Model Organism Neurospora Crassa Research Articles

The First Observation of the Filamentous Fungus Neurospora crassa Growing in the Roots of the Grass Brachypodium distachyon.

The model organism Neurospora crassa has been cultivated in laboratories since the 1920s and its saprotrophic lifestyle has been established for decades. However, beyond their role as saprotrophs, fungi engage in intricate relationships with plants, showcasing diverse connections ranging from mutualistic to pathogenic. Although N. crassa has been extensively investigated under laboratory conditions, its ecological characteristics remain largely unknown. In contrast, Brachypodium distachyon, a sweet grass closely related to significant crops, demonstrates remarkable ecological flexibility and participates in a variety of fungal interactions, encompassing both mutualistic and harmful associations. Through a comprehensive microscopic analysis using electron, fluorescence, and confocal laser scanning microscopy, we discovered a novel endophytic interaction between N. crassa and B. distachyon roots, where fungal hyphae not only thrive in the apoplastic space and vascular bundle but also may colonize plant root cells. This new and so far hidden trait of one of the most important fungal model organisms greatly enhances our view of N. crassa, opening new perspectives concerning the fungus' ecological role. In addition, we present a new tool for studying plant-fungus interspecies communication, combining two well-established model systems, which improves our possibilities of experimental design on the molecular level.

RHO-3 plays a significant role in hyphal extension rate, conidiation, and the integrity of the Spitzenkörper in Neurospora crassa

The Rho family of monomeric GTPases act as signaling proteins to establish and maintain cell polarity and other essential cellular processes. Rho3 is a GTPase of the Rho family that is exclusive of fungi that regulate cell polarity in yeast. However, studies have yet to explore its function in filamentous fungi. In this work, we investigated the role of RHO-3 in the model organism Neurospora crassa. Confocal microscopy analysis revealed that RHO-3 localizes in the outer region of the Spitzenkörper (Spk), in the plasma membrane from region II to the beginning of region III, and in the septa of mature hyphae. The phenotypic effect of the rho-3 deletion was analyzed. The results revealed that the rho-3 null strain showed severe defects in growth rate, aerial hyphae length, and conidia production. The organization of the Spk is also affected in the absence of RHO-3. Co-expression analysis of GFP-RHO-3 with glucan synthase 1 (GS-1-mChFP) and chitin synthase 1 (CHS-1-mChFP) revealed that RHO-3 localizes in the external region of the Spk in the macrovesicles zone. In summary, our results suggest that RHO-3 is not essential for the polarized growth of hyphae but plays a significant role in hyphal extension rate, conidiation, sexual reproduction and the integrity of the Spk, possibly regulating the delivery of macrovesicles to the apical dome.

Adaptation to glucose starvation is associated with molecular reorganization of the circadian clock in Neurospora crassa.

The circadian clock governs rhythmic cellular functions by driving the expression of a substantial fraction of the genome and thereby significantly contributes to the adaptation to changing environmental conditions. Using the circadian model organism Neurospora crassa, we show that molecular timekeeping is robust even under severe limitation of carbon sources, however, stoichiometry, phosphorylation and subcellular distribution of the key clock components display drastic alterations. Protein kinase A, protein phosphatase 2 A and glycogen synthase kinase are involved in the molecular reorganization of the clock. RNA-seq analysis reveals that the transcriptomic response of metabolism to starvation is highly dependent on the positive clock component WC-1. Moreover, our molecular and phenotypic data indicate that a functional clock facilitates recovery from starvation. We suggest that the molecular clock is a flexible network that allows the organism to maintain rhythmic physiology and preserve fitness even under long-term nutritional stress.

The genome organization of Neurospora crassa at high resolution uncovers principles of fungal chromosome topology.

The eukaryotic genome must be precisely organized for its proper function, as genome topology impacts transcriptional regulation, cell division, replication, and repair, among other essential processes. Disruptions to human genome topology can lead to diseases, including cancer. The advent of chromosome conformation capture with high-throughput sequencing (Hi-C) to assess genome organization has revolutionized the study of nuclear genome topology; Hi-C has elucidated numerous genomic structures, including chromosomal territories, active/silent chromatin compartments, Topologically Associated Domains, and chromatin loops. While low-resolution heatmaps can provide important insights into chromosomal level contacts, high-resolution Hi-C datasets are required to reveal folding principles of individual genes. Of particular interest are high-resolution chromosome conformation datasets of organisms modeling the human genome. Here, we report the genome topology of the fungal model organism Neurospora crassa at a high resolution. Our composite Hi-C dataset, which merges 2 independent datasets generated with restriction enzymes that monitor euchromatin (DpnII) and heterochromatin (MseI), along with our DpnII/MseI double digest dataset, provide exquisite detail for both the conformation of entire chromosomes and the folding of chromatin at the resolution of individual genes. Within constitutive heterochromatin, we observe strong yet stochastic internal contacts, while euchromatin enriched with either activating or repressive histone post-translational modifications associates with constitutive heterochromatic regions, suggesting intercompartment contacts form to regulate transcription. Consistent with this, a strain with compromised heterochromatin experiences numerous changes in gene expression. Our high-resolution Neurospora Hi-C datasets are outstanding resources to the fungal community and provide valuable insights into higher organism genome topology.

Principles underlying the complex dynamics of temperature entrainment by a circadian clock

SummaryAutonomously oscillating circadian clocks resonate with daily environmental (zeitgeber) rhythms to organize physiology around the solar day. Although entrainment properties and mechanisms have been studied widely and in great detail for light-dark cycles, entrainment to daily temperature rhythms remains poorly understood despite that they are potent zeitgebers. Here we investigate the entrainment of the chronobiological model organism Neurospora crassa, subject to thermocycles of different periods and fractions of warm versus cold phases, mimicking seasonal variations. Depending on the properties of these thermocycles, regularly entrained rhythms, period-doubling (frequency demultiplication) but also irregular aperiodic behavior occurs. We demonstrate that the complex nonlinear phenomena of experimentally observed entrainment dynamics can be understood by molecular mathematical modeling.

P.377 Rats with long learning experience showed resistance to the impairing effect of MK-801 in several cognitive assays

Accumulating evidence from both experimental and clinical investigations indicates a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and, therefore, is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa. We show that, in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in the WT, in Δras2, operation of the circadian clock was affected by glucose; compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness, the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Although glucose did not affect the subcellular distribution of FRQ in the WT, highly elevated FRQ levels were detected in the nucleus in Δras2. RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-bromo-cyclic AMP partially rescued the circadian phenotype in vivo. We therefore propose that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock.

Identification of a PRC2 Accessory Subunit Required for Subtelomeric H3K27 Methylation in Neurospora crassa.

Polycomb repressive complex 2 (PRC2) catalyzes methylation of histone H3 at lysine 27 (H3K27) in genomic regions of most eukaryotes and is critical for maintenance of the associated transcriptional repression. However, the mechanisms that shape the distribution of H3K27 methylation, such as recruitment of PRC2 to chromatin and/or stimulation of PRC2 activity, are unclear. Here, using a forward genetic approach in the model organism Neurospora crassa, we identified two alleles of a gene, NCU04278, encoding an unknown PRC2 accessory subunit (PAS). Loss of PAS resulted in losses of H3K27 methylation concentrated near the chromosome ends and derepression of a subset of associated subtelomeric genes. Immunoprecipitation followed by mass spectrometry confirmed reciprocal interactions between PAS and known PRC2 subunits, and sequence similarity searches demonstrated that PAS is not unique to N. crassa PAS homologs likely influence the distribution of H3K27 methylation and underlying gene repression in a variety of fungal lineages.

Unveiling the Impact of the Core Circadian Clock Protein FREQUENCY on Output in Neurospora crassa

Circadian rhythms are cellular oscillations that tune an organism's biology to the 24‐hour light/dark cycle on Earth. This cellular time‐keeping mechanism aids in the anticipation of environmental changes and increases organismal survival. These rhythms are maintained by a transcriptional/translational negative feedback loop, called the core circadian clock, that is evolutionarily conserved throughout most branches of life. In this core clock, positive arm proteins act to transcriptionally activate negative arm proteins. The negative arms proteins act back on the positive arm, in a time specific manner, to halt the negative arm's activation before degradation and re‐initiation, thus setting the period of the clock. Beyond this core clock, positive arm proteins also act to transcriptionally activate a variety of other genes that do not participate in the core clock mechanism in a time dependent manor, referred to as clock‐controlled genes (ccgs.) Most research into the mechanisms of circadian regulation on output has focused on transcriptional activation by the positive arm proteins. However, recently published work has demonstrated that there are many oscillating proteins for which the corresponding mRNA is not rhythmic. This has led to the theory that the regulation of circadian output is also accomplished via post‐translational mechanisms, which we hypothesize are imparted by the negative arm proteins.In the model organism Neurospora crassa, the negative arm comprises FREQUENCY (FRQ), Frequency‐Interacting RNA Helicase (FRH), and Casein Kinase 1 (CK‐1). Importantly, FRQ has been computationally and experimentally proven to be an intrinsically disordered protein (IDP). Aside from lacking a single, predetermined three‐dimensional structure, IDPs play key roles in cellular signaling and regulation due, in part, to the transient but highly specific and varied interacting partners they have. These attributes make FRQ a prime candidate to impart post‐translational circadian regulation. The aim of this study is to characterize protein complexes, centered around FRQ, that change in a time specific manner, to determine the impact these interactions could impart post‐translationally on circadian regulatory pathways. To this end, Neurospora samples where crosslinked and purified through co‐immunoprecipitation before protein identification through liquid chromatography mass spectrometry. Initial results suggest that FRQ plays a role in post‐translational regulation on metabolism, the cell cycle, and a variety of other functions.Support or Funding InformationThis work was funded through the support of the NIGMS/NIH R35GM128687 grant and the Rensselaer Polytechnic Institue Start Up Fund.This abstract is from the Experimental Biology 2019 Meeting. There is no full text article associated with this abstract published in The FASEB Journal.

Unveiling the Impact of the Negative Arm of the Circadian Clock on Output in Neurospora crassa

Circadian rhythms are cellular oscillations that tune biology to Earth's 24-hour light/dark cycle, aiding in the anticipation of environmental changes. These rhythms are maintained by an evolutionarily conserved transcriptional/translational negative feedback loop, referred to as the core circadian clock. In this core clock, positive arm proteins act to transcriptionally activate negative arm proteins that act back on the positive arm to halt the negative arm's activation before degradation and re-initiation, thus setting the period of the clock. Most research into circadian regulation on output has focused on transcriptional activation by the positive arm proteins. However, recently published work has demonstrated that there are many oscillating proteins for which the corresponding mRNA is not rhythmic. This has led to the theory that the post-translational regulation of circadian output exists, which we hypothesize is imparted by the negative arm proteins. In the model organism Neurospora crassa, the negative arm includes FREQUENCY (FRQ). FRQ has been computationally and experimentally proven to be an intrinsically disordered protein (IDP). Aside from lacking a predetermined three-dimensional structure, IDPs play key roles in cellular signaling and regulation due, in part, to the transient but highly specific and varied interacting partners they have. These attributes make FRQ a prime candidate to impart circadian regulation post-translationally. The aim of this study is to characterize protein complexes, centered around FRQ, that change in a time specific manor to determine the impact these interactions could impart on circadian regulatory pathways. To this end, Neurospora samples where crosslinked and purified through co-immunoprecipitation before protein identification through liquid chromatography mass spectrometry. Initial results suggest that FRQ plays a role in regulation on metabolism, the cell cycle, and a variety of other functions through novel protein interactions.

The small G protein RAS2 is involved in the metabolic compensation of the circadian clock in the circadian model Neurospora crassa

Accumulating evidence from both experimental and clinical investigations indicates a tight interaction between metabolism and circadian timekeeping; however, knowledge of the underlying mechanism is still incomplete. Metabolic compensation allows circadian oscillators to run with a constant speed at different substrate levels and, therefore, is a substantial criterion of a robust rhythm in a changing environment. Because previous data have suggested a central role of RAS2-mediated signaling in the adaptation of yeast to different nutritional environments, we examined the involvement of RAS2 in the metabolic regulation of the clock in the circadian model organism Neurospora crassa We show that, in a ras2-deficient strain, the period is longer than in the control. Moreover, unlike in the WT, in Δras2, operation of the circadian clock was affected by glucose; compared with starvation conditions, the period was longer and the oscillation of expression of the frequency (frq) gene was dampened. In constant darkness, the delayed phosphorylation of the FRQ protein and the long-lasting accumulation of FRQ in the nucleus were in accordance with the longer period and the less robust rhythm in the mutant. Although glucose did not affect the subcellular distribution of FRQ in the WT, highly elevated FRQ levels were detected in the nucleus in Δras2 RAS2 interacted with the RAS-binding domain of the adenylate cyclase in vitro, and the cAMP analogue 8-bromo-cyclic AMP partially rescued the circadian phenotype in vivo We therefore propose that RAS2 acts via a cAMP-dependent pathway and exerts significant metabolic control on the Neurospora circadian clock.

The Impact of IDPs on Circadian Rhythms

Through the ideal programming of behavior, it is believed that our circadian clock, our internal molecular time keeper, enhances fitness by ensuring that many organismal functions are optimally timed with the phase of the circadian day. As clock regulation is extensive, controlling everything from blood pressure levels to sleep, disruptions of the clock can be detrimental; chronic disturbance of the clock leads to an increased risk for heart attack, cancer, diabetes, and mental health disorders among a host of other maladies. My work has shown that the highly conserved mechanism that regulates circadian timing, a molecular transcription-translation negative feedback loop, is profoundly affected by IDPs. The core clock complex in the clock model organism Neurospora crassa consists of two sets of protein pairs, the positive arm (comprised of the White Collar 1/ White Collar 2 Complex or WCC) and the negative arm (comprised of the Frequency (FRQ)/Frequency-Interacting RNA Helicase (FRH) complex or FFC). The WCC drives the expression of the FFC, which then regulates its own transcription by inhibiting the activity of the WCC. The FFC is then targeted for degradation, allowing for the reactivation of the WCC, restarting the cycle. I have demonstrated that FRQ is an Intrinsically Disordered Protein (IDP); currently, the reason for the IDP nature of FRQ is unknown. In addition, FRH stabilizes FRQ and mediates the interactions between the core clock proteins using its own disordered N-terminus. This intrinsic disorder in core clock genes is conserved from Neurospora to higher eukaryotes and my labs seeks to identify the reason for the conservation of a high level of protein disorder in the circadian clock.

Intrinsic Disorder in the Highly-Ordered Circadian Clock

The circadian clock, our internal molecular time keeper, regulates everything from blood pressure levels to sleep. Through the ideal programming of behavior, it is believed that the clock enhances fitness by ensuring that many organismal functions are optimally timed with the phase of the circadian day. Conversely, due to the extent of circadian regulation, disruptions of the clock can be harmful; chronic disturbance of the clock leads to an increased risk for heart attack, cancer, diabetes, and mental health disorders among a host of other maladies. The highly conserved mechanism which regulates circadian timing is a molecular transcription-translation negative feedback loop. The core clock complex in the clock model organism Neurospora crassa consists of two sets of protein pairs, the positive arm (comprised of the White Collar 1/ White Collar 2 Complex or WCC) and the negative arm (comprised of the Frequency (FRQ)/Frequency-Interacting RNA Helicase (FRH) complex or FFC). The WCC drives the expression of the FFC, which then regulates its own transcription by inhibiting the activity of the WCC. The FFC is then targeted for degradation, allowing for the reactivation of the WCC, restarting the cycle. My work has shown that intrinsic protein disorder is essential for the proper functioning of this circadian circuit. I have demonstrated that FRQ is an Intrinsically Disordered Protein (IDP); currently, the reason for the IDP nature of FRQ is unknown. In complex with FRQ, FRH stabilizes FRQ and mediates the interactions between the core clock proteins using its disordered N-terminus. All core clock genes in Neurospora, as well as in higher eukaryotes, are predicted to be intrinsically disordered in some proportion and my labs seeks to identify the reason for the conservation of a high level of protein disorder in the circadian clock.

Transcription factor Xpp1 is a switch between primary and secondary fungal metabolism

Fungi can produce a wide range of chemical compounds via secondary metabolism. These compounds are of major interest because of their (potential) application in medicine and biotechnology and as a potential source for new therapeutic agents and drug leads. However, under laboratory conditions, most secondary metabolism genes remain silent. This circumstance is an obstacle for the production of known metabolites and the discovery of new secondary metabolites. In this study, we describe the dual role of the transcription factor Xylanase promoter binding protein 1 (Xpp1) in the regulation of both primary and secondary metabolism of Trichoderma reesei Xpp1 was previously described as a repressor of xylanases. Here, we provide data from an RNA-sequencing analysis suggesting that Xpp1 is an activator of primary metabolism. This finding is supported by our results from a Biolog assay determining the carbon source assimilation behavior of an xpp1 deletion strain. Furthermore, the role of Xpp1 as a repressor of secondary metabolism is shown by gene expression analyses of polyketide synthases and the determination of the secondary metabolites of xpp1 deletion and overexpression strains using an untargeted metabolomics approach. The deletion of Xpp1 resulted in the enhanced secretion of secondary metabolites in terms of diversity and quantity. Homologs of Xpp1 are found among a broad range of fungi, including the biocontrol agent Trichoderma atroviride, the plant pathogens Fusarium graminearum and Colletotrichum graminicola, the model organism Neurospora crassa, the human pathogen Sporothrix schenckii, and the ergot fungus Claviceps purpurea.

Analysis of Circadian Rhythms in the Basal Filamentous Ascomycete Pyronema confluens.

Many organisms use circadian clocks to adapt to daily changes in the environment. Major insights into the molecular mechanisms of circadian oscillators have been gained through studies of the model organism Neurospora crassa; however, little is known about molecular components of circadian clocks in other fungi. An important part of the N. crassa circadian clock is the frequency (frq) gene, homologs of which can be found in Sordariomycetes, Dothideomycetes, and Leotiomycetes, but not Eurotiomycetes. Recently, we identified a frq homolog in Pyronema confluens, a member of the early-diverging Pezizomycete lineage of filamentous ascomycetes. The P. confluens FRQ shares many conserved domains with the N. crassa FRQ. However, there is no known morphological phenotype showing overt circadian rhythmicity in P. confluens. To investigate whether a molecular clock is present, we analyzed frq transcription in constant darkness, and found circadian oscillation of frq with a peak in the subjective morning. This rhythm was temperature compensated. To identify additional clock-controlled genes, we performed RNA sequencing of two time points (subjective morning and evening). Circadian expression of two morning-specific genes was verified by reverse transcription quantitative polymerase chain reaction (RT-qPCR) over a full time course, whereas expression of two putative morning-specific and five putative evening-specific genes could not be verified as circadian. frq expression was synchronized, but not entrained by light. In summary, we have found evidence for two of the three main properties of circadian rhythms (free-running rhythm, temperature compensation) in P. confluens, suggesting that a circadian clock with rhythmically expressed frq is present in this basal filamentous ascomycete.

Linear Discriminant Analysis Identifies Mitochondrially Localized Proteins in Neurospora crassa.

Besides their role as powerhouses, mitochondria play a pivotal role in the spatial organization of numerous enzymatic functions. They are connected to the ER, and many pathways are organized through the mitochondrial membranes. Thus, the precise definition of mitochondrial proteomes remains a challenging task. Here, we have established a proteomic strategy to accurately determine the mitochondrial localization of proteins from the fungal model organism Neurospora crassa. This strategy relies on both highly pure mitochondria as well as the quantitative monitoring of mitochondrial components along their consecutive enrichment. Pure intact mitochondria were obtained by a multistep approach combining differential and density Percoll (ultra) centrifugations. When compared with three other intermediate enrichment stages, peptide sequencing and quantitative profiling of pure mitochondrial fractions revealed prototypic regulatory profiles of per se mitochondrial components. These regulatory profiles constitute a distinct cluster defining the mitochondrial compartment and support linear discriminant analyses, which rationalized the annotation process. In total, this approach experimentally validated the mitochondrial localization of 512 proteins including 57 proteins that had not been reported for N. crassa before.

Global Analysis of Serine/Threonine and Tyrosine Protein Phosphatase Catalytic Subunit Genes inNeurospora crassaReveals 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 defects in at least one of these traits, whereas 29% possessed phenotypes in all three. Chemical sensitivity screens were conducted to reveal additional phenotypes for the mutants. This resulted in the identification of at least one chemical sensitivity phenotype for 17 phosphatase knockout mutants, including novel chemical sensitivities for two phosphatase mutants lacking a growth or developmental phenotype. Hence, chemical sensitivity or growth/developmental phenotype was observed for all 24 viable mutants. We investigated p38 mitogen-activated protein kinase (MAPK) phosphorylation profiles in the phosphatase mutants and identified nine potential candidates for regulators of the p38 MAPK. We demonstrated that the PP2C class phosphatase pph-8 (NCU04600) is an important regulator of female sexual development in N. crassa. In addition, we showed that the Δcsp-6 (ΔNCU08380) mutant exhibits a phenotype similar to the previously identified conidial separation mutants, Δcsp-1 and Δcsp-2, that lack transcription factors important for regulation of conidiation and the circadian clock.

Activation of a TRP-like channel and intracellular Ca2+ dynamics during phospholipase-C-mediated cell death.

ABSTRACTThe model organism Neurospora crassa undergoes programmed cell death when exposed to staurosporine. Here, we show that staurosporine causes defined changes in cytosolic free Ca2+ ([Ca2+]c) dynamics and a distinct Ca2+ signature that involves Ca2+ influx from the external medium and internal Ca2+ stores. We investigated the molecular basis of this Ca2+ response by using [Ca2+]c measurements combined with pharmacological and genetic approaches. Phospholipase C was identified as a pivotal player during cell death, because modulation of the phospholipase C signaling pathway and deletion of PLC-2, which we show to be involved in hyphal development, results in an inability to trigger the characteristic staurosporine-induced Ca2+ signature. Using Δcch-1, Δfig-1 and Δyvc-1 mutants and a range of inhibitors, we show that extracellular Ca2+ entry does not occur through the hitherto described high- and low-affinity Ca2+ uptake systems, but through the opening of plasma membrane channels with properties resembling the transient receptor potential (TRP) family. Partial blockage of the response to staurosporine after inhibition of a putative inositol-1,4,5-trisphosphate (IP3) receptor suggests that Ca2+ release from internal stores following IP3 formation combines with the extracellular Ca2+ influx.

Coordination of K + Transporters in Neurospora: TRK1 Is Scarce and Constitutive, while HAK1 Is Abundant and Highly Regulated

Fungi, plants, and bacteria accumulate potassium via two distinct molecular machines not directly coupled to ATP hydrolysis. The first, designated TRK, HKT, or KTR, has eight transmembrane helices and is folded like known potassium channels, while the second, designated HAK, KT, or KUP, has 12 transmembrane helices and resembles MFS class proteins. One of each type functions in the model organism Neurospora crassa, where both are readily accessible for biochemical, genetic, and electrophysiological characterization. We have now determined the operating balance between Trk1p and Hak1p under several important conditions, including potassium limitation and carbon starvation. Growth measurements, epitope tagging, and quantitative Western blotting have shown the gene HAK1 to be much more highly regulated than is TRK1. This conclusion follows from three experimental results: (i) Trk1p is expressed constitutively but at low levels, and it is barely sensitive to extracellular [K(+)] and/or the coexpression of HAK1; (ii) Hak1p is abundant but is markedly depressed by elevated extracellular concentrations of K(+) and by coexpression of TRK1; and (iii) Carbon starvation slowly enhances Hak1p expression and depresses Trk1p expression, yielding steady-state Hak1p:Trk1p ratios of ∼500:1, viz., 10- to 50-fold larger than that in K(+)- and carbon-replete cells. Additionally, it appears that both potassium transporters can adjust kinetically to sustained low-K(+) stress by means of progressively increasing transporter affinity for extracellular K(+). The underlying observations are (iv) that K(+) influx via Trk1p remains nearly constant at ∼9 mM/h when extracellular K(+) is progressively depleted below 0.05 mM and (v) that K(+) influx via Hak1p remains at ∼3 mM/h when extracellular K(+) is depleted below 0.1 mM.

Reactive oxygen species can modulate circadian phase and period in Neurospora crassa

Reactive oxygen species (ROS) may serve as signals coupling metabolism to other cell functions. In addition to being by-products of normal metabolism, they are generated at elevated levels under environmental stress situations. We analyzed how reactive oxygen species affect the circadian clock in the model organism Neurospora crassa. In light/dark cycles, an increase in the levels of reactive oxygen species advanced the phase of both the conidiation rhythm and the expression of the clock gene frequency. Our results indicate a dominant role of the superoxide anion in the control of the phase. Elevation of superoxide production resulted in the activation of protein phosphatase 2A, a regulator of the positive element of the circadian clock. Our data indicate that even under nonstress conditions, reactive oxygen species affect circadian timekeeping. Reduction of their basal levels results in a delay of the phase in light/dark cycles and a longer period under constant conditions. We show that under entrained conditions the phase depends on the temperature and reactive oxygen species contribute to this effect. Our results suggest that the superoxide anion is an important factor controlling the circadian oscillator and is able to reset the clock most probably by activating protein phosphatase 2A, thereby modulating the activity of the White Collar complex.

Light-Inducible System for Tunable Protein Expression inNeurospora crassa

Filamentous fungi are important model systems for understanding eukaryotic cellular processes, including the study of protein expression. A salient feature of fungi is the ability of the protein-processing machinery to perform all of the extensive posttranslational modifications needed in the complex world of eukaryotic organisms, making them great hosts for production of eukaryotic proteins. In the model organism Neurospora crassa, several regulatable promoters have been used for heterologous gene expression but all suffer from leaky expression absent stimuli or an inability to induce protein expression at levels greater than those seen in vivo. To increase and better control in vivo protein expression in Neurospora, we have harnessed the light-induced vvd promoter. vvd promoter-driven mRNA expression is dependent upon light, shows a graded response, and is rapidly shut off when returned to the dark. The vvd promoter is a highly tunable and regulatable system, which could be a useful instrument for those interested in efficient and controllable gene expression.