Disordered clock protein interactions and charge blocks turn an hourglass into a persistent circadian oscillator.

Organismal physiology is widely regulated by the molecular circadian clock, a feedback loop composed of protein complexes whose members are enriched in intrinsically disordered regions. These regions can mediate protein-protein interactions via SLiMs, but the contribution of these disordered regions to clock protein interactions had not been elucidated. To determine the functionality of these disordered regions, we applied a synthetic peptide microarray approach to the disordered clock protein FRQ in Neurospora crassa. We identified residues required for FRQ's interaction with its partner protein FRH, the mutation of which demonstrated FRH is necessary for persistent clock oscillations but not repression of transcriptional activity. Additionally, the microarray demonstrated an enrichment of FRH binding to FRQ peptides with a net positive charge. We found that positively charged residues occurred in significant "blocks" within the amino acid sequence of FRQ and that ablation of one of these blocks affected both core clock timing and physiological clock output. Finally, we found positive charge clusters were a commonly shared molecular feature in repressive circadian clock proteins. Overall, our study suggests a mechanistic purpose for positive charge blocks and yielded insights into repressive arm protein roles in clock function.

Phosphorylation, disorder, and phase separation govern the behavior of Frequency in the fungal circadian clock.

Circadian clocks are composed of transcription-translation negative feedback loops that pace rhythms of gene expression to the diurnal cycle. In the filamentous fungus Neurospora crassa, the proteins Frequency (FRQ), the FRQ-interacting RNA helicase (FRH), and Casein-Kinase I (CK1) form the FFC complex that represses expression of genes activated by the white-collar complex (WCC). FRQ orchestrates key molecular interactions of the clock despite containing little predicted tertiary structure. Spin labeling and pulse-dipolar electron spin resonance spectroscopy provide domain-specific structural insights into the 989-residue intrinsically disordered FRQ and the FFC. FRQ contains a compact core that associates and organizes FRH and CK1 to coordinate their roles in WCC repression. FRQ phosphorylation increases conformational flexibility and alters oligomeric state, but the changes in structure and dynamics are non-uniform. Full-length FRQ undergoes liquid-liquid phase separation (LLPS) to sequester FRH and CK1 and influence CK1 enzymatic activity. Although FRQ phosphorylation favors LLPS, LLPS feeds back to reduce FRQ phosphorylation by CK1 at higher temperatures. Live imaging of Neurospora hyphae reveals FRQ foci characteristic of condensates near the nuclear periphery. Analogous clock repressor proteins in higher organisms share little position-specific sequence identity with FRQ; yet, they contain amino acid compositions that promote LLPS. Hence, condensate formation may be a conserved feature of eukaryotic clocks.

Natural oscillations known as circadian rhythms influence many processes in humans and other animals including sleep, eating, brain activity and body temperature. These rhythms allow us to anticipate and prepare for regular changes in our environment including day-night cycles and the temperature of our surroundings. Circadian clocks in animals, fungi and other 'eukaryotic' organisms rely on networks of components that repress their own production to generate oscillations in their levels in cells over the course of a 24-hour period. The components in animal and fungus circadian clocks are different but there are strong similarities in their properties and how the networks operate. As a result, a type of fungus known as Neurospora crassa is often used as a model to study how circadian rhythms work in animals. A central component in the N. crassa circadian clock is a protein known as Frequency (FRQ). It is a large protein that, unlike most proteins, lacks a well-defined, three-dimensional structure. Despite this, it is able to bind to and regulate other proteins to repress its own production. One of its protein partners known as CK1 attaches small tags known as phosphate groups to FRQ to set the length of the circadian rhythm. However, it remains unclear how FRQ interacts with its protein partners or what effect the phosphate groups have on its activity. To address this question, Tariq, Maurici et al. used biochemical approaches to study the structure of FRQ. The experiments revealed that it contains a compact core that is able to bind to CK1 and other protein partners. The way FRQ regulates its protein partners is unusual: it undergoes a chemical process known as liquid-liquid phase separation to sequester other circadian clock proteins and modulate their enzymatic activities. In this process, a solution containing molecules of FRQ separates into two distinct components (known as phases), one of which contains FRQ and its partners in a concentrated liquid-like mixture. Evidence for such mixtures has also been found in living fungal cells. Further experiments suggest that liquid-liquid phase separation of FRQ may allow the clock to compensate for changes in temperature to maintain a regular rhythm. The circadian clocks of animals and other organisms all have proteins that perform similar roles as FRQ and maintain sequence properties that promote liquid-liquid phase separation. Therefore, it is possible that liquid-liquid phase separation may be a common feature of circadian rhythms in nature.

CDF family of zinc transporters ZRC-1, MSC-2, and ZRG-17 are involved in survival at high zinc conditions, vegetative development, and cellulase utilization in Neurospora crassa.

The cation diffusion facilitator (CDF) family of zinc transporters plays a crucial role in zinc homeostasis in eukaryotes, including fungi. Here, we investigated the cell functions and genetic interactions of CDF zinc transporters zrc-1 and msc-2 in Neurospora crassa. The Δzrc-1 mutant could not grow in a high-zinc environment, indicating that the zinc transporter protein ZRC-1 was essential for growth in high-zinc conditions. However, the deletion of msc-2 did not show any severe phenotypic defects. Furthermore, we studied the genetic interactions of the zinc transporters using the CDF double mutants. Previously, zrg-17 was reported to be critical, where the Δzrg-17 mutant showed defects in both vegetative development and asexual sporulation. Interestingly, the Δmsc-2;Δzrg-17 double mutant showed phenotypes similar to the wild type, and restored the phenotypic defects of the Δzrg-17 mutation. However, the Δzrc-1;Δmsc-2 and Δzrc-1;Δzrg-17 double mutants continue to display phenotypic defects like their parental single mutants. The double mutant Δzrc-1;Δzrg-17 showed severe vegetative growth defects, including slow growth, short aerial hyphae, narrowed septation, and defective asexual sporulation. In addition, aerial hyphae development of the Δzrc-1;Δmsc-2 and Δzrc-1;Δzrg-17 double mutants were reduced under endoplasmic reticulum stress. Thus, this study revealed the cell functions and genetic interactions of zrc-1, msc-2, and zrg-17 for vegetative development and tolerance to stress conditions in N. crassa.

Conversion of Deproteinized Cheese Whey to Lactobionate by an Engineered Neurospora crassa Strain F5.

We report a novel production process for lactobionic acid (LBA) production using an engineered Neurospora crassa strain F5. The wild-type N. crassa strain produces cellobiose dehydrogenase (CDH) and uses lactose as a carbon source. N. crassa strain F5, which was constructed by deleting six out of the seven ß-glucosidases in the wild type, showed a much slower lactose utilization rate and produced a much higher level of cellobiose dehydrogenase (CDH) than the wild type. Strain N. crassa F5 produced CDH and laccase simultaneously on the pretreated wheat straw with 3 µM of cycloheximide added as the laccase inducer. The deproteinized cheese whey was added directly to the shake flasks with the fungus present to achieve LBA production. Strain F5 produced about 37 g/L of LBA from 45 g/L of lactose in 27 h since deproteinized cheese whey addition. The yield of LBA from consumed lactose was about 85%, and the LBA productivity achieved was about 1.37 g/L/h.

A rapid PCR-based method to determine the Neurospora crassa mating type.

So far mating type determination in Neurospora crassa requires test crosses with strains of known mating type. We present a simple, quick, and reliable polymerase chain reaction-based method for mating type determination in N. crassa.

Cloning and expression of Neurospora crassa cellobiohydrolase II in Pichia pastoris.

A major cellobiohydrolase of Neurospora crassa CBH2 was successfully expressed in Pichia pastoris. The maximum Avicelase activity in shake flask among seven transformants which selected on 4.0 g/L G418 plates was 0.61 U/mL. The optimal pH and temperature for Avicelase activity of the recombinant CBH2 were determined to be 4.8 and 60 °C, respectively. The new CBH2 maintained 63.5 % Avicelase activity in the range of pH 4.0-10.4, and 60.2 % Avicelase activity in the range of 30-90 °C. After incubation at 70-90 °C for 1 h, the Avicelase activity retained 60.5 % of its initial activity. The presence of Zn2+, Ca2+ or Cd2+ enhanced the Avicelase activity of the CBH2, of which Cd2+ at 10 mM causing the highest increase. The recombinant CBH2 was used to enhance the Avicel hydrolysis by improving the exo-exo-synergism between CBH2 and CBH1 in N.crassa cellulase. The enzymatic hydrolysis yield was increased by 38.1 % by adding recombinant CBH2 and CBH1, and the yield was increased by 215.4 % when the temperature is raised to 70 °C. This work provided a CBH2 with broader pH range and better heat resistance, which is a potential enzyme candidate in food, textile, pulp and paper industries, and other industrial fields.

Recombination hotspots in Neurospora crassa controlled by idiomorphic sequences and meiotic silencing.

Genes regulating recombination in specific chromosomal intervals of Neurospora crassa were described in the 1960s, but the mechanism is still unknown. For each of the rec-1, rec-2, and rec-3 genes, a single copy of the putative dominant allele, for example, rec-2SL found in St Lawrence OR74 A wild type, reduces recombination in chromosomal regions specific to that gene. However, when we sequenced the recessive allele, rec-2LG (derived from the Lindegren 1A wild type), we found that a 10 kb region in rec-2SL strains was replaced by a 2.7 kb unrelated sequence, making the "alleles" idiomorphs. When we introduced sad-1, a mutant lacking the RNA-dependent RNA polymerase that silences unpaired coding regions during meiosis into crosses heterozygous rec-2SL/rec-2LG, it increased recombination, indicating that meiotic silencing of a gene promoting recombination is responsible for dominant suppression of recombination. Consistent with this, mutation of rec-2LG by Repeat-Induced Point mutation generated an allele with multiple stop codons in the predicted rec-2 gene, which does not promote recombination and is recessive to rec-2LG. Sad-1 also relieves suppression of recombination in relevant target regions, in crosses heterozygous for rec-1 alleles and in crosses heterozygous for rec-3 alleles. We conclude that for all 3 known rec genes, 1 allele appears dominant only because meiotic silencing prevents the product of the active, "recessive," allele from stimulating recombination during meiosis. In addition, the proposed amino acid sequence of REC-2 suggests that regulation of recombination in Neurospora differs from any currently known mechanism.

Circadian clock- and temperature-associated genes contribute to overall genomic differentiation along elevation in lichenized fungi.

Circadian regulation is linked to local environmental adaptation, and many species with broad climatic niches display variation in circadian genes. Here, we hypothesize that lichenizing fungi occupying different climate zones tune their metabolism to local environmental conditions with the help of their circadian systems. We study two species of the genus Umbilicaria occupying similar climatic niches (Mediterranean and the cold temperate) in different continents. Using homology to Neurospora crassa genes, we identify gene sets associated with circadian rhythms (11 core, 39 peripheral genes) as well as temperature response (37 genes). Nucleotide diversity of these genes is significantly correlated with mean annual temperature, minimum temperature of the coldest month and mean temperature of the coldest quarter. Furthermore, we identify altitudinal clines in allele frequencies in several non-synonymous substitutions in core clock components, for example, white collar-like, frh-like and various ccg-like genes. A dN/dS approach revealed a few significant peripheral clock- and temperature-associated genes (e.g. ras-1-like, gna-1-like) that may play a role in fine-tuning the circadian clock and temperature-response machinery. An analysis of allele frequency changes demonstrated the strongest evidence for differentiation above the genomic background in the clock-associated genes in U. pustulata. These results highlight the likely relevance of the circadian clock in environmental adaptation, particularly frost tolerance, of lichens. Whether or not the fungal clock modulates the symbiotic interaction within the lichen consortium remains to be investigated. We corroborate the finding of genetic variation in clock components along altitude-not only latitude-as has been reported in other species.

Histone deacetylation and cytosine methylation compartmentalize heterochromatic regions in the genome organization of Neurospora crassa.

Chromosomes must correctly fold in eukaryotic nuclei for proper genome function. Eukaryotic organisms hierarchically organize their genomes, including in the fungus Neurospora crassa, where chromatin fiber loops compact into Topologically Associated Domain-like structures formed by heterochromatic region aggregation. However, insufficient data exist on how histone posttranslational modifications (PTMs), including acetylation, affect genome organization. In Neurospora, the HCHC complex [composed of the proteins HDA-1, CDP-2 (Chromodomain Protein-2), Heterochromatin Protein-1, and CHAP (CDP-2 and HDA-1 Associated Protein)] deacetylates heterochromatic nucleosomes, as loss of individual HCHC members increases centromeric acetylation, and alters the methylation of cytosines in DNA. Here, we assess whether the HCHC complex affects genome organization by performing Hi-C in strains deleted of the cdp-2 or chap genes. CDP-2 loss increases intra- and interchromosomal heterochromatic region interactions, while loss of CHAP decreases heterochromatic region compaction. Individual HCHC mutants exhibit different patterns of histone PTMs genome-wide, as CDP-2 deletion increases heterochromatic H4K16 acetylation, yet smaller heterochromatic regions lose H3K9 trimethylation and gain interheterochromatic region interactions; CHAP loss produces minimal acetylation changes but increases heterochromatic H3K9me3 enrichment. Loss of both CDP-2 and the DIM-2 DNA methyltransferase causes extensive genome disorder as heterochromatic-euchromatic contacts increase despite additional H3K9me3 enrichment. Our results highlight how the increased cytosine methylation in HCHC mutants ensures genome compartmentalization when heterochromatic regions become hyperacetylated without HDAC activity.

The transcriptional factor Clr-5 is involved in cellulose degradation through regulation of amino acid metabolism in Neurospora crassa.

BACKGROUND: Filamentous fungi are efficient degraders of plant biomass and the primary producers of commercial cellulolytic enzymes. While the transcriptional regulation mechanisms of cellulases have been continuously explored in lignocellulolytic fungi, the induction of cellulase production remains a complex multifactorial system, with several aspects still largely elusive. RESULTS: In this study, we identified a Zn2Cys6 transcription factor, designated as Clr-5, which regulates the expression of cellulase genes by influencing amino acid metabolism in Neurospora crassa during growth on cellulose. The deletion of clr-5 caused a significant decrease in secreted protein and cellulolytic enzyme activity of N. crassa, which was partially alleviated by supplementing with yeast extract. Transcriptomic profiling revealed downregulation of not only the genes encoding main cellulases but also those related to nitrogen metabolism after disruption of Clr-5 under Avicel condition. Clr-5 played a crucial role in the utilization of multiple amino acids, especially leucine and histidine. When using leucine or histidine as the sole nitrogen source, the Δclr-5 mutant showed significant growth defects on both glucose and Avicel media. Comparative transcriptomic analysis revealed that the transcript levels of most genes encoding carbohydrate-active enzymes and those involved in the catabolism and uptake of histidine, branched-chain amino acids, and aromatic amino acids, were remarkably reduced in strain Δclr-5, compared with the wild-type N. crassa when grown in Avicel medium with leucine or histidine as the sole nitrogen source. These findings underscore the important role of amino acid metabolism in the regulation of cellulase production in N. crassa. Furthermore, the function of Clr-5 in regulating cellulose degradation is conserved among ascomycete fungi. CONCLUSIONS: These findings regarding the novel transcription factor Clr-5 enhance our comprehension of the regulatory connections between amino acid metabolism and cellulase production, offering fresh prospects for the development of fungal cell factories dedicated to cellulolytic enzyme production in bio-refineries.

Lineage-specific genes are clustered with HET-domain genes and respond to environmental and genetic manipulations regulating reproduction in Neurospora.

Lineage-specific genes (LSGs) have long been postulated to play roles in the establishment of genetic barriers to intercrossing and speciation. In the genome of Neurospora crassa, most of the 670 Neurospora LSGs that are aggregated adjacent to the telomeres are clustered with 61% of the HET-domain genes, some of which regulate self-recognition and define vegetative incompatibility groups. In contrast, the LSG-encoding proteins possess few to no domains that would help to identify potential functional roles. Possible functional roles of LSGs were further assessed by performing transcriptomic profiling in genetic mutants and in response to environmental alterations, as well as examining gene knockouts for phenotypes. Among the 342 LSGs that are dynamically expressed during both asexual and sexual phases, 64% were detectable on unusual carbon sources such as furfural, a wildfire-produced chemical that is a strong inducer of sexual development, and the structurally-related furan 5-hydroxymethyl furfural (HMF). Expression of a significant portion of the LSGs was sensitive to light and temperature, factors that also regulate the switch from asexual to sexual reproduction. Furthermore, expression of the LSGs was significantly affected in the knockouts of adv-1 and pp-1 that regulate hyphal communication, and expression of more than one quarter of the LSGs was affected by perturbation of the mating locus. These observations encouraged further investigation of the roles of clustered lineage-specific and HET-domain genes in ecology and reproduction regulation in Neurospora, especially the regulation of the switch from the asexual growth to sexual reproduction, in response to dramatic environmental conditions changes.

The sensor histidine kinase (SLN1) and acetyl-CoA carboxylase (ACC1) coordinately regulate the response of Neurospora crassa to the springtail Sinella curviseta (Collembola: Entomobryidae) attack.

IMPORTANCE: Understanding the regulatory pathways by which fungi respond to environmental signals through interlinked genes provides insights into the interactions between fungi and insects. The coordinated optimization of the regulatory networks is necessary for fungi to adapt to their habitats. We demonstrated that the synergistic regulation of sensor histidine kinase (SLN1) and acetyl-CoA carboxylase (ACC1) plays a critical role in regulating the fungal response to Sinella curviseta stress. Furthermore, we found that the enhanced production of trehalose, carotenoids, and 5-MTHF plays crucial role in the resistance to the fungivore. Our results provide insights into the understanding of the adaptation of N. crassa to environmental stimuli.

The cell functions of phospholipase C-1, Ca2+/H+ exchanger-1, and secretory phospholipase A2 in tolerance to stress conditions and cellulose degradation in Neurospora crassa.

We investigated the cell functions of the Ca2+ signaling genes phospholipase C-1 (plc-1), Ca2+/H+ exchanger (cpe-1), and secretory phospholipase A2 (splA2) for stress responses and cellulose utilization in Neurospora crassa. The Δplc-1, Δcpe-1, and ΔsplA2 mutants displayed increased sensitivity to the alkaline pH and reduced survival during induced thermotolerance. The ΔsplA2 mutant also exhibited hypersensitivity to the DTT-induced endoplasmic reticulum (ER) stress, increased microcrystalline cellulose utilization, increased protein secretion, and glucose accumulation in the culture supernatants. Moreover, the ΔsplA2 mutant could not grow on microcrystalline cellulose during ER stress. Furthermore, plc-1, cpe-1, and splA2 synthetically regulate the acquisition of thermotolerance induced by heat shock, responses to alkaline pH and ER stress, and utilization of cellulose and other alternate carbon sources in N. crassa. In addition, expression of the alkaline pH regulator, pac-3, and heat shock proteins, hsp60, and hsp80 was reduced in the Δplc-1, Δcpe-1, and ΔsplA2 single and double mutants. The expression of the unfolded protein response (UPR) markers grp-78 and pdi-1 was also significantly reduced in the mutants showing growth defect during ER stress. The increased cellulolytic activities of the ΔsplA2 and Δcpe-1; ΔsplA2 mutants were due to increased cbh-1, cbh-2, and endo-2 expression in N. crassa. Therefore, plc-1, cpe-1, and splA2 are involved in stress responses and cellulose utilization in N. crassa.

Permissiveness and competition within and between Neurospora crassa syncytia.

A multinucleate syncytium is a common growth form in filamentous fungi. Comprehensive functions of the syncytial state remain unknown, but it likely allows for a wide range of adaptations to enable filamentous fungi to coordinate growth, reproduction, responses to the environment, and to distribute nuclear and cytoplasmic elements across a colony. Indeed, the underlying mechanistic details of how syncytia regulate cellular and molecular processes spatiotemporally across a colony are largely unexplored. Here, we implemented a strategy to analyze the relative fitness of different nuclear populations in syncytia of Neurospora crassa, including nuclei with loss-of-function mutations in essential genes, based on production of multinucleate asexual spores using flow cytometry of pairings between strains with differentially fluorescently tagged nuclear histones. The distribution of homokaryotic and heterokaryotic asexual spores in pairings was assessed between different auxotrophic and morphological mutants, as well as with strains that were defective in somatic cell fusion or were heterokaryon incompatible. Mutant nuclei were compartmentalized into both homokaryotic and heterokaryotic asexual spores, a type of bet hedging for maintenance and evolution of mutational events, despite disadvantages to the syncytium. However, in pairings between strains that were blocked in somatic cell fusion or were heterokaryon incompatible, we observed a "winner-takes-all" phenotype, where asexual spores originating from paired strains were predominantly one genotype. These data indicate that syncytial fungal cells are permissive and tolerate a wide array of nuclear functionality, but that cells/colonies that are unable to cooperate via syncytia formation actively compete for resources.

Parental effects in a filamentous fungus: Phenotype, fitness and mechanism.

In nature, organisms have to cope with constantly changing environments. In certain conditions, it may be advantageous for the parents to pass on information about the environment, or resources to their offspring. Such transfers are known as parental effects, and they are well documented in plants and animals, but not in other eukaryotes, such as fungi. Many fungi disperse through spores, and fungal spores can potentially carry information or resources to the next generation. Understanding parental effects and their evolutionary consequences in fungi is of vital importance as they perform crucial ecosystem functions. In this study, we investigated whether parental effects are present in the filamentous fungus Neurospora crassa, how long do they last, whether the effects are adaptive, and what is their mechanism. We performed a fully factorial match/mismatch experiment for a good and a poor quality environment, in which we measured the initial growth of strains that experienced either a matched or mismatched environment in their previous generation. We found a strong silver-spoon effect in initial mycelium growth, which lasted for one generation, and increased fitness during competition experiments. By using deletion mutants that lacked key genes in epigenetic processes, we show that epigenetic mechanisms are not involved in this effect. Instead, we show that spore glycogen content, glucose availability and a radical transcription shift in spores are the main mechanisms behind this parental effect.

Multiple calcium signaling genes play a role in the circadian period of Neurospora crassa.

The Ca2+ signaling genes cpe-1, plc-1, ncs-1, splA2, camk-1, camk-2, camk-3, camk-4, cmd, and cnb-1 are necessary for a normal circadian period length in Neurospora crassa. In addition, the Q10 values ranged between 0.8 and 1.2 for the single mutants lacking cpe-1, splA2, camk-1, camk-2, camk-3, camk-4, and cnb-1, suggesting that the circadian clock exhibits standard temperature compensation. However, the Q10 value for the ∆plc-1 mutant was 1.41 at 25 and 30 °C, 1.53 and 1.40 for the ∆ncs-1 mutant at 20 and 25 °C, and at 20 and 30 °C, respectively, suggesting a partial loss of temperature compensation in these two mutants. Moreover, expression of frq, a regulator of the circadian period, and the blue light receptor wc-1, were increased >2-fold in the Δplc-1, ∆plc-1; ∆cpe-1, and the ∆plc-1; ∆splA2 mutants at 20 °C. The frq mRNA level was increased >2-fold in the Δncs-1 mutant compared to the ras-1bd strain at 20 °C. Therefore, multiple Ca2+ signaling genes regulate the circadian period, by influencing expression of the frq and wc-1 genes that are critical for maintaining the normal circadian period length in N. crassa.

Domains required for the interaction of the central negative element FRQ with its transcriptional activator WCC within the core circadian clock of Neurospora.

In the negative feedback loop composing the Neurospora circadian clock, the core element, FREQUENCY (FRQ), binds with FRQ-interacting RNA helicase (FRH) and casein kinase 1 to form the FRQ-FRH complex (FFC) which represses its own expression by interacting with and promoting phosphorylation of its transcriptional activators White Collar-1 (WC-1) and WC-2 (together forming the White Collar complex, WCC). Physical interaction between FFC and WCC is a prerequisite for the repressive phosphorylations, and although the motif on WCC needed for this interaction is known, the reciprocal recognition motif(s) on FRQ remains poorly defined. To address this, we assessed FFC-WCC in a series of frq segmental-deletion mutants, confirming that multiple dispersed regions on FRQ are necessary for its interaction with WCC. Biochemical analysis shows that interaction between FFC and WCC but not within FFC or WCC can be disrupted by high salt, suggesting that electrostatic forces drive the association of the two complexes. As a basic sequence on WC-1 was previously identified as a key motif for WCC-FFC assembly, our mutagenetic analysis targeted negatively charged residues of FRQ, leading to identification of three Asp/Glu clusters in FRQ that are indispensable for FFC-WCC formation. Surprisingly, in several frq Asp/Glu-to-Ala mutants that vastly diminish FFC-WCC interaction, the core clock still oscillates robustly with an essentially wildtype period, indicating that the interaction between the positive and negative elements in the feedback loop is required for the operation of the circadian clock but is not a determinant of the period length.

The nutrient-sensing GCN2 signaling pathway is essential for circadian clock function by regulating histone acetylation under amino acid starvation.

Circadian clocks are evolved to adapt to the daily environmental changes under different conditions. The ability to maintain circadian clock functions in response to various stresses and perturbations is important for organismal fitness. Here, we show that the nutrient-sensing GCN2 signaling pathway is required for robust circadian clock function under amino acid starvation in Neurospora. The deletion of GCN2 pathway components disrupts rhythmic transcription of clock gene frq by suppressing WC complex binding at the frq promoter due to its reduced histone H3 acetylation levels. Under amino acid starvation, the activation of GCN2 kinase and its downstream transcription factor CPC-1 establish a proper chromatin state at the frq promoter by recruiting the histone acetyltransferase GCN-5. The arrhythmic phenotype of the GCN2 kinase mutants under amino acid starvation can be rescued by inhibiting histone deacetylation. Finally, genome-wide transcriptional analysis indicates that the GCN2 signaling pathway maintains robust rhythmic expression of metabolic genes under amino acid starvation. Together, these results uncover an essential role of the GCN2 signaling pathway in maintaining the robust circadian clock function in response to amino acid starvation, and demonstrate the importance of histone acetylation at the frq locus in rhythmic gene expression.

Chromatin structure influences rate and spectrum of spontaneous mutations in Neurospora crassa.

Although mutation rates have been extensively studied, variation in mutation rates throughout the genome is poorly understood. To understand patterns of genetic variation, it is important to understand how mutation rates vary. Chromatin modifications may be an important factor in determining variation in mutation rates in eukaryotic genomes. To study variation in mutation rates, we performed a mutation accumulation (MA) experiment in the filamentous fungus Neurospora crassa and sequenced the genomes of the 40 MA lines that had been propagated asexually for approximately 1015 [Formula: see text] mitoses. We detected 1322 mutations in total and observed that the mutation rate was higher in regions of low GC, in domains of H3K9 trimethylation, in centromeric regions, and in domains of H3K27 trimethylation. The rate of single-nucleotide mutations in euchromatin was [Formula: see text] In contrast, the mutation rate in H3K9me3 domains was 10-fold higher: 2.43 [Formula: see text] We also observed that the spectrum of single-nucleotide mutations was different between H3K9me3 and euchromatic domains. Our statistical model of mutation rate variation predicted a moderate amount of extant genetic variation, suggesting that the mutation rate is an important factor in determining levels of natural genetic variation. Furthermore, we characterized mutation rates of structural variants, complex mutations, and the effect of local sequence context on the mutation rate. Our study highlights that chromatin modifications are associated with mutation rates, and accurate evolutionary inferences should take variation in mutation rates across the genome into account.

Antifungal azoles trigger a xenobiotic detoxification pathway and chitin synthesis in Neurospora crassa.

The presence of antifungal drugs is prompting the fungal microorganisms to react by mechanisms broader than the resistance. The fungi evolved mechanisms, by which they respond to various stress conditions, including the presence of antifungal compounds. In this work, we studied the response of model filamentous fungus Neurospora crassa to azole antifungals in the broader context of the adaptation mechanisms. We demonstrated the increase in expression of filamentous fungi-specific genes encoding cytochrome enzymes of CYP65 clan and plasma membrane-localized ABCC transporters. Azoles appear not to conjugate with glutathione. Surprisingly, the azoles caused changes in the hyphae organization and the amount of chitin in cell wall by the same manner that was thought to be echinocandin-specific. The response to individual azoles appeared to be influenced by the structure of azole compound (prochloraz - main outlier). Taken together, these findings demonstrate the importance of study of stress response mechanisms, specifically in filamentous fungi. Many aspects of the reaction within azoles seem to be similar, though specificities are occurring.