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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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
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
characterization of greenbeard genes involved in long-distance kind discrimination in a microbial eukaryote.microorganisms are capable of communication and cooperation to perform social activities. cooperation can be enforced using kind discrimination mechanisms in which individuals preferentially help or punish others, depending on genetic relatedness only at certain loci. in the filamentous fungus neurospora crassa, genetically identical asexual spores (germlings) communicate and fuse in a highly regulated process, which is associated with fitness benefits during colony establishment. recognition an ...201627077707
beyond animals and plants: dynamic maternal effects in the fungus neurospora crassa.maternal effects are widely documented in animals and plants, but not in fungi or other eukaryotes. a principal cause of maternal effects is asymmetrical parental investment in a zygote, creating greater maternal vs. paternal influence on offspring phenotypes. asymmetrical investments are not limited to animals and plants, but are also prevalent in fungi and groups including apicomplexans, dinoflagellates and red algae. evidence suggesting maternal effects among fungi is sparse and anecdotal. in ...201627062053
direct electrochemistry of nitrate reductase from the fungus neurospora crassa.we report the first direct (unmediated) catalytic electrochemistry of a eukaryotic nitrate reductase (nr). nr from the filamentous fungus neurospora crassa, is a member of the mononuclear molybdenum enzyme family and contains a mo, heme and fad cofactor which are involved in electron transfer from nad(p)h to the (mo) active site where reduction of nitrate to nitrite takes place. nr was adsorbed on an edge plane pyrolytic graphite (epg) working electrode. non-turnover redox responses were observe ...201627060250
signal exchange and integration during self-fusion in filamentous fungi.growth and propagation of filamentous ascomycete fungi commonly involves vegetative cell fusion. in the red bread mold neurospora crassa and many other ascomycete species, fusion occurs between germinating spores during colony formation and between hyphal branches in established mycelia. both fusion processes promote the development and behavior of the fungal colony as a supra-cellular network. germling and hyphal fusion in n. crassa rely on an unusual mode of cellular communication, in which th ...201627032478
chemotropism and cell fusion in neurospora crassa relies on the formation of distinct protein complexes by ham-5 and a novel protein ham-14.in filamentous fungi, communication is essential for the formation of an interconnected, multinucleate, syncytial network, which is constructed via hyphal fusion or fusion of germinated asexual spores (germlings). anastomosis in filamentous fungi is comparable to other somatic cell fusion events resulting in syncytia, including myoblast fusion during muscle differentiation, macrophage fusion, and fusion of trophoblasts during placental development. in neurospora crassa, fusion of genetically ide ...201627029735
prd-1, a component of the circadian system of neurospora crassa, is a member of the dead-box rna helicase family.the circadian rhythms found in almost all organisms are driven by molecular oscillators, including transcription/translation feedback loops (ttfls). however, ttfl-independent oscillators can drive rhythms in both eukaryotes and prokaryotes. the fungus neurospora crassa is a model organism for studying the molecular mechanism of the circadian clock. although a circadian ttfl involving the proteins frq, wc-1, and wc-2 is well-characterized in n. crassa, rhythms can still be observed in the absence ...201627029286
calcineurin subunits a and b interact to regulate growth and asexual and sexual development in neurospora crassa.calcineurin is a calcium/calmodulin dependent protein phosphatase in eukaryotes that consists of a catalytic subunit a and a regulatory subunit b. previous studies in the filamentous fungus neurospora crassa had suggested that the catalytic subunit of calcineurin might be an essential protein. we generated n. crassa strains expressing the a (cna-1) and b (cnb-1) subunit genes under the regulation of ptcu-1, a copper-responsive promoter. in these strains, addition of bathocuproinedisulfonic acid ...201627019426
the seb-1 transcription factor binds to the stre motif in neurospora crassa and regulates a variety of cellular processes including the stress response and reserve carbohydrate metabolism.when exposed to stress conditions, all cells induce mechanisms resulting in an attempt to adapt to stress that involve proteins which, once activated, trigger cell responses by modulating specific signaling pathways. in this work, using a combination of pulldown assays and mass spectrometry analyses, we identified the neurospora crassa seb-1 transcription factor that binds to the stress response element (stre) under heat stress. orthologs of seb-1 have been functionally characterized in a few fi ...201626994287
defining individual size in the model filamentous fungus neurospora crassa.it is challenging to apply the tenets of individuality to filamentous fungi: a fungal mycelium can contain millions of genetically diverse but totipotent nuclei, each capable of founding new mycelia. moreover, a single mycelium can potentially stretch over kilometres, and it is unlikely that its distant parts share resources or have the same fitness. here, we directly measure how a single mycelium of the model ascomycete neurospora crassa is patterned into reproductive units (rus), meaning subpo ...201626962146
the fast-evolving phy-2 gene modulates sexual development in response to light in the model fungus neurospora crassa.rapid responses to changes in incident light are critical to the guidance of behavior and development in most species. phytochrome light receptors in particular play key roles in bacterial physiology and plant development, but their functions and regulation are less well understood in fungi. nevertheless, genome-wide expression measurements provide key information that can guide experiments that reveal how genes respond to environmental signals and clarify their role in development. we performed ...201626956589
microtubules and associated molecular motors in neurospora crassa.the cytoskeleton provides structure, shape and movement to various cells. microtubules (mts) are tubular structures made of α and β-tubulin heterodimers organized in 13 protofilaments, forming a hollow cylinder. a vast group of mt-associated proteins determines the function, behavior and interaction of the mts with other cellular components. among these proteins, molecular motors such as the dynein-dynactin complex and kinesin superfamily play roles in mt organization and organelle transport. th ...201626951369
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
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
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
deoxycholic acid transformations catalyzed by selected filamentous fungi.more than 100 filamentous fungi strains, mostly ascomycetes and zygomycetes from different phyla, were screened for the ability to convert deoxycholic acid (dca) to valuable bile acid derivatives. along with 11 molds which fully degraded dca, several strains were revealed capable of producing cholic acid, ursocholic acid, 12-keto-lithocholic acid (12-keto-lca), 3-keto-dca, 15β-hydroxy-dca and 15β-hydroxy-12-oxo-lca as major products from dca. the last metabolite was found to be a new compound. t ...201626718089
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 fungus tremella mesenterica encodes the longest metallothionein currently known: gene, protein and metal binding characterization.fungal cu-thioneins, and among them, the paradigmatic neurospora crassa metallothionein (mt) (26 residues), were once considered as the shortest mts--the ubiquitous, versatile metal-binding proteins--among all organisms, and thus representatives of their primeval forms. nowadays, fungal mts of diverse lengths and sequence features are known, following the huge heterogeneity of the kingdom of fungi. at the opposite end of n. crassa mt, the recently reported cryptococcus neoformans cnmt1 and cnmt2 ...201626882011
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
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
functional evolution in orthologous cell-encoded rna-dependent rna polymerases.many eukaryotic organisms encode more than one rna-dependent rna polymerase (rdrp) that probably emerged as a result of gene duplication. such rdrp paralogs often participate in distinct rna silencing pathways and show characteristic repertoires of enzymatic activities in vitro however, to what extent members of individual paralogous groups can undergo functional changes during speciation remains an open question. we show that orthologs of qde-1, an rdrp component of the quelling pathway in neur ...201626907693
bio-transformation of agri-food wastes by newly isolated neurospora crassa and lactobacillus plantarum for egg production.using bio-transferred feedstuff was a cost-effective approach to improve egg quality and production; particularly, the nutritive diet came from agri-food wastes. in this study, optimization of fermentation conditions and co-cultivation of neurospora crassa with lactobacillus plantarum was performed in a simple bioreactor. the optimized fermentation of beer lees substrates through n. crassa led to the hydrolysis rates of crude fiber increasing to 43.27%. compared to that of using n. crassa alone, ...201626740129
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
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
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
[proteins from neurospora crassa. 2. immunoelectrophoretically detectable proteins and their relationship to growth time]. 20164973223
isolation, mapping, and characterization of trehalaseless mutants of neurospora crassa.mutant strains of neurospora crassa that lack trehalase and are unable to grow on trehalose were isolated, and the gene (tre) was positioned on the right arm of linkage group i. maltase and beta-galactosidase activities are almost identical in tre(-) strains, whereas that of invertase was reduced by more than half and those of acid phosphatase and amylase were somewhat increased. heterocaryons between standard and trehalaseless strains yield less than one-tenth the activity of the former. in add ...20165001211
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
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
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
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
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
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
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 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
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
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 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
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
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
correction for honda et al., dual chromatin recognition by the histone deacetylase complex hchc is required for proper dna methylation in neurospora crassa. 201728137865
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
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 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
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
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
optimization of the hyper sensor for robust real-time detection of hydrogen peroxide in the rice blast fungus.reactive oxygen species (ros) production and breakdown have been studied in detail in plant-pathogenic fungi, including the rice blast fungus, magnaporthe oryzae; however, the examination of the dynamic process of ros production in real time has proven to be challenging. we resynthesized an existing ros sensor, called hyper, to exhibit optimized codon bias for fungi, specifically neurospora crassa, and used a combination of microscopy and plate reader assays to determine whether this construct c ...201726950262
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
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
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
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 kinetochore interaction network (kin) of ascomycetes.chromosome segregation relies on coordinated activity of a large assembly of proteins, the kinetochore interaction network (kin). how conserved the underlying mechanisms driving the epigenetic phenomenon of centromere and kinetochore assembly and maintenance are remains unclear, even though various eukaryotic models have been studied. more than 50 different proteins, many in multiple copies, comprise the kin or are associated with fungal centromeres and kinetochores. proteins isolated from immun ...201726908646
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
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
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
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
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
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
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