association between codon optimality and mrna stability in schizosaccharomyces pombe.recent experiments have shown that codon optimality is a major determinant of mrna stability in saccharomyces cerevisiae and that this phenomenon may be conserved in escherichia coli and some metazoans, although work in neurospora crassa is not consistent with this model.201627825301
short communication: conversion of lactose and whey into lactic acid by engineered yeast.lactose is often considered an unwanted and wasted byproduct, particularly lactose trapped in acid whey from yogurt production. but using specialized microbial fermentation, the surplus wasted acid whey could be converted into value-added chemicals. the baker's yeast saccharomyces cerevisiae, which is commonly used for industrial fermentation, cannot natively ferment lactose. the present study describes how an engineered s. cerevisiae yeast was constructed to produce lactic acid from purified la ...201727837988
antisense transcription licenses nascent transcripts to mediate transcriptional gene silencing.in eukaryotes, antisense transcription can regulate sense transcription by induction of epigenetic modifications. we showed previously that antisense transcription triggers dicer-independent sirna (disirna) production and disirna locus dna methylation (dldm) in neurospora crassa here we show that the conserved exonuclease eri-1 (enhanced rnai-1) is a critical component in this process. antisense transcription and eri-1 binding to target rnas are necessary and sufficient to trigger dldm. converge ...201627856616
the neurospora transcription factor adv-1 transduces light signals and temporal information to control rhythmic expression of genes involved in cell fusion.light and the circadian clock have a profound effect on the biology of organisms through the regulation of large sets of genes. toward understanding how light and the circadian clock regulate gene expression, we used genome-wide approaches to identify the direct and indirect targets of the light-responsive and clock-controlled transcription factor adv-1 in neurospora crassa a large proportion of adv-1 targets were found to be light- and/or clock-controlled, and enriched for genes involved in dev ...201727856696
normal chromosome conformation depends on subtelomeric facultative heterochromatin in neurospora crassa.high-throughput chromosome conformation capture (hi-c) analyses revealed that the 3d structure of the neurospora crassa genome is dominated by intra- and interchromosomal links between regions of heterochromatin, especially constitutive heterochromatin. elimination of trimethylation of lysine 9 on histone h3 (h3k9me3) or its binding partner heterochromatin protein 1 (hp1)-both prominent features of constitutive heterochromatin-have little effect on the hi-c pattern. it remained possible that di- ...201627856763
glucose starvation alters heat shock response, leading to death of wild type cells and survival of map kinase signaling mutant.a moderate heat shock induces neurospora crassa to synthesize large quantities of heat shock proteins that are protective against higher, otherwise lethal temperatures. however, wild type cells do not survive when carbohydrate deprivation is added to heat shock. in contrast, a mutant strain defective in a stress-activated protein kinase does survive the combined stresses. in order to understand the basis for this difference in survival, we have determined the relative levels of detected proteins ...201627870869
the calmodulin gene in neurospora crassa is required for normal vegetative growth, ultraviolet survival, and sexual development.we isolated a neurospora crassa mutant of the calmodulin (cmd) gene using repeat-induced point mutation and studied its phenotypes. the cmd (rip) mutant showed a defect in growth, reduced aerial hyphae, decreased carotenoid accumulation, a severe reduction in viability upon ultraviolet (uv) irradiation, and a fertility defect. moreover, meiotic silencing of the cmd gene resulted in a barren phenotype. in addition, we also performed site-directed mutational analysis of the calcium/calmodulin-depe ...201627888323
selective enzymatic transformation to aldehydes in vivo by fungal carboxylate reductase from neurospora crassa.the enzymatic reduction of carboxylic acids is in its infancy with only a handful of biocatalysts available to this end. we have increased the spectrum of carboxylate-reducing enzymes (cars) with the sequence of a fungal car from neurospora crassa or74a (nccar). nccar was efficiently expressed in e. coli using an autoinduction protocol at low temperature. it was purified and characterized in vitro, revealing a broad substrate acceptance, a ph optimum at ph 5.5-6.0, a tm of 45 °c and inhibition b ...201627917101
identification of a novel l-rhamnose uptake transporter in the filamentous fungus aspergillus niger.the study of plant biomass utilization by fungi is a research field of great interest due to its many implications in ecology, agriculture and biotechnology. most of the efforts done to increase the understanding of the use of plant cell walls by fungi have been focused on the degradation of cellulose and hemicellulose, and transport and metabolism of their constituent monosaccharides. pectin is another important constituent of plant cell walls, but has received less attention. in relation to th ...201627984587
alternative oxidase transcription factors aod2 and aod5 of neurospora crassa control the expression of genes involved in energy production and metabolism.in neurospora crassa, blocking the function of the standard mitochondrial electron transport chain results in the induction of an alternative oxidase (aox). aox transfers electrons directly from ubiquinol to molecular oxygen. aox serves as a model of retrograde regulation since it is encoded by a nuclear gene that is regulated in response to signals from mitochondria. the n. crassa transcription factors aod2 and aod5 are necessary for the expression of the aox gene. to gain insight into the mech ...201727986792
cations optimization for protein enrichment in rice straw by mixed cultures of neurospora crassa 14-8 and candida utilis using response surface methodology.this study investigated the effect of cation optimization by mixed cultures of neurospora crassa and candida utilis on the true protein (tp) content. firstly, to enhance the nutritional contents of rice straw (rs), two fermentation parameters (effect of inoculation ratio and inoculation time) were optimized. it was found that when c. utilis was inoculated 60 h later than n. crassa with the inoculation ratio of 1:5 (n. crassa to c. utilis), the maximum tp yield was obtained. in order to further o ...201627987187
hyphal ontogeny in neurospora crassa: a model organism for all seasons.filamentous fungi have proven to be a better-suited model system than unicellular yeasts in analyses of cellular processes such as polarized growth, exocytosis, endocytosis, and cytoskeleton-based organelle traffic. for example, the filamentous fungus neurospora crassa develops a variety of cellular forms. studying the molecular basis of these forms has led to a better, yet incipient, understanding of polarized growth. polarity factors as well as rho gtpases, septins, and a localized delivery of ...201627990280
oxygen activation at the active site of a fungal lytic polysaccharide monooxygenase.lytic polysaccharide monooxygenases have attracted vast attention owing to their abilities to disrupt glycosidic bonds via oxidation instead of hydrolysis and to enhance enzymatic digestion of recalcitrant substrates including chitin and cellulose. we have determined high-resolution x-ray crystal structures of an enzyme from neurospora crassa in the resting state and of a copper(ii) dioxo intermediate complex formed in the absence of substrate. x-ray crystal structures also revealed "pre-bound" ...201728004877
the woronin body in the nematophagous fungus arthrobotrys oligospora is essential for trap formation and efficient pathogenesis.the woronin body is a unique organelle in pezizomycotina species. following the injury of hyphae, it can quickly seal the septal pores to reduce the loss of cytoplasm and promote hyphal healing. the woronin body is also considered a significant factor in efficient pathogenesis in many fungal pathogens. in this study, we identified aohex1, a homologue of neurospora crassa hex1, a main component of the woronin body in the genome of the nematode-trapping fungus arthrobotrys oligospora. to study the ...201728007213
lac-1 and lag-1 with ras-1 affect aging and the biological clock in neurospora crassa.using an automated cell counting technique developed previously (case et al., ecology and evolution 2014; 4: 3494), we explore the lifespan effects of lac-1, a ceramide synthase gene paralogous to lag-1 in neurospora crassa in conjunction with the band bd (ras-1) gene. we find that the replicative lifespan of a lac-1(ko)bd double mutants is short, about one race tube cycle, and this double mutant lacks a strong ~21-hr clock cycle as shown by race tube and fluorometer analysis of fluorescent stra ...201628031787
disruption of non-anchored cell wall protein ncw-1 promotes cellulase production by increasing cellobiose uptake in neurospora crassa.to elucidate the mechanism of cellulase signal transduction in filamentous fungi including the components of the cellulase induction pathway.201728039555
an evolutionarily conserved transcriptional activator-repressor module controls expression of genes for d-galacturonic acid utilization in aspergillus niger.the expression of genes encoding extracellular polymer-degrading enzymes and the metabolic pathways required for carbon utilization in fungi are tightly controlled. the control is mediated by transcription factors that are activated by the presence of specific inducers, which are often monomers or monomeric derivatives of the polymers. a d-galacturonic acid-specific transcription factor named gaar was recently identified and shown to be an activator for the expression of genes involved in galact ...201728049705
transcription factor xpp1 is a switch between primary and secondary fungal metabolism.fungi can produce a wide range of chemical compounds via secondary metabolism. these compounds are of major interest because of their (potential) application in medicine and biotechnology and as a potential source for new therapeutic agents and drug leads. however, under laboratory conditions, most secondary metabolism genes remain silent. this circumstance is an obstacle for the production of known metabolites and the discovery of new secondary metabolites. in this study, we describe the dual r ...201728074041
rna interference in fungi: retention and loss.rna interference (rnai) is a mechanism conserved in eukaryotes, including fungi, that represses gene expression by means of small noncoding rnas (srnas) of about 20 to 30 nucleotides. its discovery is one of the most important scientific breakthroughs of the past 20 years, and it has revolutionized our perception of the functioning of the cell. initially described and characterized in neurospora crassa, the rnai is widespread in fungi, suggesting that it plays important functions in the fungal k ...201628087943
identification of glutaminyl cyclase genes involved in pyroglutamate modification of fungal lignocellulolytic enzymes.the breakdown of plant biomass to simple sugars is essential for the production of second-generation biofuels and high-value bioproducts. currently, enzymes produced from filamentous fungi are used for deconstructing plant cell wall polysaccharides into fermentable sugars for biorefinery applications. a post-translational n-terminal pyroglutamate modification observed in some of these enzymes occurs when n-terminal glutamine or glutamate is cyclized to form a five-membered ring. this modificatio ...201728096492
deletion of ph regulator pac-3 affects cellulase and xylanase activity during sugarcane bagasse degradation by neurospora crassa.microorganisms play a vital role in bioethanol production whose usage as fuel energy is increasing worldwide. the filamentous fungus neurospora crassa synthesize and secrete the major enzymes involved in plant cell wall deconstruction. the production of cellulases and hemicellulases is known to be affected by the environmental ph; however, the regulatory mechanisms of this process are still poorly understood. in this study, we investigated the role of the ph regulator pac-3 in n. crassa during t ...201728107376
identification and characterization of the glucose dual-affinity transport system in neurospora crassa: pleiotropic roles in nutrient transport, signaling, and carbon catabolite repression.the glucose dual-affinity transport system (low- and high-affinity) is a conserved strategy used by microorganisms to cope with natural fluctuations in nutrient availability in the environment. the glucose-sensing and uptake processes are believed to be tightly associated with cellulase expression regulation in cellulolytic fungi. however, both the identities and functions of the major molecular components of this evolutionarily conserved system in filamentous fungi remain elusive. here, we syst ...201728115989
correction for honda et al., dual chromatin recognition by the histone deacetylase complex hchc is required for proper dna methylation in neurospora crassa. 201728137865
crystallization of a fungal lytic polysaccharide monooxygenase expressed from glycoengineered pichia pastoris for x-ray and neutron diffraction.lytic polysaccharide monooxygenases (lpmos) are carbohydrate-disrupting enzymes secreted by bacteria and fungi that break glycosidic bonds via an oxidative mechanism. fungal lpmos typically act on cellulose and can enhance the efficiency of cellulose-hydrolyzing enzymes that release soluble sugars for bioethanol production or other industrial uses. the enzyme pmo-2 from neurospora crassa (ncpmo-2) was heterologously expressed in pichia pastoris to facilitate crystallographic studies of the funga ...201728177316
a deletion variant partially complements a porin-less strain of neurospora crassa.mitochondrial porin, the voltage-dependent anion channel, plays an important role in metabolism and other cellular functions within eukaryotic cells. to further the understanding of porin structure and function, neurospora crassa wild-type porin was replaced with a deletion variant lacking residues 238-242 (238porin). 238porin was assembled in the mitochondrial outer membrane, but the steady state levels were only about 3% of those of the wild-type protein. the strain harbouring 238porin display ...201728177769
the nuclear cap-binding complex mediates meiotic silencing by unpaired dna.in the filamentous fungus neurospora crassa, cross walls between individual cells are normally incomplete, making the entire fungal network vulnerable to attack by viruses and selfish dnas. accordingly, several genome surveillance mechanisms are maintained to help the fungus combat these repetitive elements. one of these defense mechanisms is called meiotic silencing by unpaired dna (msud), which identifies and silences unpaired genes during meiosis. utilizing common rna interference (rnai) prot ...201728179391
engineering neurospora crassa for cellobionate production directly from cellulose without any enzyme addition.in a cellulosic biorefinery, the cellulase enzymes needed for hydrolysis are one of the major contributors to high processing costs, while the hydrolysis product, cellobiose, has strong inhibition to the cellulases. in this study, we report engineering recombinant neurospora crassa strains which are able to produce cellobionate, an organic acid, from cellulose without any enzyme addition. recombinant strains were constructed by heterologously expressing laccase genes from different sources under ...201728193328
the inhibition of mating in phycomyces blakesleeanus by light is dependent on the mada-madb complex that acts in a sex-specific manner.light is an environmental signal that influences reproduction in the mucoromycotina fungi, as it does in many other species of fungi. mating in phycomyces blakesleeanus is inhibited by light, but the molecular mechanisms for this inhibition are uncharacterized. in this analysis, the role of the light-sensing mada-madb complex in mating was tested. the mada-madb complex is homologous to the neurospora crassa white collar complex. three genes required for cell type determination in the sex locus o ...201728214601
the dynamics of signal complex formation mediating germling fusion in neurospora crassa.colony initiation of filamentous fungi commonly involves fusion of germinating vegetative spores. studies in neurospora crassa revealed an unusual cell-cell communication mechanism mediating this process, in which the fusion partners coordinately alternate between two physiological stages, probably related to signal sending and receiving. this "cell dialog" involves the alternating, oscillatory recruitment of the so protein and the mak-2 map kinase module to the apical plasma membrane of growing ...201728216441
neurospora tetrasperma crosses heterozygous for hybrid translocation strains produce rare eight-spored asci-bearing heterokaryotic ascospores.during ascogenesis in neurospora, the ascospores are partitioned at the eight-nucleus stage that follows meiosis and a post-meiotic mitosis, and the ascospores that form in eight-spored asci are usually homokaryotic. we had previously created novel tnt strains by introgressing four neurospora crassa insertional translocations (eb4, ibj5, uk14-1, and b362i) into n. tetrasperma. we now show that crosses of all the tnt strains with single-mating-type derivatives of the standard n. tetrasperma pseud ...201728229961
the pleiotropic vegetative and sexual development phenotypes of neurospora crassa arise from double mutants of the calcium signaling genes plc-1, spla2, and cpe-1.we investigated phenotypes of the double mutants of the calcium (ca(2+)) signaling genes plc-1, spla2, and cpe-1 encoding for a phospholipase c1 (plc-1), a secretory phospholipase a2 (spla2), and a ca(2+)/h(+) exchanger (cpe-1), respectively, to understand the cell functions regulated by their genetic interactions. mutants lacking plc-1 and either spla2 or cpe-1 exhibited numerous defects including reduced colonial growth, stunted aerial hyphae, premature conidiation on plates with delayed germi ...201728265741
genetic and biochemical characterization of the gh72 family of cell wall transglycosylases in neurospora crassa.the neurospora crassa genome encodes five gh72 family transglycosylases, and four of these enzymes (gel-1, gel-2, gel-3 and gel-5) have been found to be present in the cell wall proteome. we carried out an extensive genetic analysis on the role of these four transglycosylases in cell wall biogenesis and demonstrated that the transglycosylases are required for the formation of a normal cell wall. as suggested by the proteomic analysis, we found that multiple transglycosylases were being expressed ...201728285007
establishment of crispr/cas9 in alternaria alternata.the filamentous fungus alternaria alternata is a potent producer of many secondary metabolites, some of which like alternariol or alternariol-methyl ether are toxic and/or cancerogenic. many alternaria species do not only cause post-harvest losses of food and feed, but are aggressive plant pathogens. despite the great economic importance and the large number of research groups working with the fungus, the molecular toolbox is rather underdeveloped. gene deletions often result in heterokaryotic s ...201728286319
structural studies of neurospora crassa lpmo9d and redox partner cdhiia using neutron crystallography and small-angle scattering.sensitivity to hydrogen/deuterium and lack of observable radiation damage makes cold neutrons an ideal probe the structural studies of proteins with highly photosensitive groups such as the copper center of lytic polysaccharide monooxygenases (lpmos) and flavin adenine dinucleotide (fad) and heme redox cofactors of cellobiose dehydrogenases (cdhs). here, neutron crystallography and small-angle neutron scattering are used to investigate neurospora crassa lpmo9d (nclpmo9d) and cdhiia (nccdhiia), r ...201728291519
a ras gtpase associated protein is involved in the phototropic and circadian photobiology responses in fungi.light is an environmental signal perceived by most eukaryotic organisms and that can have major impacts on their growth and development. the madc protein in the fungus phycomyces blakesleeanus (mucoromycotina) has been postulated to form part of the photosensory input for phototropism of the fruiting body sporangiophores, but the madc gene has remained unidentified since the 1960s when madc mutants were first isolated. in this study the madc gene was identified by positional cloning. all madc mu ...201728322269
xylitol production by saccharomyces cerevisiae overexpressing different xylose reductases using non-detoxified hemicellulosic hydrolysate of corncob.xylitol production was compared in fed batch fermentation by saccharomyces cerevisiae strains overexpressing xylose reductase (xr) genes from candida tropicalis, pichia stipitis, neurospora crassa, and an endogenous gene gre3. the gene encoding a xylose specific transporter (sut1) from p. stipitis was cloned to improve xylose transport and fed batch fermentation was used with glucose as a cosubstrate to regenerate nadph. xylitol yield was near theoretical for all the strains in fed batch ferment ...201628330197
fluorescent protein as a tool for investigating meiotic recombination in neurospora.we have built a series of neurospora crassa strains containing alleles of green fluorescent protein (gfp) to provide a visual phenotype for investigating meiotic recombination. these strains provide a convenient means of screening the neurospora knockout library for genes involved in genetic recombination. they permit rapid analysis of recombination outcomes by allowing visualization of segregation patterns in a large number of octads from crosses heterozygous for gfp. using this system the effe ...201728349393
extracellular calcium triggers unique transcriptional programs and modulates staurosporine-induced cell death in neurospora crassa.alterations in the intracellular levels of calcium are a common response to cell death stimuli in animals and fungi and, particularly, in the neurospora crassa response to staurosporine. we highlight the importance of the extracellular availability of ca(2+) for this response. limitation of the ion in the culture medium further sensitizes cells to the drug and results in increased accumulation of reactive oxygen species (ros). conversely, an approximately 30-fold excess of external ca(2+) leads ...201428357255
a new proteinaceous pathogen-associated molecular pattern (pamp) identified in ascomycete fungi induces cell death in solanaceae.pathogen-associated molecular patterns (pamps) are detected by plant pattern recognition receptors (prrs), which gives rise to pamp-triggered immunity (pti). we characterized a novel fungal pamp, cell death inducing 1 (rccdi1), identified in the rhynchosporium commune transcriptome sampled at an early stage of barley (hordeum vulgare) infection. the ability of rccdi1 and its homologues from different fungal species to induce cell death in nicotiana benthamiana was tested following agroinfiltrati ...201728386988
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