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genetically engineered photoinducible homodimerization system with improved dimer-forming efficiency.vivid (vvd) is a photoreceptor derived from neurospora crassa that rapidly forms a homodimer in response to blue light. although vvd has several advantages over other photoreceptors as photoinducible homodimerization system, vvd has a critical limitation in its low dimer-forming efficiency. to overcome this limitation of wild-type vvd, here we conduct site-directed saturation mutagenesis in the homodimer interface of vvd. we have found that the ile52cys mutation of vvd (vvd-52c) substantially im ...201424428544
the proteome and phosphoproteome of neurospora crassa in response to cellulose, sucrose and carbon starvation.improving cellulolytic enzyme production by plant biomass degrading fungi holds great potential in reducing costs associated with production of next-generation biofuels generated from lignocellulose. how fungi sense cellulosic materials and respond by secreting enzymes has mainly been examined by assessing function of transcriptional regulators and via transcriptional profiling. here, we obtained global proteomic and phosphoproteomic profiles of the plant biomass degrading filamentous fungus neu ...201424881580
an asymmetric pan3 dimer recruits a single pan2 exonuclease to mediate mrna deadenylation and decay.the pan2-pan3 complex functions in general and microrna-mediated mrna deadenylation. however, mechanistic insight into pan2 and its complex with the asymmetric pan3 dimer is lacking. here, we describe crystal structures that show that neurospora crassa pan2 comprises two independent structural units: a c-terminal catalytic unit and an n-terminal assembly unit that engages in a bipartite interaction with pan3 dimers. the catalytic unit contains the exonuclease domain in an intimate complex with a ...201424880343
heterochromatin controls γh2a localization in neurospora crassa.in response to genotoxic stress, atr and atm kinases phosphorylate h2a in fungi and h2ax in animals on a c-terminal serine. the resulting modified histone, called γh2a, recruits chromatin-binding proteins that stabilize stalled replication forks or promote dna double-strand-break repair. to identify genomic loci that might be prone to replication fork stalling or dna breakage in neurospora crassa, we performed chromatin immunoprecipitation (chip) of γh2a followed by next-generation sequencing (c ...201424879124
secret lifestyles of neurospora crassa.neurospora crassa has a long history as an excellent model for genetic, cellular, and biochemical research. although this fungus is known as a saprotroph, it normally appears on burned vegetations or trees after forest fires. however, due to a lack of experimental evidence, the nature of its association with living plants remains enigmatic. here we report that scots pine (pinus sylvestris) is a host plant for n. crassa. the endophytic lifestyle of n. crassa was found in its interaction with scot ...201424875794
experimental evolution of bet hedging under manipulated environmental uncertainty in neurospora crassa.all organisms are faced with environmental uncertainty. bet-hedging theory expects unpredictable selection to result in the evolution of traits that maximize the geometric-mean fitness even though such traits appear to be detrimental over the shorter term. despite the centrality of fitness measures to evolutionary analysis, no direct test of the geometric-mean fitness principle exists. here, we directly distinguish between predictions of competing fitness maximization principles by testing cohen ...201424870047
dynamics of transcriptome evolution in the model eukaryote neurospora.mounting evidence indicates that changes in the transcriptome contribute significantly to the phenotypic differentiation of closely related species. nonetheless, further genome-wide studies, spanning a broad range of organisms, are needed to decipher the factors driving transcriptome evolution. the model neurospora (ascomycota) comprises a simple system for empirically studying the evolutionary dynamics of the transcriptome. here, we studied the evolution of gene expression in neurospora crassa ...201424848562
neurospora crassa 1,3-α-glucan synthase, ags-1, is required for cell wall biosynthesis during macroconidia development.the neurospora crassa genome encodes two 1,3-α-glucan synthases. one of these 1,3-α-glucan synthase genes, ags-1, was shown to be required for the synthesis of 1,3-α-glucan in the aerial hyphae and macroconidia cell walls. 1,3-α-glucan was found in the conidia cell wall, but was absent from the vegetative hyphae cell wall. deletion of ags-1 affected conidial development. δags-1 produced only 5 % as many conidia as the wt and most of the conidia produced by δags-1 were not viable. the ags-1 upstr ...201424847001
regulated dna methylation and the circadian clock: implications in cancer.since the cloning and discovery of dna methyltransferases (dnmt), there has been a growing interest in dna methylation, its role as an epigenetic modification, how it is established and removed, along with the implications in development and disease. in recent years, it has become evident that dynamic dna methylation accompanies the circadian clock and is found at clock genes in neurospora, mice and cancer cells. the relationship among the circadian clock, cancer and dna methylation at clock gen ...201425198253
light-mediated participation of the vivid-like protein of fusarium fujikuroi vvda in pigmentation and development.the phytopathogen fusarium fujikuroi is a model fungus for the production of different secondary metabolites. these include the acidic xanthophyll neurosporaxanthin, produced through the activity of the enzymes encoded by the car genes, of which carra and carb play a major role. expression of these genes is induced by light but, in contrast to other fungi, the induction is not impaired under continuous illumination in null mutants of the f. fujikuroi wc-1-like gene, wcoa. therefore, we investiga ...201425154020
efficient detection of unpaired dna requires a member of the rad54-like family of homologous recombination proteins.meiotic silencing by unpaired dna (msud) is a process that detects unpaired regions between homologous chromosomes and silences them for the duration of sexual development. while the phenomenon of msud is well recognized, the process that detects unpaired dna is poorly understood. in this report, we provide two lines of evidence linking unpaired dna detection to a physical search for dna homology. first, we have found that a putative snf2-family protein (sad-6) is required for efficient msud in ...201425146971
vib1, a link between glucose signaling and carbon catabolite repression, is essential for plant cell wall degradation by neurospora crassa.filamentous fungi that thrive on plant biomass are the major producers of hydrolytic enzymes used to decompose lignocellulose for biofuel production. although induction of cellulases is regulated at the transcriptional level, how filamentous fungi sense and signal carbon-limited conditions to coordinate cell metabolism and regulate cellulolytic enzyme production is not well characterized. by screening a transcription factor deletion set in the filamentous fungus neurospora crassa for mutants una ...201425144221
structural and functional organization of growing tips of neurospora crassa hyphae.data are presented on a variety of intracellular structures of the vegetative hyphae of the filamentous fungus neurospora crassa and the involvement of these structures in the tip growth of the hyphae. current ideas on the molecular and genetic mechanisms of tip growth and regulation of this process are considered. on the basis of comparison of data on behaviors of mitochondria and microtubules and data on the electrical heterogeneity of the hyphal apex, a hypothesis is proposed about a possible ...201425108323
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
the evolutionary history of the genes involved in the biosynthesis of the antioxidant ergothioneine.ergothioneine (egt) is a histidine betaine derivative that exhibits antioxidant action in humans. egt is primarily synthesized by fungal species and a number of bacterial species. a five-gene cluster (egta, egtb, egtc, egtd &egte) responsible for egt production in mycobacteria smegmatis has recently been identified. the first fungal biosynthetic egt gene (ncegt-1) has also been identified in neurospora crassa. ncegt-1 contains domains similar to those found in m. smegmatis egtb and egtd. egt is ...201425068406
genetic control of anastomosis in podospora anserina.we developed a new microscopy procedure to study anastomoses in the model ascomycete podospora anserina and compared it with the previous method involving the formation of balanced heterokaryons. both methods showed a good correlation. heterokaryon formation was less quantifiable, but enabled to observe very rare events. microscopic analysis evidenced that anastomoses were greatly influence by growth conditions and were severely impaired in the idc mutants of the pampk1, pampk2, idc1 and panox1 ...201425064063
genome-wide characterization of light-regulated genes in neurospora crassa.the filamentous fungus neurospora crassa responds to light in complex ways. to thoroughly study the transcriptional response of this organism to light, rna-seq was used to analyze capped and polyadenylated mrna prepared from mycelium grown for 24 hr in the dark and then exposed to light for 0 (control) 15, 60, 120, and 240 min. more than three-quarters of all defined protein coding genes (79%) were expressed in these cells. the increased sensitivity of rna-seq compared with previous microarray s ...201425053707
activation of a trp-like channel and intracellular ca2+ dynamics during phospholipase-c-mediated cell death.the model organism neurospora crassa undergoes programmed cell death when exposed to staurosporine. here, we show that staurosporine causes defined changes in cytosolic free ca(2+) ([ca(2+)]c) dynamics and a distinct ca(2+) signature that involves ca(2+) influx from the external medium and internal ca(2+) stores. we investigated the molecular basis of this ca(2+) response by using [ca(2+)]c measurements combined with pharmacological and genetic approaches. phospholipase c was identified as a piv ...201425037570
snowyowl: accurate prediction of fungal genes by using rna-seq and homology information to select among ab initio models.locating the protein-coding genes in novel genomes is essential to understanding and exploiting the genomic information but it is still difficult to accurately predict all the genes. the recent availability of detailed information about transcript structure from high-throughput sequencing of messenger rna (rna-seq) delineates many expressed genes and promises increased accuracy in gene prediction. computational gene predictors have been intensively developed for and tested in well-studied animal ...201424980894
heterologous expression and kinetic characterisation of neurospora crassa β-xylosidase in pichia pastoris.to degrade plant hemicelluloses fungi employ β-xylosidases to hydrolyse xylooligosaccharides, released by endo-xylanases, into xylose. we have expressed the β-xylosidase from neurospora crassa in pichia pastoris under the control of alcohol oxidase 1 (aox1) promoter. the recombinant enzyme is optimally active at 50 °c and ph 5.0 with km and vmax values of 8.9 mm and 1052 μmol min⁻¹ mg⁻¹ respectively against 4-nitrophenyl β-xylopyranoside. xylose is a non-competitive inhibitor with a k(i) of 1.72 ...201424629269
natural antisense transcripts and long non-coding rna in neurospora crassa.the prevalence of long non-coding rnas (lncrna) and natural antisense transcripts (nats) has been reported in a variety of organisms. while a consensus has yet to be reached on their global importance, an increasing number of examples have been shown to be functional, regulating gene expression at the transcriptional and post-transcriptional level. here, we use rna sequencing data from the abi solid platform to identify lncrna and nats obtained from samples of the filamentous fungus neurospora c ...201424621812
evidence of a critical role for cellodextrin transporte 2 (cdt-2) in both cellulose and hemicellulose degradation and utilization in neurospora crassa.cdt-1 and cdt-2 are two cellodextrin transporters discovered in the filamentous fungus neurospora crassa. previous studies focused on characterizing the role of these transporters in only a few conditions, including cellulose degradation, and the function of these two transporters is not yet completely understood. in this study, we show that deletion of cdt-2, but not cdt-1, results in growth defects not only on avicel but also on xylan. cdt-2 can be highly induced by xylan, and this mutant has ...201424586693
transcriptional comparison of the filamentous fungus neurospora crassa growing on three major monosaccharides d-glucose, d-xylose and l-arabinose.d-glucose, d-xylose and l-arabinose are the three major monosaccharides in plant cell walls. complete utilization of all three sugars is still a bottleneck for second-generation cellulolytic bioethanol production, especially for l-arabinose. however, little is known about gene expression profiles during l-arabinose utilization in fungi and a comparison of the genome-wide fungal response to these three major monosaccharides has not yet been reported.201424581151
[improving cellulases production with neurospora crassa by morphology mutants screening].filamentous fungi are widely used for large-scale production of cellulases. morphological characteristics of mycelia under submerged condition are closely correlated with cellulases productivity. in order to find out the critical genes involved in the mycelial morphology development and cellulases production in liquid fermentation, 95 neurospora crassa morphological mutants (named as szy1-95) were screened for cellulases production. compared with the wild type, cellulases production in four muta ...201424818479
septum development in neurospora crassa: the septal actomyosin tangle.septum formation in neurospora crassa was studied by fluorescent tagging of actin, myosin, tropomyosin, formin, fimbrin, bud-4, and chs-1. in chronological order, we recognized three septum development stages: 1) septal actomyosin tangle (sat) assembly, 2) contractile actomyosin ring (car) formation, 3) car constriction together with plasma membrane ingrowth and cell wall construction. septation began with the assembly of a conspicuous tangle of cortical actin cables (sat) in the septation site ...201424800890
analysis of the antimicrobial effects of nonthermal plasma on fungal spores in ionic solutions.the antimicrobial efficiency of reactive species-based control strategies is significantly affected by the dynamics of reactive species in the biological environment. atmospheric-pressure nonthermal plasma is an ionized gas in which various reactive species are produced. the various levels of antimicrobial activity may result from the dynamic interaction of the plasma-generated reactive species with the environment. however, the nature of the interaction between plasma and environments is poorly ...201424794411
cdc-42 and rac-1 regulate opposite chemotropisms in neurospora crassa.cell polarization and fusion are crucial developmental processes that occur in response to intracellular and extracellular signals. asexual spores (conidia) of the mold neurospora crassa differentiate two types of polarized cell protrusions, germ tubes and conidial anastomosis tubes (cats), which exhibit negative and positive chemotropism, respectively. we provide the first evidence that shared and separate functions of the rho-type gtpases cdc-42 and rac-1 regulate these opposite chemotropisms. ...201424790223
directed evolution of a cellodextrin transporter for improved biofuel production under anaerobic conditions in saccharomyces cerevisiae.introduction of a cellobiose utilization pathway consisting of a cellodextrin transporter and a β-glucosidase into saccharomyces cerevisiae enables co-fermentation of cellobiose and xylose. cellodextrin transporter 1 (cdt1) from neurospora crassa has been established as an effective transporter for the engineered cellobiose utilization pathways. however, cellodextrin transporter 2 (cdt2) from the same species is a facilitator and has the potential to be more efficient than cdt1 under anaerobic c ...201424519319
identification and characterization of a galacturonic acid transporter from neurospora crassa and its application for saccharomyces cerevisiae fermentation processes.pectin-rich agricultural wastes potentially represent favorable feedstocks for the sustainable production of alternative energy and bio-products. their efficient utilization requires the conversion of all major constituent sugars. the current inability of the popular fermentation host saccharomyces cerevisiae to metabolize the major pectic monosaccharide d-galacturonic acid (d-gala) significantly hampers these efforts. while it has been reasoned that the optimization of cellular d-gala uptake wi ...201424502254
bright to dim oscillatory response of the neurospora circadian oscillator.the fungus neurospora crassa constitutes an important model system extensively used in chronobiology. several studies have addressed how environmental cues, such as light, can reset or synchronize a circadian system. by means of an optimized firefly luciferase reporter gene and a controllable lighting system, we show that neurospora can display molecular circadian rhythms in dim light when cultures receive bright light prior to entering dim light conditions. we refer to this behavior as the "bri ...201424492882
a kinetic study of the effects of light on circadian rhythmicity of the frq promoter of neurospora crassa.the role of the frq gene in the neurospora crassa circadian rhythm has been widely studied, but technical limitations have hindered a thorough analysis of frq circadian expression waveform. through our experiments, we have shown an improved precision in defining neurospora's circadian rhythm kinetics using a codon optimized firefly luciferase gene reporter linked to a frq promoter. in vivo examination of this real-time reporter has allowed for a better understanding of the relationship of the li ...201424492881
photo morphogenesis and photo response of the blue-light receptor gene cmwc-1 in different strains of cordyceps militaris.light is a necessary environmental factor for stroma formation and development of cordyceps militaris, a well-known edible and medicinal fungus. in this study, photo morphogenesis and the blue-light receptor gene were studied using five representative strains of c. militaris. the results suggest that light was essential for colony pigmentation and could promote conidia production. cmwc-1, the homologe of the blue-light photoreceptor of neurospora crassa, was cloned from the genome of c. militari ...201424484244
circadian rhythms synchronize mitosis in neurospora crassa.the cell cycle and the circadian clock communicate with each other, resulting in circadian-gated cell division cycles. alterations in this network may lead to diseases such as cancer. therefore, it is critical to identify molecular components that connect these two oscillators. however, molecular mechanisms between the clock and the cell cycle remain largely unknown. a model filamentous fungus, neurospora crassa, is a multinucleate system used to elucidate molecular mechanisms of circadian rhyth ...201424474764
the importance of pyroglutamate in cellulase cel7a.the commercialization of lignocellulosic biofuels relies in part on the ability to engineer cellulase enzymes to have properties compatible with practical processing conditions. the cellulase cel7a has been a common engineering target because it is present in very high concentrations in commercial cellulase cocktails. significant effort has thus been focused on its recombinant expression. in particular, the yeast saccharomyces cerevisiae has often been used both in the engineering and basic stud ...201424375151
calcium/calmodulin-dependent kinases are involved in growth, thermotolerance, oxidative stress survival, and fertility in neurospora crassa.calcium/calmodulin-dependent kinases (ca(2+)/camks) are ser/thr protein kinases that respond to change in cytosolic free ca(2+) ([ca(2+)]c) and play multiple cellular roles in organisms ranging from fungi to humans. in the filamentous fungus neurospora crassa, four ca(2+)/cam-dependent kinases, ca(2+)/camk-1 to 4, are encoded by the genes ncu09123, ncu02283, ncu06177, and ncu09212, respectively. we found that camk-1 and camk-2 are essential for full fertility in n. crassa. the survival of ∆camk- ...201424570326
a new variant of self-excising β-recombinase/six cassette for repetitive gene deletion and homokaryon purification in neurospora crassa.in a previous study, we developed a cassette employing a bacterial β-recombinase acting on six recognition sequences (β-rec/six), which allowed repetitive site-specific gene deletion and marker recycling in neurospora crassa. however, only one positive selection marker was used in the cassette. a tedious subsequent procedure was needed to purify homokaryons due to the lack of a negative selection after cassette eviction. additionally, the endoxylanase xylp promoter from penicillium chrysogenum u ...201424556286
the neurospora crassa exocyst complex tethers spitzenkörper vesicles to the apical plasma membrane during polarized growth.fungal hyphae are among the most highly polarized cells. hyphal polarized growth is supported by tip-directed transport of secretory vesicles, which accumulate temporarily in a stratified manner in an apical vesicle cluster, the spitzenkörper. the exocyst complex is required for tethering of secretory vesicles to the apical plasma membrane. we determined that the presence of an octameric exocyst complex is required for the formation of a functional spitzenkörper and maintenance of regular hyphal ...201424523289
an in vitro peptide complementation assay for cyt-18-dependent group i intron splicing reveals a new role for the n-terminus.the mitochondrial tyrosyl trna synthetase from neurospora crassa (cyt-18 protein) is a bifunctional group i intron splicing cofactor. cyt-18 is capable of splicing multiple group i introns from a wide variety of sources by stabilizing the catalytically active intron structures. cyt-18 and mt tyrrss from related fungal species have evolved to assist in group i intron splicing in part by the accumulation of three n-terminal domain insertions. biochemical and structural analysis indicate that the n ...201424520960
analysis of cellodextrin transporters from neurospora crassa in saccharomyces cerevisiae for cellobiose fermentation.saccharomyces cerevisiae can be engineered to ferment cellodextrins produced by cellulases as a product of cellulose hydrolysis. direct fermentation of cellodextrins instead of glucose is advantageous because glucose inhibits cellulase activity and represses the fermentation of non-glucose sugars present in cellulosic hydrolyzates. to facilitate cellodextrin utilization by s. cerevisiae, a fungal cellodextrin-utilizing pathway from neurospora crassa consisting of a cellodextrin transporter and a ...201424190499
molecular cloning and expression of fungal cellobiose transporters and β-glucosidases conferring efficient cellobiose fermentation in saccharomyces cerevisiae.cellobiose was once regarded as a byproduct that should be removed from biomass hydrolysates because of its inhibitory activity to cellulases. it was revealed, however, that cellobiose could serve as a co-substrate for xylose fermentation by engineered saccharomyces cerevisiae. despite its advantages, to date, little is known about cellodextrin transporters that endow s. cerevisiae with cellobiose transporting ability. in this study, engineered s. cerevisiae strains capable of fermenting cellobi ...201424184384
determinants of regioselective hydroxylation in the fungal polysaccharide monooxygenases.the ubiquitous fungal polysaccharide monooxygenases (pmos) (also known as gh61 proteins, lpmos, and aa9 proteins) are structurally related but have significant variation in sequence. a heterologous expression method in neurospora crassa was developed as a step toward connecting regioselectivity of the chemistry to pmo phylogeny. activity assays, as well as sequence and phylogenetic analyses, showed that the majority of fungal pmos fall into three major groups with distinctive active site surface ...201424350607
global analysis of serine/threonine and tyrosine protein phosphatase catalytic subunit genes in neurospora crassa reveals interplay between phosphatases and the p38 mitogen-activated protein kinase.protein phosphatases are integral components of the cellular signaling machinery in eukaryotes, regulating diverse aspects of growth and development. the genome of the filamentous fungus and model organism neurospora crassa encodes catalytic subunits for 30 protein phosphatase genes. in this study, we have characterized 24 viable n. crassa phosphatase catalytic subunit knockout mutants for phenotypes during growth, asexual development, and sexual development. we found that 91% of the mutants had ...201424347630
evidence for transceptor function of cellodextrin transporters in neurospora crassa.neurospora crassa colonizes burnt grasslands and metabolizes both cellulose and hemicellulose from plant cell walls. when switched from a favored carbon source to cellulose, n. crassa dramatically up-regulates expression and secretion of genes encoding lignocellulolytic enzymes. however, the means by which n. crassa and other filamentous fungi sense the presence of cellulose in the environment remains unclear. previously, we have shown that a n. crassa mutant carrying deletions of three β-glucos ...201424344125
a c4-oxidizing lytic polysaccharide monooxygenase cleaving both cellulose and cello-oligosaccharides.lignocellulosic biomass is a renewable resource that significantly can substitute fossil resources for the production of fuels, chemicals, and materials. efficient saccharification of this biomass to fermentable sugars will be a key technology in future biorefineries. traditionally, saccharification was thought to be accomplished by mixtures of hydrolytic enzymes. however, recently it has been shown that lytic polysaccharide monooxygenases (lpmos) contribute to this process by catalyzing oxidati ...201424324265
the gene cuta of fusarium fujikuroi, encoding a protein of the haloacid dehalogenase family, is involved in osmotic stress and glycerol metabolism.survival of micro-organisms in natural habitats depends on their ability to adapt to variations in osmotic conditions. we previously described the gene cut-1 of neurospora crassa, encoding a protein of the haloacid dehalogenase family with an unknown function in the osmotic stress response. here we report on the functional analysis of cuta, the orthologous gene in the phytopathogenic fungus fusarium fujikuroi. cuta mrna levels increased transiently after exposure to 0.68 m nacl and were reduced ...201424129845
evolution of rna-protein interactions: non-specific binding led to rna splicing activity of fungal mitochondrial tyrosyl-trna synthetases.the neurospora crassa mitochondrial tyrosyl-trna synthetase (mttyrrs; cyt-18 protein) evolved a new function as a group i intron splicing factor by acquiring the ability to bind group i intron rnas and stabilize their catalytically active rna structure. previous studies showed: (i) cyt-18 binds group i introns by using both its n-terminal catalytic domain and flexibly attached c-terminal anticodon-binding domain (ctd); and (ii) the catalytic domain binds group i introns specifically via multiple ...201425536042
the aging biological clock in neurospora crassa.the biological clock affects aging through ras-1 (bd) and lag-1, and these two longevity genes together affect a clock phenotype and the clock oscillator in neurospora crassa. using an automated cell-counting technique for measuring conidial longevity, we show that the clock-associated genes lag-1 and ras-1 (bd) are true chronological longevity genes. for example, wild type (wt) has an estimated median life span of 24 days, while the double mutant lag-1, ras-1 (bd) has an estimated median life s ...201425535564
[lah-3, a transcriptional factor involved in osmotic regulation in neurospora crassa].[objective] in order to identify the function of lah-3 in osmotic regulation, we generated lah-3 deletion strain and analyzed its phenotype by osmostress treatment. [methods] we used homologous recombination to replace lah-3gene by hph gene and treated these cells with 4% nacl and 1 m sorbitol to analyze the phenotype. northern blot was used to detect the expressions of osmoresponsing genes. western blot was used to examine the phosphorylation level of lah-3 and os-2 and the expression of os-2. ...201425522587
selection and evaluation of reference genes for expression studies with quantitative pcr in the model fungus neurospora crassa under different environmental conditions in continuous culture.neurospora crassa has served as a model organism for studying circadian pathways and more recently has gained attention in the biofuel industry due to its enhanced capacity for cellulase production. however, in order to optimize n. crassa for biotechnological applications, metabolic pathways during growth under different environmental conditions must be addressed. reverse-transcription quantitative pcr (rt-qpcr) is a technique that provides a high-throughput platform from which to measure the ex ...201425474155
nothing special in the specialist? draft genome sequence of cryomyces antarcticus, the most extremophilic fungus from antarctica.the draft genome of the antarctic endemic fungus cryomyces antarcticus is presented. this rock inhabiting, microcolonial fungus is extremely stress tolerant and it is a model organism for exobiology and studies on stress resistance in eukaryots. since this fungus is a specialist in the most extreme environment of the earth, the analysis of its genome is of important value for the understanding of fungal genome evolution and stress adaptation. a comparison with neurospora crassa as well as with o ...201425296285
role of a remote leucine residue in the catalytic function of polyol dehydrogenase.studies on the protein-metal binding sites have mainly focused on the residues immediately surrounding the reacting substrate, cofactors, and metal ions. the contribution of residues in remote layers to the highly optimized microenvironments of catalytic active sites is not well understood. to improve our understanding, the present study examined the role of remote residues on the structure and function of zinc-dependent polyol dehydrogenases. we used an integrated computational and biochemical ...201425292207
biomineralization of metal carbonates by neurospora crassa.in this research, the urease-positive fungus neurospora crassa was investigated for the biomineralization of calcium carbonate and its potential application in metal biorecovery and/or bioremediation. after 12 d incubation at 25 °c in urea and calcium-containing medium, extensive biomineralization of fungal filaments was observed. energy dispersive x-ray analysis of crystalline precipitates on the hyphae of n. crassa showed that the main elements present in the crystals were ca, c, and o. x-ray ...201425423300
endogenous ergothioneine is required for wild type levels of conidiogenesis and conidial survival but does not protect against 254 nm uv-induced mutagenesis or kill.ergothioneine, a histidine derivative, is concentrated in conidia of ascomycetous fungi. to investigate the function of ergothioneine, we crossed the wild type neurospora crassa (egt(+)) and an ergothioneine non-producer (egt(-), δegt-1, a knockout in ncu04343.5) and used the egt(+) and egt(-) progeny strains for phenotypic analyses. compared to the egt(+) strains, egt(-) strains had a 59% reduction in the number of conidia produced on vogel's agar. after storage of egt(+) and egt(-) conidia at ...201425446508
the histone h3 lysine 9 methyltransferase dim-5 modifies chromatin at frequency and represses light-activated gene expression.the transcriptional program controlling the circadian rhythm requires coordinated regulation of chromatin. characterization of the chromodomain helicase dna-binding enzyme chd1 revealed dna methylation in the promoter of the central clock gene frequency (frq) in neurospora crassa. in this report, we show that the dna methylation at frq is not only dependent on the dna methyltransferase dim-2 but also on the h3k9 methyltransferase dim-5 and hp1. histone h3 lysine 9 trimethylation (h3k9me3) occurs ...201425429045
characterization of the neurospora crassa cell fusion proteins, ham-6, ham-7, ham-8, ham-9, ham-10, amph-1 and whi-2.intercellular communication of vegetative cells and their subsequent cell fusion is vital for different aspects of growth, fitness, and differentiation of filamentous fungi. cell fusion between germinating spores is important for early colony establishment, while hyphal fusion in the mature colony facilitates the movement of resources and organelles throughout an established colony. approximately 50 proteins have been shown to be important for somatic cell-cell communication and fusion in the mo ...201425279949
the combined structural and kinetic characterization of a bacterial nitronate monooxygenase from pseudomonas aeruginosa pao1 establishes nmo class i and ii.nitronate monooxygenase (nmo) oxidizes the mitochondrial toxin propionate 3-nitronate (p3n) to malonate semialdehyde. the enzyme has been previously characterized biochemically in fungi, but no structural information is available. based on amino acid similarity 4,985 genes are annotated in the genbank(tm) as nmo. of these, 4,424 (i.e. 89%) are bacterial genes, including several pseudomonads that have been shown to use p3n as growth substrate. here, we have cloned and expressed the gene pa4202 of ...201425002579
dead-box protein cyt-19 is activated by exposed helices in a group i intron rna.dead-box proteins are nonprocessive rna helicases and can function as rna chaperones, but the mechanisms of their chaperone activity remain incompletely understood. the neurospora crassa dead-box protein cyt-19 is a mitochondrial rna chaperone that promotes group i intron splicing and has been shown to resolve misfolded group i intron structures, allowing them to refold. building on previous results, here we use a series of tertiary contact mutants of the tetrahymena group i intron ribozyme to d ...201425002474
bioinformatic and biochemical characterizations of c-s bond formation and cleavage enzymes in the fungus neurospora crassa ergothioneine biosynthetic pathway.ergothioneine is a histidine thiol derivative. its mycobacterial biosynthetic pathway has five steps (egta-e catalysis) with two novel reactions: a mononuclear nonheme iron enzyme (egtb) catalyzed oxidative c-s bond formation and a plp-mediated c-s lyase (egte) reaction. our bioinformatic and biochemical analyses indicate that the fungus neurospora crassa has a more concise ergothioneine biosynthetic pathway because its nonheme iron enzyme, egt1, makes use of cysteine instead of γ-glu-cys as the ...201425275953
characterizing the roles of cryphonectria parasitica rna-dependent rna polymerase-like genes in antiviral defense, viral recombination and transposon transcript accumulation.an inducible rna-silencing pathway, involving a single dicer protein, dcl2, and a single argonaute protein, agl2, was recently shown to serve as an effective antiviral defense response in the chestnut blight fungus cryphonectria parasitica. eukaryotic rna-dependent rna polymerases (rdrps) are frequently involved in transcriptional and posttranscriptional gene silencing and antiviral defense. we report here the identification and characterization of four rdrp genes (rdr1-4) in the c. parasitica g ...201425268858
a protein kinase screen of neurospora crassa mutant strains reveals that the snf1 protein kinase promotes glycogen synthase phosphorylation.glycogen functions as a carbohydrate reserve in a variety of organisms and its metabolism is highly regulated. the activities of glycogen synthase and glycogen phosphorylase, the rate-limiting enzymes of the synthesis and degradation processes, respectively, are regulated by allosteric modulation and reversible phosphorylation. to identify the protein kinases affecting glycogen metabolism in neurospora crassa, we performed a screen of 84 serine/threonine kinase knockout strains. we identified mu ...201425253091
neurospora crassa as a model organism to explore the interconnected network of the cell cycle and the circadian clock.budding and fission yeast pioneered uncovering molecular mechanisms of eukaryotic cell division cycles. however, they do not possess canonical circadian clock machinery that regulates physiological processes with a period of about 24h. on the other hand, neurospora crassa played a critical role in elucidating molecular mechanisms of circadian rhythms, but have not been utilized frequently for cell cycle studies. recent findings demonstrate that there exists a conserved coupling between the cell ...201425239547
a high-throughput gene disruption methodology for the entomopathogenic fungus metarhizium robertsii.systematic gene disruption is a direct way to interrogate a fungal genome to functionally characterize the full suite of genes involved in various biological processes. metarhizium robertsii is extraordinarily versatile, and it is a pathogen of arthropods, a saprophyte and a beneficial colonizer of rhizospheres. thus, m. robertsii can be used as a representative to simultaneously study several major lifestyles that are not shared by the "model" fungi saccharomyces cerevisiae and neurospora crass ...201425222118
reaction-diffusion systems for spatio-temporal intracellular protein networks: a beginner's guide with two examples.spatio-temporal dynamics of a variety of proteins is, among other things, regulated by post-translational modifications of these proteins. such modifications can thus influence stability and biochemical activities of the proteins, activity and stability of their upstream targets within specific signalling pathways. commonly used mathematical tools for such protein-protein (and/or protein-mrna) interactions in single cells, namely, michaelis-menten and hill kinetics, yielding a system of ordinary ...201425210594
efficient gene targeting in ligase iv-deficient monascus ruber m7 by perturbing the non-homologous end joining pathway.inactivating the non-homologous end joining (nhej) pathway is a well established method to increase gene replacement frequency (grf) in filamentous fungi because nhej is predominant for the repair of dna double strand breaks (dsbs), while gene targeting is based on homologous recombination (hr). dna ligase iv, a component of the nhej system, is strictly required for the nhej in saccharomyces cerevisiae and neurospora crassa. to enhance the grf in monascus ruber m7, we deleted the mrlig4 gene enc ...201425209642
a family of starch-active polysaccharide monooxygenases.the recently discovered fungal and bacterial polysaccharide monooxygenases (pmos) are capable of oxidatively cleaving chitin, cellulose, and hemicelluloses that contain β(1→4) linkages between glucose or substituted glucose units. they are also known collectively as lytic pmos, or lpmos, and individually as aa9 (formerly gh61), aa10 (formerly cbm33), and aa11 enzymes. pmos share several conserved features, including a monocopper center coordinated by a bidentate n-terminal histidine residue and ...201425201969
isolation, characterization and application of a cellulose-degrading strain neurospora crassa s1 from oil palm empty fruit bunch.oil palm empty fruit bunch (efb) is a lignocellulosic waste produced in palm oil industry. efb mainly consists of cellulose, hemicellulose (mainly xylan) and lignin and has a great potential to be reused. converting efb to fermentable sugars and value-added chemicals is a much better choice than treating efb as waste.201425384340
cellulose surface degradation by a lytic polysaccharide monooxygenase and its effect on cellulase hydrolytic efficiency.lytic polysaccharide monooxygenase (lpmo) represents a unique principle of oxidative degradation of recalcitrant insoluble polysaccharides. used in combination with hydrolytic enzymes, lpmo appears to constitute a significant factor of the efficiency of enzymatic biomass depolymerization. lpmo activity on different cellulose substrates has been shown from the slow release of oxidized oligosaccharides into solution, but an immediate and direct demonstration of the enzyme action on the cellulose s ...201425361767
the transcription factor ste12 mediates the regulatory role of the tmk1 map kinase in mycoparasitism and vegetative hyphal fusion in the filamentous fungus trichoderma atroviride.mycoparasitic species of the fungal genus trichoderma are potent antagonists able to combat plant pathogenic fungi by direct parasitism. an essential step in this mycoparasitic fungus-fungus interaction is the detection of the fungal host followed by activation of molecular weapons in the mycoparasite by host-derived signals. the trichoderma atroviride map kinase tmk1, a homolog of yeast fus3/kss1, plays an essential role in regulating the mycoparasitic host attack, aerial hyphae formation and c ...201425356841
gene expression differences among three neurospora species reveal genes required for sexual reproduction in neurospora crassa.many fungi form complex three-dimensional fruiting bodies, within which the meiotic machinery for sexual spore production has been considered to be largely conserved over evolutionary time. indeed, much of what we know about meiosis in plant and animal taxa has been deeply informed by studies of meiosis in saccharomyces and neurospora. nevertheless, the genetic basis of fruiting body development and its regulation in relation to meiosis in fungi is barely known, even within the best studied mult ...201425329823
genetic characterization of the neurospora crassa molybdenum cofactor biosynthesis.molybdenum (mo) is a trace element that is essential for important cellular processes. to gain biological activity, mo must be complexed in the molybdenum cofactor (moco), a pterin derivative of low molecular weight. moco synthesis is a multi-step pathway that involves a variable number of genes in eukaryotes, which are assigned to four steps of eukaryotic moco biosynthesis. moco biosynthesis mutants lack any moco-dependent enzymatic activities, including assimilation of nitrate (plants and fung ...201424569084
the transcription factor bcltf1 regulates virulence and light responses in the necrotrophic plant pathogen botrytis cinerea.botrytis cinerea is the causal agent of gray mold diseases in a range of dicotyledonous plant species. the fungus can reproduce asexually by forming macroconidia for dispersal and sclerotia for survival; the latter also participate in sexual reproduction by bearing the apothecia after fertilization by microconidia. light induces the differentiation of conidia and apothecia, while sclerotia are exclusively formed in the absence of light. the relevance of light for virulence of the fungus is not o ...201424415947
fungal type iii polyketide synthases.this article covers the literature on fungal type iii polyketide synthases (pkss) published from 2005 to 2014. since the first discovery of fungal type iii pks genes in aspergillus oryzae, reported in 2005, putative genes for type iii pkss have been discovered in fungal genomes. compared with type i pkss, type iii pkss are much less abundant in fungi. however, type iii pkss could have some critical roles in fungi. this article summarizes the studies on fungal type iii pks functional analysis, in ...201425182423
a large nonconserved region of the tethering protein leashin is involved in regulating the position, movement, and function of woronin bodies in aspergillus oryzae.the woronin body is a pezizomycotina-specific organelle that is typically tethered to the septum, but upon hyphal wounding, it plugs the septal pore to prevent excessive cytoplasmic loss. leashin (lah) is a large woronin body tethering protein that contains highly conserved n- and c-terminal regions and a long (∼2,500-amino-acid) nonconserved middle region. as the involvement of the nonconserved region in woronin body function has not been investigated, here, we functionally characterized indivi ...201424813188
sensitivity of neurospora crassa to a marine-derived aspergillus tubingensis anhydride exhibiting antifungal activity that is mediated by the mas1 protein.the fungus aspergillus tubingensis (strain oy907) was isolated from the mediterranean marine sponge ircinia variabilis. extracellular extracts produced by this strain were found to inhibit the growth of several fungi. among the secreted extract components, a novel anhydride metabolite, tubingenoic anhydride a (1) as well as the known 2-carboxymethyl-3-hexylmaleic acid anhydride, asperic acid, and campyrone a and c were purified and their structure elucidated. compound 1 and 2-carboxymethyl-3-hex ...201425257783
interdependence of the actin and the microtubule cytoskeleton during fungal growth.cell polarization is a theme in biology conserved from bacteria to man. one of the most extremely polarized cells in nature is the hyphae of filamentous fungi. a continuous flow of secretion vesicles from the hyphal cell body to the tip is essential for cell wall and membrane extension. microtubules (mts) and actin, along with their corresponding motor proteins, are involved in the secretion process. therefore, the arrangement of the cytoskeleton is a crucial step to establish and maintain polar ...201424879477
regulation of vectorial supply of vesicles to the hyphal tip determines thigmotropism in neurospora crassa.thigmotropism is the ability of an organism to respond to a topographical stimulus by altering its axis of growth. the thigmotropic response of the model fungus neurospora crassa was quantified using microfabricated glass slides with ridges of defined height. we show that the polarity machinery at the hyphal tip plays a role in the thigmotropic response of n. crassa. deletion of n. crassa genes encoding the formin, bni-1, and the rho-gtpase, cdc-42, an activator of bni-1 in yeast, cdc-24, its gu ...201424607352
predicting essential genes for identifying potential drug targets in aspergillus fumigatus.aspergillus fumigatus (af) is a ubiquitous and opportunistic pathogen capable of causing acute, invasive pulmonary disease in susceptible hosts. despite current therapeutic options, mortality associated with invasive af infections remains unacceptably high, increasing 357% since 1980. therefore, there is an urgent need for the development of novel therapeutic strategies, including more efficacious drugs acting on new targets. thus, as noted in a recent review, "the identification of essential ge ...201424569026
nitrogen regulation of fungal secondary metabolism in fungi.fungi occupy diverse environments where they are constantly challenged by stressors such as extreme ph, temperature, uv exposure, and nutrient deprivation. nitrogen is an essential requirement for growth, and the ability to metabolize a wide variety of nitrogen sources enables fungi to colonize different environmental niches and survive nutrient limitations. favored nitrogen sources, particularly ammonium and glutamine, are used preferentially, while the expression of genes required for the use ...201425506342
potential conservation of circadian clock proteins in the phylum nematoda as revealed by bioinformatic searches.although several circadian rhythms have been described in c. elegans, its molecular clock remains elusive. in this work we employed a novel bioinformatic approach, applying probabilistic methodologies, to search for circadian clock proteins of several of the best studied circadian model organisms of different taxa (mus musculus, drosophila melanogaster, neurospora crassa, arabidopsis thaliana and synechoccocus elongatus) in the proteomes of c. elegans and other members of the phylum nematoda. wi ...201425396739
efficient formation of heterokaryotic sclerotia in the filamentous fungus aspergillus oryzae.heterokaryon formation by hyphal fusion occurs during a sexual/parasexual cycle in filamentous fungi, and therefore, it is biotechnologically important for crossbreeding. in the industrial filamentous fungus aspergillus oryzae, a parasexual cycle has been reported, and it was recently suggested that sexual reproduction should be possible. however, as a. oryzae enters into hyphal fusion with a much lower frequency than neurospora crassa, the process of heterokaryon formation has not been extensiv ...201424201891
the aspergillus nidulans zn(ii)2cys6 transcription factor an5673/rhar mediates l-rhamnose utilization and the production of α-l-rhamnosidases.various plant-derived substrates contain l-rhamnose that can be assimilated by many fungi and its liberation is catalyzed by α-l-rhamnosidases. initial data obtained in our laboratory focussing on two aspergillus nidulans α-l-rhamnosidase genes (rhaa and rhae) showed α-l-rhamnosidase production to be tightly controlled at the level of transcription by the carbon source available. whilst induction is effected by l-rhamnose, unlike many other glycosyl hydrolase genes repression by glucose and othe ...201425416526
neurospora crassa female development requires the pacc and other signal transduction pathways, transcription factors, chromatin remodeling, cell-to-cell fusion, and autophagy.using a screening protocol we have identified 68 genes that are required for female development in the filamentous fungus neurospora crassa. we find that we can divide these genes into five general groups: 1) genes encoding components of the pacc signal transduction pathway, 2) other signal transduction pathway genes, including genes from the three n. crassa map kinase pathways, 3) transcriptional factor genes, 4) autophagy genes, and 5) other miscellaneous genes. complementation and rip studies ...201425333968
bem46 shows eisosomal localization and association with tryptophan-derived auxin pathway in neurospora crassa.bem46 proteins are evolutionarily conserved, but their functions remain elusive. we reported previously that the bem46 protein in neurospora crassa is targeted to the endoplasmic reticulum (er) and is essential for ascospore germination. in the present study, we established a bem46 knockout strain of n. crassa. this δbem46 mutant exhibited a level of ascospore germination lower than that of the wild type but much higher than those of the previously characterized bem46-overexpressing and rna inte ...201424928924
cold-induced increase of glycerol kinase activity in neurospora crassa: rapid inactivation of the enzyme in vivo.the glycerol kinase activity induced by incubation of neurospora crassa at low temperatures was rapidly lost when cultures were returned to 26 c. after a short lag, the activity disappeared irreversibly with a half-life of approximately 15 min. the loss of activity was not due to a change in the level of an inhibitor or activator. glycerol reduced the activity loss but did not completely prevent it, which was an effect that was dependent on protein synthesis. the cold-induced activity was also a ...20144281774
induction of neurospora crassa laccase with protein synthesis inhibitors.rapidly growing neurospora crassa does not produce laccase (p-diphenol:oxygen oxidoreductase; ec 1.10.3.2). low concentrations of cycloheximide induce the production of this enzyme, most of which is secreted into the media. in general, limited inhibition of protein synthesis seems to derepress laccase synthesis since actinomycin d and, to a limited extent, puromycin also induce laccase production. similarities in the conditions of laccase and tyrosinase induction, plus investigations with two ty ...20144278535
cyanide-resistant respiration in neurospora crassa.cell respiration in wild type and poky was studied as part of a long-term investigation of cyanide-resistant respiration in neurospora. respiration in wild type proceeds via a cytochrome chain which is similar to that of higher organisms; it is sensitive to antimycin a or cyanide. poky, on the other hand, respires by means of two alternative oxidase systems. one of these is analogous to the wild-type cytochrome chain in that it can be inhibited by antimycin a or cyanide; this system accounts for ...20144333318
interaction of galactosaminoglycan with neurospora conidia.the inactivation of neurospora crassa conidia by galactosaminoglycan isolated from cultures of this organism was followed by measuring colony-forming ability and ability to take up radiolabeled metabolites. when kinetic data on the loss of transport function and on killing were analyzed by use of target theory, it appeared that few "hits" are required for inactivation. however, studies with radio-labeled galactosaminoglycan mucopolysaccharides showed that cells receiving a single lethal hit have ...20144281775
phosphate starvation in fungi induces the replacement of phosphatidylcholine with the phosphorus-free betaine lipid diacylglyceryl-n,n,n-trimethylhomoserine.diacylglyceryl-n,n,n-trimethylhomoserine (dgts) is a phosphorus-free betaine-lipid analog of phosphatidylcholine (ptdcho) synthesized by many soil bacteria, algae, and nonvascular plants. synthesis of dgts and other phosphorus-free lipids in bacteria occurs in response to phosphorus (p) deprivation and results in the replacement of phospholipids by nonphosphorous lipids. the genes encoding dgts biosynthetic enzymes have previously been identified and characterized in bacteria and the alga chlamy ...201424728191
molecular evolution and functional characterisation of an ancient phenylalanine ammonia-lyase gene (nnpal1) from nelumbo nucifera: novel insight into the evolution of the pal family in angiosperms.phenylalanine ammonia-lyase (pal; e.c.4.3.1.5) is a key enzyme of the phenylpropanoid pathway in plant development, and it catalyses the deamination of phenylalanine to trans-cinnamic acid, leading to the production of secondary metabolites. this enzyme has been identified in many organisms, ranging from prokaryotes to higher plants. because nelumbo nucifera is a basal dicot rich in many secondary metabolites, it is a suitable candidate for research on the phenylpropanoid pathway.201424884360
czt-1 is a novel transcription factor controlling cell death and natural drug resistance in neurospora crassa.we pinpoint czt-1 (cell death-activated zinc cluster transcription factor) as a novel transcription factor involved in tolerance to cell death induced by the protein kinase inhibitor staurosporine in neurospora crassa. transcriptional profiling of staurosporine-treated wild-type cells by rna-sequencing showed that genes encoding the machinery for protein synthesis are enriched among the genes repressed by the drug. functional category enrichment analyses also show that genes encoding components ...201424717808
role of the alternaria alternata blue-light receptor lrea (white-collar 1) in spore formation and secondary metabolism.alternaria alternata is a filamentous fungus that causes considerable loss of crops of economically important feed and food worldwide. it produces more than 60 different secondary metabolites, among which alternariol (aoh) and altertoxin (atx) are the most important mycotoxins. we found that mycotoxin production and spore formation are regulated by light in opposite ways. whereas spore formation was largely decreased under light conditions, the production of aoh was stimulated 2- to 3-fold. atx ...201424532063
spin-labeled mitochondrial lipids in neurospora crassa. 20144301148
activation of membrane-bound adenylate cyclase by glucagon in neurospora crassa.membrane-bound adenylate cyclase in neurospora crassa is activated by glucagon. half-maximal effect is observed at hormone concentrations of about 10 nm. after solubilization of the enzyme with lubrol-px, the glucagon effect is lost. incubation of neurospora cells with glucagon leads to a decrease in the activity of glycogen synthetase (ec 2.4.1.11) and to an increase in the activity of glycogen phosphorylase (ec 2.4.1.1) and in the rate of glycogenolysis.20144263508
[effect of some orotaldehyde derivatives as orotic acid antagonists in neurospora crassa]. 20144391358
preparation of mitochondrial membrane proteins from neurospora crassa: prevention of lipid autoxidation damage by an antioxidant. 20144266161
genetic alterations at the molecular level in x-ray induced ad-3b mutants of neurospora crassa. 20144265447
cytochrome b in neurospora crassa mitochondria. site of translation of the heme protein. 20144273944
[isolation and properties of plasma membranes of neurospora crassa mycelium]. 20144264117
genetic recombination in neurospora crassa affected by changes in the supply of cellular energy. 20144281764
genetic analysis of a suppressor of urease-deficiency in neurospora crassa. 20144281763
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