Fungus Neurospora Crassa Research Articles

An Inactivation Switch Enables Rhythms in a Neurospora Clock Model.

Autonomous endogenous time-keeping is ubiquitous across many living organisms, known as the circadian clock when it has a period of about 24 h. Interestingly, the fundamental design principle with a network of interconnected negative and positive feedback loops is conserved through evolution, although the molecular components differ. Filamentous fungus Neurospora crassa is a well-established chrono-genetics model organism to investigate the underlying mechanisms. The core negative feedback loop of the clock of Neurospora is composed of the transcription activator White Collar Complex (WCC) (heterodimer of WC1 and WC2) and the inhibitory element called FFC complex, which is made of FRQ (Frequency protein), FRH (Frequency interacting RNA Helicase) and CK1a (Casein kinase 1a). While exploring their temporal dynamics, we investigate how limit cycle oscillations arise and how molecular switches support self-sustained rhythms. We develop a mathematical model of 10 variables with 26 parameters to understand the interactions and feedback among WC1 and FFC elements in nuclear and cytoplasmic compartments. We performed control and bifurcation analysis to show that our novel model produces robust oscillations with a wild-type period of 22.5 h. Our model reveals a switch between WC1-induced transcription and FFC-assisted inactivation of WC1. Using the new model, we also study the possible mechanisms of glucose compensation. A fairly simple model with just three nonlinearities helps to elucidate clock dynamics, revealing a mechanism of rhythms’ production. The model can further be utilized to study entrainment and temperature compensation.

Conidiation in Neurospora crassa: vegetative reproduction by a model fungus.

Asexual development, conidiation, in the filamentous fungus Neurospora crassa is a simple developmental process that starts with the growth of aerial hyphae. Then, the formation of constrictions and subsequent maturation gives rise to the mature conidia that are easily dispersed by air currents. Conidiation is regulated by environmental factors such as light, aeration and nutrient limitation, and by the circadian clock. Different regulatory proteins acting at different stages of conidiation have been described. The role of transcription factors such as FL, and components of signal transduction pathways such as the cAMP phosphodiesterase ACON-2 suggest a complex interplay between differential transcription and signal transduction pathways. Comparisons between the molecular basis of conidiation in N. crassa and other filamentous fungi will help to identify common regulatory elements.

Solution structure and novel insights into phylogeny and mode of action of the Neosartorya (Aspergillus) fischeri antifungal protein (NFAP)

Small, cysteine-rich and cationic antifungal proteins from natural sources are promising candidates for the development of novel treatment strategies to prevent and combat infections caused by drug-resistant fungi. However, limited information about their structure and antifungal mechanism hampers their future applications. In the present study, we determined the solution structure, dynamics and associated solvent areas of the Neosartorya (Aspergillus) fischeri antifungal protein NFAP. Genome mining within the genus revealed the presence of orthologous genes in N. fischeri and Neosartorya spathulata, and genes encoding closely related proteins can be found in Penicillium brasiliensis and Penicillium oxalicum. We show that the tertiary structure of these putative proteins can be resolved using the structure of NFAP as reliable template for in silico prediction. Localization studies with fluorescence-labelled protein pointed at an energy-dependent uptake mechanism of NFAP in the sensitive model fungus Neurospora crassa and subsequent cytoplasmic localization coincided with cell-death induction. The presented results contribute to a better understanding of the structure/function relationship of NFAP and related proteins and pave the way towards future antifungal drug development.

Integration of Self and Non-self Recognition Modulates Asexual Cell-to-Cell Communication in Neurospora crassa.

Cells rarely exist alone, which drives the evolution of diverse mechanisms for identifying and responding appropriately to the presence of other nearby cells. Filamentous fungi depend on somatic cell-to-cell communication and fusion for the development and maintenance of a multicellular, interconnected colony that is characteristic of this group of organisms. The filamentous fungus Neurospora crassa is a model for investigating the mechanisms of somatic cell-to-cell communication and fusion. N. crassa cells chemotropically grow toward genetically similar cells, which ultimately make physical contact and undergo cell fusion. Here, we describe the development of a Pprm1-luciferase reporter system that differentiates whether genes function upstream or downstream of a conserved MAP kinase (MAPK) signaling complex, by using a set of mutants required for communication and cell fusion. The vast majority of these mutants are deficient for self-fusion and for fusion when paired with wild-type cells. However, the Δham-11 mutant is unique in that it fails to undergo self-fusion, but chemotropic interactions and cell fusion are restored in Δham-11 + wild-type interactions. In genetically dissimilar cells, chemotropic interactions are regulated by genetic differences at doc-1 and doc-2, which regulate prefusion non-self recognition; cells with dissimilar doc-1 and doc-2 alleles show greatly reduced cell-fusion frequencies. Here, we show that HAM-11 functions in parallel with the DOC-1 and DOC-2 proteins to regulate the activity of the MAPK signaling complex. Together, our data support a model of integrated self and non-self recognition processes that modulate somatic cell-to-cell communication in N. crassa.

Degradation of anti‐nutritional factors and reduction of immunoreactivity of tempeh by co‐fermentation with Rhizopus oligosporus RT‐3 and Actinomucor elegans DCY‐1

SummaryThe effects of solid‐state fermentation with four filamentous fungi (Rhizopus oligosporus, Actinomucor elegans, Neurospora crassa and Rhizopus oryzae) and their combination on the degradation of protein allergens and anti‐nutritional factors (ANFs) of tempeh were evaluated. Glycinin, β‐conglycinin, trypsin inhibitor and flatulence‐causing oligosaccharides were significantly hydrolysed by all assayed strains but the level of reduction of ANFs depended on the strain. ELISA was conducted to evaluate IgE immunoreactivity using sera from soy sensitive individuals. Results revealed that co‐fermentation of R. oligosporus and A. elegans resulted in the highest reduction in IgE immunoreactivity for all sera used and showed greatest degradations on α’, α, β, acidic subunits (100% hydrolysed) and extensive hydrolysis of flatulence‐causing oligosaccharides. Tempeh fermented with R. oligosporus and A. elegans displayed high level of soluble protein and peptide (<10 kD) content. Co‐fermentation of R. oligosporus and A. elegans was proved to be effective in producing hypoallergenic tempeh.

Nucleosome Positioning by an Evolutionarily Conserved Chromatin Remodeler Prevents Aberrant DNA Methylation in Neurospora.

In the filamentous fungus Neurospora crassa, constitutive heterochromatin is marked by tri-methylation of histone H3 lysine 9 (H3K9me3) and DNA methylation. We identified mutations in the Neurospora defective in methylation-1 (dim-1) gene that cause defects in cytosine methylation and implicate a putative AAA-ATPase chromatin remodeler. Although it was well-established that chromatin remodelers can affect transcription by influencing DNA accessibility with nucleosomes, little was known about the role of remodelers on chromatin that is normally not transcribed, including regions of constitutive heterochromatin. We found that dim-1 mutants display both reduced DNA methylation in heterochromatic regions as well as increased DNA methylation and H3K9me3 in some intergenic regions associated with highly expressed genes. Deletion of dim-1 leads to atypically spaced nucleosomes throughout the genome and numerous changes in gene expression. DIM-1 localizes to both heterochromatin and intergenic regions that become hyper-methylated in dim-1 strains. Our findings indicate that DIM-1 normally positions nucleosomes in both heterochromatin and euchromatin and that the standard arrangement and density of nucleosomes is required for the proper function of heterochromatin machinery.

Structural studies of a glycoside hydrolase family 3 β-glucosidase from the model fungus Neurospora crassa.

The glycoside hydrolase family 3 (GH3) β-glucosidases are a structurally diverse family of enzymes. Cel3A from Neurospora crassa (NcCel3A) belongs to a subfamily of key enzymes that are crucial for industrial biomass degradation. β-Glucosidases hydrolyse the β-1,4 bond at the nonreducing end of cellodextrins. The hydrolysis of cellobiose is of special importance as its accumulation inhibits other cellulases acting on crystalline cellulose. Here, the crystal structure of the biologically relevant dimeric form of NcCel3A is reported. The structure has been refined to 2.25 Å resolution, with an Rcryst and Rfree of 0.18 and 0.22, respectively. NcCel3A is an extensively N-glycosylated glycoprotein that shares 46% sequence identity with Hypocrea jecorina Cel3A, the structure of which has recently been published, and 61% sequence identity with the thermophilic β-glucosidase from Rasamsonia emersonii. NcCel3A is a three-domain protein with a number of extended loops that deepen the active-site cleft of the enzyme. These structures characterize this subfamily of GH3 β-glucosidases and account for the high cellobiose specificity of this subfamily.

Asexual reproduction and growth rate: independent and plastic life history traits in Neurospora crassa

Trade-offs among traits influencing fitness are predicted by life history theory because resources allocated to one function are unavailable to another. Here we examine the relationship between two such traits, asexual reproduction and growth rate, in the filamentous fungus Neurospora crassa, where shared genetic and physiological factors and a source–sink energetic relationship between growth and reproduction may constrain the evolution of these traits. To test growth–reproduction relationships in this species, we independently selected on mycelial growth rate or asexual spore production in a heterogeneous lab-derived population and evaluated the response of the non-selected traits. Combined with phenotypes for the 20 wild strains used to produce the heterogeneous population and the genome-wide genotypes of 468 strains, these data show that growth and reproduction are highly plastic in N. crassa and do not trade off either among wild strains or after laboratory selection in two environments. Rather, we find no predictable growth–reproduction relationship in the environments tested, indicating an effective absence of genetic constraint between these traits. Our results suggest that growth rate and asexual reproduction may not respond predictably to environmental change and suggest that reliance on a single trait as a proxy for fitness in fungal studies may be inadvisable.

A conserved fungal hub protein involved in adhesion and drug resistance in the human pathogen Candida albicans

Drug resistance and cellular adhesion are two key elements of both dissemination and prevalence of the human fungal pathogen Candida albicans. Smi1 belongs to a family of hub proteins conserved among the fungal kingdom whose functions in cellular signaling affect morphogenesis, cell wall synthesis and stress resistance. The data presented here indicate that C. albicans SMI1 is a functional homolog of Saccharomyces cerevisiae KNR4 and is involved in the regulation of cell wall synthesis. Expression of SMI1 in S. cerevisiae knr4Δ null mutants rescued their sensitivity to caspofungin and to heat stress. Deletion of SMI1 in C. albicans resulted in sensitivity to the cell-wall-perturbing compounds Calcofluor White and Caspofungin. Analysis of wild-type and mutant cells by Atomic Force Microscopy showed that the Young’s Modulus (stiffness) of the cell wall was reduced by 85% upon deletion of SMI1, while cell surface adhesion measured by Force Spectroscopy showed that the surface expression of adhesive molecules was also reduced in the mutant. Over-expression of SMI1, on the contrary, increased cell surface adhesion by 6-fold vs the control strain. Finally, Smi1-GFP localized as cytoplasmic patches and concentrated spots at the sites of new cell wall synthesis including the tips of growing hyphae, consistent with a role in cell wall regulation. Thus, Smi1 function appears to be conserved across fungi, including the yeast S. cerevisiae, the yeast and hyphal forms of C. albicans and the filamentous fungus Neurospora crassa.

Circadian rhythms, metabolic oscillators, and the target of rapamycin (TOR) pathway: the Neurospora connection.

Circadian (24-h) rhythmicity is a fundamental property of eukaryotic cells, and it is not surprising that it intersects with fundamental metabolic processes. Many links between these two processes have been documented, and speculation has been growing that there may be circadian "metabolic oscillators" that interact with and exist independently of the well-known circadian transcription/translation feedback loops (TTFLs) that have been extensively studied. This review takes a critical look at the evidence for the existence of metabolic oscillators at the cellular level, attempting to answer these questions: does metabolism affect circadian rhythmicity, and vice versa? Is metabolism rhythmic, and if so, is that rhythmicity cell autonomous? Systems displaying "non-canonical rhythmicity" in the absence of functional TTFLs provide opportunities for identifying metabolic oscillators, and this review emphasizes the fungus Neurospora crassa as a model system. Recent papers describing links between the target of rapamycin (TOR) signaling pathway and circadian rhythmicity are highlighted, suggesting the potential for TOR signaling in generating rhythmicity independent of TTFLs.

Calcium binding of the antifungal protein PAF: Structure, dynamics and function aspects by NMR and MD simulations.

Calcium ions (Ca2+) play an important role in the toxicity of the cysteine-rich and cationic antifungal protein PAF from Penicillium chrysogenum: high extracellular Ca2+ levels reduce the toxicity of PAF in the sensitive model fungus Neurospora crassa in a concentration dependent way. However, little is known about the mechanistic details of the Ca2+ ion impact and the Ca2+ binding capabilities of PAF outside the fungal cell, which might be the reason for the activity loss. Using nuclear magnetic resonance (NMR), isothermal titration calorimetry and molecular dynamics (MD) simulations we demonstrated that PAF weakly, but specifically binds Ca2+ ions. MD simulations of PAF predicted one major Ca2+ binding site at the C-terminus involving Asp53 and Asp55, while Asp19 was considered as putative Ca2+ binding site. The exchange of Asp19 to serine had little impact on the Ca2+ binding, however caused the loss of antifungal activity, as was shown in our recent study. Now we replaced the C-terminal aspartates and expressed the serine variant PAFD53S/D55S. The specific Ca2+ binding affinity of PAFD53S/D55S decreased significantly if compared to PAF, whereas the antifungal activity was retained. To understand more details of Ca2+ interactions, we investigated the NMR and MD structure/dynamics of the free and Ca2+-bound PAF and PAFD53S/D55S. Though we found some differences between these protein variants and the Ca2+ complexes, these effects cannot explain the observed Ca2+ influence. In conclusion, PAF binds Ca2+ ions selectively at the C-terminus; however, this Ca2+ binding does not seem to play a direct role in the previously documented modulation of the antifungal activity of PAF.

LET dependence on killing effect and mutagenicity in the model filamentous fungus Neurospora crassa

Purpose: To assess the unique biological effects of different forms of ionizing radiation causing DNA double-strand breaks (DSBs), we compared the killing effect, mutagenesis frequency, and mutation type spectrum using the model filamentous fungus Neurospora.Materials and methods: Asexual spores of wild-type Neurospora and two DSB repair-deficient strains [one homologous recombination- and the other non-homologous end-joining (NHEJ) pathway-deficient] were irradiated with argon (Ar)-ion beams, ferrous (Fe)-ion beams, or X-rays. Relative biological effectiveness (RBE), forward mutation frequencies at the ad-3 loci, and mutation spectra at the ad-3B gene were determined.Results: The canonical NHEJ (cNHEJ)-deficient strain showed resistance to higher X-ray doses, while other strains showed dose-dependent sensitivity. In contrast, the killing effects of Ar-ion and Fe-ion beam irradiation were dose-dependent in all strains tested. The rank order of RBE was Ar-ion > Fe-ion > C-ion. Deletion mutations were the most common, but deletion size incremented with the increasing value of linear energy transfer (LET).Conclusions: We found marked differences in killing effect of a cNHEJ-deficient mutant between X-ray and high-LET ion beam irradiations (Ar and Fe). The mutation spectra also differed between irradiation types. These differences may be due to the physical properties of each radiation and the repair mechanism of induced damage in Neurospora crassa. These results may guide the choice of irradiation beam to kill or mutagenize fungi for agricultural applications or further research.

A-to-I mRNA editing in fungi: occurrence, function, and evolution.

A-to-I RNA editing is an important post-transcriptional modification that converts adenosine (A) to inosine (I) in RNA molecules via hydrolytic deamination. Although editing of mRNAs catalyzed by adenosine deaminases acting on RNA (ADARs) is an evolutionarily conserved mechanism in metazoans, organisms outside the animal kingdom lacking ADAR orthologs were thought to lack A-to-I mRNA editing. However, recent discoveries of genome-wide A-to-I mRNA editing during the sexual stage of the wheat scab fungus Fusarium graminearum, model filamentous fungus Neurospora crassa, Sordaria macrospora, and an early diverging filamentous ascomycete Pyronema confluens indicated that A-to-I mRNA editing is likely an evolutionarily conserved feature in filamentous ascomycetes. More importantly, A-to-I mRNA editing has been demonstrated to play crucial roles in different sexual developmental processes and display distinct tissue- or development-specific regulation. Contrary to that in animals, the majority of fungal RNA editing events are non-synonymous editing, which were shown to be generally advantageous and favored by positive selection. Many non-synonymous editing sites are conserved among different fungi and have potential functional and evolutionary importance. Here, we review the recent findings about the occurrence, regulation, function, and evolution of A-to-I mRNA editing in fungi.

Fabrication of In-Channel High-Aspect Ratio Sensing Pillars for Protrusive Force Measurements on Fungi and Oomycetes

This paper reports the fabrication and application of a Lab-on-a-Chip platform containing single-elastomeric micropillars in channel constrictions, which enable the measurement of protrusive forces exerted by individual fungal hyphae. We show the device design, the fabrication process, and photoresist optimization required to adapt the microfluidic platform to relatively thin hyphae. To demonstrate the applicability of the devices, the oomycete Achlya bisexualis and the fungus Neurospora crassa were cultured on PDMS chips. Devices were combined with confocal imaging to study the interaction of A. bisexualis hyphae with the measurement pillars. The force exerted by individual hyphae of N. crassa was measured and compared with a hyphal growth rate and diameter. The platform provides a new tool to help understand the molecular processes that underlie protrusive growth and this may present new ways to tackle the pathogenic growth of these organisms and thus combat the loss of diversity that they cause. This paper is based on the conference proceedings presented at the 31st IEEE International Conference on Micro Electro Mechanical Systems (MEMS 2018), Belfast. [2018-0090]

Effects of acclimation time and epigenetic mechanisms on growth of Neurospora in fluctuating environments.

Reaction norms or tolerance curves have often been used to predict how organisms deal with fluctuating environments. A potential drawback is that reaction norms measured in different constant environments may not capture all aspects of organismal responses to fluctuating environments. We examined growth of the filamentous fungus Neurospora crassa in fluctuating temperatures and tested if growth in fluctuating temperatures can be explained simply by the growth in different constant temperatures or if more complex models are needed. In addition, as previous studies on fluctuating environments have revealed that past temperatures that organisms have experienced can affect their response to current temperature, we tested the roles of different epigenetic mechanisms in response to fluctuating environments using different mutants. We found that growth of Neurospora can be predicted in fluctuating temperatures to some extent if acclimation times are taken into account in the model. Interestingly, while fluctuating environments have been linked with epigenetic responses, we found only some evidence of involvement of epigenetic mechanisms on tolerating fluctuating temperatures. Mutants which lacked H3K4 or H3K36 methylation had slightly impaired response to temperature fluctuations, in addition the H3K4 methylation mutant and a mutant in the RNA interference pathway had altered acclimation times.

Fungal Light-Oxygen-Voltage Domains for Optogenetic Control of Gene Expression and Flocculation in Yeast.

ABSTRACTOptogenetic switches permit accurate control of gene expression upon light stimulation. These synthetic switches have become a powerful tool for gene regulation, allowing modulation of customized phenotypes, overcoming the obstacles of chemical inducers, and replacing their use by an inexpensive resource: light. In this work, we implemented FUN-LOV, an optogenetic switch based on the photon-regulated interaction of WC-1 and VVD, two LOV (light-oxygen-voltage) blue-light photoreceptors from the fungus Neurospora crassa. When tested in yeast, FUN-LOV yields light-controlled gene expression with exquisite temporal resolution and a broad dynamic range of over 1,300-fold, as measured by a luciferase reporter. We also tested the FUN-LOV switch for heterologous protein expression in Saccharomyces cerevisiae, where Western blot analysis confirmed strong induction upon light stimulation, surpassing by 2.5 times the levels achieved with a classic GAL4/galactose chemical-inducible system. Additionally, we utilized FUN-LOV to control the ability of yeast cells to flocculate. Light-controlled expression of the flocculin-encoding gene FLO1, by the FUN-LOV switch, yielded flocculation in light (FIL), whereas the light-controlled expression of the corepressor TUP1 provided flocculation in darkness (FID). Altogether, the results reveal the potential of the FUN-LOV optogenetic switch to control two biotechnologically relevant phenotypes such as heterologous protein expression and flocculation, paving the road for the engineering of new yeast strains for industrial applications. Importantly, FUN-LOV’s ability to accurately manipulate gene expression, with a high temporal dynamic range, can be exploited in the analysis of diverse biological processes in various organisms.

Light induces oxidative damage and protein stability in the fungal photoreceptor Vivid.

Flavin-binding photoreceptor proteins sense blue-light (BL) in diverse organisms and have become core elements in recent optogenetic applications. The light-oxygen-voltage (LOV) protein Vivid (VVD) from the filamentous fungus Neurospora crassa is a classic BL photoreceptor, characterized by effecting a photocycle based on light-driven formation and subsequent spontaneous decay of a flavin-cysteinyl adduct. Here we report that VVD presents alternative outcomes to light exposure that result in protein self-oxidation and, unexpectedly, rise of stability through kinetic control. Using optical absorbance and mass spectrometry we show that purified VVD develops amorphous aggregates with the presence of oxidized residues located at the cofactor binding pocket. Light exposure increases oxidative levels in VVD and specific probe analysis identifies singlet oxygen production by the flavin. These results indicate that VVD acts alternatively as a photosensitizer, inducing self-oxidative damage and subsequent aggregation. Surprisingly, BL illumination has an additional, opposite effect in VVD. We show that light-induced adduct formation establishes a stable state, delaying protein aggregation until photoadduct decay occurs. In accordance, repeated BL illumination suppresses VVD aggregation altogether. Furthermore, photoadduct formation confers VVD stability against chemical denaturation. Analysis of the aggregation kinetics and testing of stabilizers against aggregation reveal that aggregation in VVD proceeds through light-dependent kinetic control and dimer formation. These results uncover the aggregation pathway of a photosensor, where light induces a remarkable interplay between protein damage and stability.

Expression of putative circadian clock components in the arbuscular mycorrhizal fungus Rhizoglomus irregulare.

Arbuscular mycorrhizal fungi (AMF) are obligatory plant symbionts that live underground, so few studies have examined their response to light. Responses to blue light by other fungi can be mediated by White Collar-1 (WC-1) and WC-2 proteins. These wc genes, together with the frequency gene (frq), also form part of the endogenous circadian clock. The clock mechanism has never been studied in AMF, although circadian growth of their hyphae in the field has been reported. Using both genomic and transcriptomic data, we have found homologs of wc-1, wc-2, and frq and related circadian clock genes in the arbuscular mycorrhizal fungus Rhizoglomus irregulare (synonym Rhizophagus irregularis). Gene expression of wc-1, wc-2, and frq was analyzed using RT-qPCR on RNA extracted from germinating spores and from fungal material cultivated in vitro with transformed carrot roots. We found that all three core clock genes were expressed in both pre- and post-mycorrhizal stages of R. irregulare growth. Similar to the model fungus Neurospora crassa, the core circadian oscillator gene frq was induced by brief light stimulation. The presence of circadian clock and output genes in R. irregulare opens the door to the study of circadian clocks in the fungal partner of plant-AMF symbiosis. Our finding also provides new insight into the evolution of the circadian frq gene in fungi.

Disruption of gul-1 decreased the culture viscosity and improved protein secretion in the filamentous fungus Neurospora crassa

BackgroundThe cellulolytic fungus Neurospora crassa is considered a potential host for enzyme and bioethanol production. However, large scale applications are hindered by its filamentous growth. Although previous investigations have shown that mycelial morphology in submerged culture can be controlled by altering physical factors, there is little knowledge available about the potential for morphology control by genetic modification.ResultsIn this study, we screened morphological mutants in the filamentous fungus N. crassa. Of the 90 morphological mutants screened, 14 mutants exhibited considerably higher viscosity compared with that of the wild type strain, and only two mutants showed low-viscosity morphologies in submerged culture. We observed that disruption of gul-1 (NCU01197), which encodes an mRNA binding protein involved in cell wall remodeling, caused pellet formation as the fermentation progressed, and resulted in the most significant decrease in viscosity of culture broth. Moreover, over-expression of gul-1 caused dramatically increased viscosity, suggesting that the gul-1 had an important function in mycelial morphology during submerged cultivation. Transcriptional profiling showed that expression of genes encoding eight GPI-anchored cell wall proteins was lowered in Δgul-1 while expression of genes associated with two non-anchored cell wall proteins was elevated. Meanwhile, the expression levels of two hydrophobin genes were also significantly altered. These results suggested that GUL-1 affected the transcription of cell wall-related genes, thereby influencing cell wall structure and mycelial morphology. Additionally, the deletion of gul-1 caused increased protein secretion, probably due to a defect in cell wall integrity, suggesting this as an alternative strategy of strain improvement for enzyme production. To confirm practical applications, deletion of gul-1 in the hyper-cellulase producing strain (∆ncw-1∆Ncap3m) significantly reduced the viscosity of culture broth.ConclusionsUsing the model filamentous fungus N. crassa, genes that affect mycelial morphology in submerged culture were explored through systematic screening of morphological mutants. Disrupting several candidate genes altered viscosities in submerged culture. This work provides an example for controlling fungal morphology in submerged fermentation by genetic engineering, and will be beneficial for industrial fungal strain improvement.

Mus-52 disruption and metabolic regulation in Neurospora crassa: Transcriptional responses to extracellular phosphate availability.

Advances in the understanding of molecular systems depend on specific tools like the disruption of genes to produce strains with the desired characteristics. The disruption of any mutagen sensitive (mus) genes in the model fungus Neurospora crassa, i.e. mus-51, mus-52, or mus-53, orthologous to the human genes KU70, KU80, and LIG4, respectively, provides efficient tools for gene targeting. Accordingly, we used RNA-sequencing and reverse transcription-quantitative polymerase chain reaction amplification techniques to evaluate the effects of mus-52 deletion in N. crassa gene transcriptional modulation, and thus, infer its influence regarding metabolic response to extracellular availability of inorganic phosphate (Pi). Notably, the absence of MUS-52 affected the transcription of a vast number of genes, highlighting the expression of those coding for transcription factors, kinases, circadian clocks, oxi-reduction balance, and membrane- and nucleolus-related proteins. These findings may provide insights toward the KU molecular mechanisms, which have been related to telomere maintenance, apoptosis, DNA replication, and gene transcription regulation, as well as associated human conditions including immune system disorders, cancer, and aging.