Conidiation Rhythm Research Articles

Methylxanthines Modulate Circadian Period Length Independently of the Action of Phosphodiesterase.

In Neurospora crassa, caffeine and other methylxanthines are known to inhibit phosphodiesterase (PDE) activity, leading to augmented cAMP levels. In this organism, it has also been shown that the addition of these drugs significantly lengthens the circadian period, as seen by conidiation rhythms. Utilizing in vivo bioluminescence reporters, pharmacological inhibitors, and cAMP analogs, we revisited the effect of methylxanthines and the role of cAMP signaling in the Neurospora clockworks. We observed that caffeine, like all tested methylxanthines, led to significant period lengthening, visualized with both core-clock transcriptional and translational reporters. Remarkably, this phenotype is still observed when phosphodiesterase (PDE) activity is genetically or chemically (via 3-isobutyl-1-methylxanthine) abrogated. Likewise, methylxanthines still exert a period effect in several cAMP signaling pathway mutants, including adenylate cyclase (cr-1) and protein kinase A (PKA) (Δpkac-1) mutants, suggesting that these drugs lead to circadian phenotypes through mechanisms different from the canonical PDE-cAMP-PKA signaling axis. Thus, this study highlights the strong impact of methylxanthines on circadian period in Neurospora, albeit the exact mechanisms somehow remain elusive. IMPORTANCE Evidence from diverse organisms show that caffeine causes changes in the circadian clock, causing period lengthening. The fungus Neurospora crassa is no exception; here, several methylxanthines such as caffeine, theophylline, and aminophylline cause period lengthening in a concentration-dependent manner. Although methylxanthines are expected to inhibit phosphodiesterase activity, we were able to show by genetic and pharmacological means that these drugs exert their effects through a different mechanism. Moreover, our results indicate that increases in cAMP levels and changes in PKA activity do not impact the circadian period and therefore are not part of underlying effects of methylxanthine. These results set the stage for future analyses dissecting the molecular mechanisms by which these drugs dramatically modify the circadian period.

The Resonance and Adaptation of Neurospora crassa Circadian and Conidiation Rhyth ms to Short Light-Dark Cycles.

Circadian clocks control the physiological and behavioral rhythms to adapt to the environment with a period of ~24 h. However, the influences and mechanisms of the extreme light/dark cycles on the circadian clock remain unclear. We showed that, in Neurospora crassa, both the growth and the microconidia production contribute to adaptation in LD12:12 (12 h light/12 h dark, periodically). Mathematical modeling and experiments demonstrate that in short LD cycles, the expression of the core clock protein FREQUENCY was entrained to the LD cycles when LD > 3:3 while it free ran when T ≤ LD3:3. The conidial rhythmicity can resonate with a series of different LD conditions. Moreover, we demonstrate that the existence of unknown blue light photoreceptor(s) and the circadian clock might promote the conidiation rhythms that resonate with the environment. The ubiquitin E3 ligase FWD-1 and the previously described CRY-dependent oscillator system were implicated in regulating conidiation under short LD conditions. These findings shed new light on the resonance of Neurospora circadian clock and conidiation rhythms to short LD cycles, which may benefit the understandings of both the basic regulatory aspects of circadian clock and the adaptation of physiological rhythms to the extreme conditions.

The resonance and adaptation of Neurospora crassa circadian and conidiation rhythms to short light-dark cycles

The resonance and adaptation of Neurospora crassa circadian and conidiation rhythms to short light-dark cycles

Shared Components of the FRQ-Less Oscillator and TOR Pathway Maintain Rhythmicity in Neurospora.

Molecular models for the endogenous oscillators that drive circadian rhythms in eukaryotes center on rhythmic transcription/translation of a small number of “clock genes.” Although substantial evidence supports the concept that negative and positive transcription/translation feedback loops (TTFLs) are responsible for regulating the expression of these clock genes, certain rhythms in the filamentous fungus Neurospora crassa continue even when clock genes (frq, wc-1, and wc-2) are not rhythmically expressed. Identification of the rhythmic processes operating outside of the TTFL has been a major unresolved area in circadian biology. Our lab previously identified a mutation (vta) that abolishes FRQ-less rhythmicity of the conidiation rhythm and also affects rhythmicity when FRQ is functional. Further studies identified the vta gene product as a component of the TOR (Target of Rapamycin) nutrient-sensing pathway that is conserved in eukaryotes. We now report the discovery of TOR pathway components including GTR2 (homologous to the yeast protein Gtr2, and RAG C/D in mammals) as binding partners of VTA through co-immunoprecipitation (IP) and mass spectrometry analysis using a VTA-FLAG strain. Reciprocal IP with GTR2-FLAG found VTA as a binding partner. A Δgtr2 strain was deficient in growth responses to amino acids. Free-running conidiation rhythms in a FRQ-less strain were abolished in Δgtr2. Entrainment of a FRQ-less strain to cycles of heat pulses demonstrated that Δgtr2 is defective in entrainment. In all of these assays, Δgtr2 is similar to Δvta. In addition, expression of GTR2 protein was found to be rhythmic across two circadian cycles, and functional VTA was required for GTR2 rhythmicity. FRQ protein exhibited the expected rhythm in the presence of GTR2 but the rhythmic level of FRQ dampened in the absence of GTR2. These results establish association of VTA with GTR2, and their role in maintaining functional circadian rhythms through the TOR pathway.

Regulation of the Neurospora Circadian Clock by the Spliceosome Component PRP5.

Increasing evidence has pointed to the connection between pre-mRNA splicing and the circadian clock; however, the underlying mechanisms of this connection remain largely elusive. In the filamentous fungus Neurospora crassa, the core circadian clock elements comprise White Collar 1 (WC-1), WC-2 and FREQUENCY (FRQ), which form a negative feedback loop to control the circadian rhythms of gene expression and physiological processes. Previously, we have shown that in Neurospora, the pre-mRNA splicing factors Pre-mRNA-processing ATP-dependent RNA helicase 5 (PRP5), protein arginine methyl transferase 5 (PRMT5) and snRNA gene U4-2 are involved in the regulation of splicing of frq transcripts, which encode the negative component of the circadian clock system. In this work we further demonstrated that repression of spliceosomal component sRNA genes, U5, U4-1, and prp5, affected the circadian conidiation rhythms. In a prp5 knockdown strain, the molecular rhythmicity was dampened. The expression of a set of snRNP genes including prp5 was up-regulated in a mutant strain lacking the clock component wc-2, suggesting that the function of spliceosome might be under the circadian control. Among these snRNP genes, the levels of prp5 RNA and PRP5 protein oscillated. The distribution of PRP5 in cytosol was rhythmic, suggesting a dynamic assembly of PRP5 in the spliceosome complex in a circadian fashion. Silencing of prp5 caused changes in the transcription and splicing of NCU09649, a clock-controlled gene. Moreover, in the clock mutant frq9, the rhythmicity of frq I-6 splicing was abolished. These data shed new lights on the regulation of circadian clock by the pre-RNA splicing, and PRP5 may link the circadian clock and pre-RNA splicing events through mediating the assembly and function of the spliceosome complex.

진핵 미생물에서의 COP9 signalosome의 역할

COP9 signalosome (CSN), which is originally identified as the regulator of the photomorphogenic development in plant, is highly conserved protein complex in diverse eukaryotic organisms. Most eukaryotic CSN complex is composed of 8 subunits, which is structurally and functionally similar to the lid subunit of 26S proteasome and eIF3 translation initiation complex. CSN play important functions in the regulation of cell cycle and checkpoint response by controlling Cullin-Ring E3 ubiquitin ligases (CRL) activities. CSN exhibits an isopeptidase activity which cleaves the neddylated moiety of cullin components. In fission yeast, S-phase cell cycle progression was delayed and the sensitivity to g-ray or UV was increased in CSN1 and CSN2 deletion mutants, indicating that yeast CSN is also involved in the checkpoint regulation. CSN in fungal system more closely resembles that of the higher organisms in the structure and assembly of their components. Functionally, CSN is associated with the regulation of conidiation rhythms in Neurospora crassa and the sexual development in Aspsergillus nidulans. Recent studies also revealed that CSN functions as an essential cell cycle regulator, playing key roles in the regulation of DNA replication and DNA damage response in Aspergillus. Overall, CSN of microorganisms, such as fission yeast and fungi, share functionally common aspects with higher organisms, implying that they can be useful tools to study the role of CSN in the CRL-mediated diverse cellular activities.

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.

A New Mutation Affecting FRQ-Less Rhythms in the Circadian System of Neurospora crassa

We are using the fungus Neurospora crassa as a model organism to study the circadian system of eukaryotes. Although the FRQ/WCC feedback loop is said to be central to the circadian system in Neurospora, rhythms can still be seen under many conditions in FRQ-less (frq knockout) strains. To try to identify components of the FRQ-less oscillator (FLO), we carried out a mutagenesis screen in a FRQ-less strain and selected colonies with altered conidiation (spore-formation) rhythms. A mutation we named UV90 affects rhythmicity in both FRQ-less and FRQ-sufficient strains. The UV90 mutation affects FRQ-less rhythms in two conditions: the free-running long-period rhythm in choline-depleted chol-1 strains becomes arrhythmic, and the heat-entrained rhythm in the frq10 knockout is severely altered. In a FRQ-sufficient background, the UV90 mutation causes damping of the free-running conidiation rhythm, reduction of the amplitude of the FRQ protein rhythm, and increased phase-resetting responses to both light and heat pulses, consistent with a decreased amplitude of the circadian oscillator. The UV90 mutation also has small but significant effects on the period of the conidiation rhythm and on growth rate. The wild-type UV90 gene product appears to be required for a functional FLO and for sustained, high-amplitude rhythms in FRQ-sufficient conditions. The UV90 gene product may therefore be a good candidate for a component of the FRQ-less oscillator. These results support a model of the Neurospora circadian system in which the FRQ/WCC feedback loop mutually interacts with a single FLO in an integrated circadian system.

Activation and localization of protein kinase C in Neurospora crassa

The Neurospora crassa protein kinase C (NPKC) is reported to be a regulator of light responsive genes. It phosphorylates the light receptor WC-1 and regulates the levels of the circadian clock protein FRQ and transcription of the light-induced albino-2 gene. In mammals, the conventional and novel isoforms of PKC are activated by diacylglycerol (DAG), which induces PKC translocation from the cytoplasm to membranes. To investigate the interaction of NPKC and DAG in Neurospora, we constructed a strain that expresses a PKC-GFP fusion protein. We found that NPKC localizes to growing tips and sub-apical plasma membrane in actively growing hyphae, and actively participates in septum development. NPKC is activated by exogenous DAG and phorbol esters, and translocates to the plasma membrane from the cytoplasm. We have previously reported that choline depletion of the chol-1 mutant of Neurospora increases DAG levels and lengthens the period of the circadian rhythm of conidiation. We have found that the activity of NPKC is rhythmic, and that NPKC levels are increased on choline depletion. However, over-expression of NPKC did not lengthen the conidiation period, indicating that PKC in Neurospora may not be responsible for the lengthened period in low choline cultures.

Effects ofprdCircadian Clock Mutations on FRQ-Less Rhythms inNeurospora

Rhythmic conidiation (spore formation) in Neurospora crassa provides a model system for investigating the molecular mechanisms of circadian rhythmicity. A feedback loop involving the frq, wc-1, and wc-2 gene products (FRQ/ WCC) is an important component of the mechanism; however, rhythmic conidiation can still be observed when these gene products are absent. The nature of the oscillator(s) that drives this FRQ-less rhythmicity (FLO) is an important question in Neurospora circadian biology. We have looked for interactions between FRQ/WCC and FLO by assaying the effects on FRQ-less rhythms of mutations known to affect the period in the presence of FRQ. We assayed 4 prd mutations (prd-1, prd-2, prd-3, and prd-4) under 2 conditions in frq(null) strains: long-period free-running rhythms in chol-1 strains grown without choline, and heat-entrainable rhythms in choline-sufficient conditions. We found effects of all 4 mutations on both types of FRQ-less rhythms. The greatest effects were seen with prd-1 and prd-2, which abolished free-running rhythms in the chol-1; frq(10) backgrounds and significantly affected entrained peak timing under heat-entrainment conditions in frq( 10) backgrounds. The prd-3 and prd-4 mutations had more subtle effects on period and stability of free-running rhythms in the chol-1; frq(10) backgrounds and had little effect on peak timing under heat-entrainment conditions in frq(10) backgrounds. These results, along with previously published evidence for effects of prd mutations on other FRQ-less rhythms, suggest that either there are common components shared between the FRQ/WCC oscillator and several FRQ-less oscillators or that there is a single oscillator driving all conidiation rhythms. We favor a model of the Neurospora circadian system in which a single FRQ-less oscillator drives conidiation and interacts with the FRQ/WCC feedback loop; the output or amplitude of the FRQ-less oscillator can be affected by many gene products and metabolic conditions that reveal FRQ-less rhythmicity. We propose that prd-1 and prd-2 are good candidates for components of the FRQ-less oscillator and that prd-3 and prd-4 act on the system mainly through effects on FRQ/WCC.

Rhythmic Conidiation in Constant Light inVividMutants ofNeurospora crassa

In Neurospora crassa, a circadian rhythm of conidiation (asexual spore formation) can be seen on the surface of agar media. This rhythm has a period of 22 hr in constant darkness (D/D). Under constant illumination (L/L), no rhythm is visible and cultures show constant conidiation. However, here we report that strains with a mutation in the vivid (vvd) gene, previously shown to code for the photoreceptor involved in photo-adaptation, exhibit conidiation rhythms in L/L as well as in D/D. The period of the rhythm of vvd strains ranges between 6 and 21 hr in L/L, depending upon the intensity of the light, the carbon source, and the presence of other mutations. Temperature compensation of the period also depends on light intensity. Dark pulses given in L/L shift the phase of the rhythm. Shifts from L/L to D/D show unexpected after effects; i.e., the short period of a vvd strain in L/L gradually lengthens over 2-3 days in D/D. The rhythm in L/L requires the white collar (wc-1) gene, but not the frequency (frq) gene. FRQ protein shows no rhythm in L/L in a vvd strain. The conidiation rhythm in L/L in vvd is therefore driven by a FRQ-less oscillator (FLO).

Conidiation rhythm and light entrainment in superoxide dismutase mutant in Neurospora crassa

Conidial formation in the filamentous fungus Neurospora crassa is regulated by nutritional conditions, light, and the circadian clock. We found that a sod-1 mutant, with a defective superoxide dismutase catalyzing the conversion of superoxide to hydrogen peroxide, had a slightly shorter period length than the wild type and clear conidial banding similar to a mutant of band (bd). However, unlike the bd mutant, the sod-1 mutant could sustain conidial banding with light pulses on a nutrient-rich medium, which involved an enhancement of the light-induced transcription of frequency (frq). sod-1 was hypersensitive to entrainment of the conidiation rhythm by light in race tubes. Furthermore, a frq(10); sod-1 double mutant showed conidiation rhythm in darkness and could be synchronized to light/dark cycles by the masking effect of light. These genetic analyses suggested that intracellular reactive oxygen species (ROS) act on circadian conidiation via multiple circadian clocks and output pathways.

The effects of temperature change on the circadian clock of Neurospora.

The phase resetting of the circadian oscillatory system by pulses of increased temperature (zeitgebers) and the temperature compensation of its period length during longer exposures are major features of the system, but are not well understood in molecular terms. In Neurospora crassa, the effects of pulses of increased temperature on the circadian rhythm of conidiation were determined and possible inputs to the oscillatory system tested, including changes in cyclic 3',5'-adenosine monophosphate (cAMP), inositol 1,4,5-trisphosphate and H+ concentrations, as well as changes of phosphorylation, synthesis and degradation of proteins. Following the kinetics of these parameters during exposure to increased temperature showed transient changes. Experimental manipulation of cAMP, Ca2+ and H+ levels, and of the synthesis and, possibly, degradation of proteins, resulted in phase shifts of the oscillatory system. It is assumed that the temperature signal affects the oscillator(s) by multiple pathways and shifts the whole state of the oscillatory system. Second messenger levels, protein synthesis and protein degradation show adaptation to longer exposures to elevated temperature which may be involved in the temperature compensation of the period length. The temperature compensation is also proposed to involve a shift in the state of all or most oscillator variables.

Kinetics of circadian band development in Neurospora crassa

The circadian rhythm of Neurospora crassa can be seen as a conidiation rhythm that produces concentric rings of bands (conidiating regions) alternating with interbands (non-conidiating regions) on the surface of an agar medium. To follow quantitatively this rhythm, densitometric analysis, gravimetric procedures, and video microscopy were employed. The circadian behavior of N. crassa is commonly monitored by cultivation in race tubes; in this work we report different growth kinetics during cultivation in conventional Petri dish cultures. Two different growth parameters were measured: total colony mass (true growth rate) and distance (colony radial expansion or hyphal elongation). Determinations of cellular mass revealed a dramatic circadian oscillation with a marked drop in growth rate during new interband formation followed by a sharp increase during the development of a new conidiation band. On the other hand, we found that the radial expansion of the colony previously reported to decrease periodically seemed unaffected by the circadian clock. Densitometric analysis showed no initial difference in the expanding margin of the colony, independent of whether that area was destined to be a band or an interband. The band areas increased rapidly in density for about 15 h whereas the interband areas maintained an equally rapid rate of increase for only 6 h. The density of band areas kept increasing slowly for almost 40 h, along with an increase in the amount of conidia. Video microscopy showed the importance of cytoplasmic flow in colony development with continuous forward flow to support hyphal morphogenesis and reverse flow to support an extended period of conidiogenesis. Our results indicate that the circadian system of Neurospora can be expressed at the level of cellular mass formation, not just as the developmental conidiation rhythm.

Circadian clock genes frequency and white collar-1 are not essential for entrainment to temperature cycles in Neurospora crassa

The fungus Neurospora crassa is a model system for investigating the mechanism of circadian rhythmicity, and the core of its circadian oscillator is thought to be a transcription/translation feedback loop involving the products of the frq (frequency), wc-1 (white-collar-1) and wc-2 (white-collar-2) genes. Several reports of rhythmicity in frq and wc null mutants have raised questions about how central the FRQ/WC loop is to the circadian system of Neurospora. Several research groups have attempted to answer this question by looking for entrainment of the conidiation banding rhythm in frq null mutants. Because the frq mutants are blind to light and cannot be entrained to light/dark cycles, these groups have used symmetric temperature cycles of equal-duration cool and warm phases to entrain the rhythm. Under these conditions, the direct effects of temperature on conidiation (masking effects) can compromise observations of the endogenous rhythm. I have reexamined this question by using short heat pulses to clearly separate masking from endogenous rhythms, and I have assayed entrainment in both frq and wc-1 null mutants. I found similar patterns of entrainment in the wild type and both mutant strains. Strong masking effects were found in the frq mutant but not in the wc-1 mutant. I conclude that a rapidly damping temperature-entrainable oscillator is present in the null mutants. A single temperature-entrainable oscillator may drive the conidiation rhythm in all strains, and additional properties such as light sensitivity and temperature compensation may be conferred by the intact FRQ/WC loop in the WT strain.

The COP9 signalosome regulates the Neurospora circadian clock by controlling the stability of the SCFFWD-1 complex

The COP9 signalosome (CSN) promotes the function of SCF-type cullin-based ubiquitin ligase complexes in vivo. Paradoxically, removal of the Nedd8 modification of cullins by CSN inhibits the ubiquitin ligase activity of SCF complexes in vitro. Ubiquitination-mediated degradation of the Neurospora circadian clock protein FREQUENCY (FRQ) is critical for clock function. Ubiquitination of FRQ requires FWD-1, the substrate-recruiting subunit of an SCF complex. Here we show that disruption of a subunit of CSN (csn-2) impairs the degradation of FRQ and compromises its normal circadian expression. A FRQ-independent oscillator drives conidiation in the csn-2 mutant, resulting in a 2-d conidiation rhythm that persists in constant darkness (DD), constant light (LL), light-to-dark (LD) transitions, and temperature cycles. Strikingly, the levels of FWD-1 are drastically reduced in csn-2 mutant, explaining the impaired degradation of FRQ. Reduction of FWD-1 levels in the mutant requires its F-box, suggesting that its degradation is due to autoubiquitination. In addition, SKP-1 and CUL-1 of the SCF(FWD-1) complex are also unstable in the mutant. Therefore, our results establish an important role of CSN in the circadian clock of Neurospora. Our findings also reconcile the CSN paradox and suggest that a major function of CSN is to maintain the stability of SCF ubiquitin ligases in vivo.

Light-independent Phosphorylation of WHITE COLLAR-1 Regulates Its Function in the Neurospora Circadian Negative Feedback Loop

Phosphorylation is a major regulatory mechanism controlling circadian clocks. In the Neurospora circadian clock, the PER-ARNT-SIM (PAS) domain-containing transcription factor, WHITE COLLAR (WC)-1, acts both as the blue light photoreceptor of the clock and as a positive element in the circadian negative feedback loop in constant darkness, by activating the transcription of the frequency (frq) gene. To understand the role of WC-1 phosphorylation, five in vivo WC-1 phosphorylation sites, located immediately downstream of the WC-1 zinc finger DNA binding domain, were identified by tandem mass spectrometry using biochemically purified endogenous WC-1 protein. Mutations of these phosphorylation sites suggest that they are major WC-1 phosphorylation sites under constant conditions but are not responsible for the light-induced hyperphosphorylation of WC-1. Although phosphorylation of these sites does not affect the light function of WC-1, strains carrying mutations of these sites show short period, low amplitude, or arrhythmic conidiation rhythms in constant darkness. Furthermore, normal or slightly higher levels of frq mRNA and FRQ proteins were observed in a mutant strain containing mutations of all five sites despite its low WC-1 levels. Together, these data suggest that phosphorylation of these sites negatively regulates the function of WC-1 in the circadian negative feedback loop and is important for the function of the Neurospora circadian clock.

Circadian rhythms in Neurospora crassa: farnesol or geraniol allow expression of rhythmicity in the otherwise arrhythmic strains frq10, wc-1, and wc-2.

In Neurospora crassa, the circadian rhythm can be seen in the bd (band) strain as a series of "bands" or conidiation (spore-forming) regions on the surface of an agar medium. Certain mutations at 3 different genes (frq, wc-1, or wc-2) lead to the loss of the circadian rhythm. In this study, it was found that the addition of 10(-4) to 10(-5) M of geraniol or farnesol restored rhythmic banding to strains that lack a circadian rhythm due to mutations in any 1 of these 3 genes. These 3 conditionally arrhythmic strains now exhibited robust, free-running conidiation rhythms. Their rhythms were neither temperature-compensated nor obviously sensitive to light, so the full properties of a circadian rhythm were not restored. At 20 degrees C, in growth tubes, farnesol treatment gave periods of 28, 26, and 22 h for the frq10, wc-1, and wc-2 strains, respectively. Geraniol treatment at 20 degrees C gave periods of 23, 25.5, and 24.5 h for the frq10, wc-1, and wc-2 strains, respectively. A PRC for temperature pulses (1 h, 20 to 40 degrees C) for the frq10 strain grown in the presence of geraniol showed strong resetting (type 0), suggesting that a temperature-sensitive oscillator was present. Farnesol and geraniol are related to known intermediates in the steroid (or mevalonate) pathway. These data are interpreted in terms of a 2-oscillator model, in which farnesol/geraniol activate or amplify a remaining oscillator (a postulated frq-less oscillator).

A new wc-1 mutant of Neurospora crassa shows unique light sensitivity in the circadian conidiation rhythm.

A new clock mutant ( rhy-2) was isolated by DNA insertion mutagenesis using a plasmid that contains a region located upstream of the cmd gene in the genome of Neurospora crassa. This mutant is arrhythmic with regard to conidiation in continuous darkness but rhythmic under a light-dark cycle. After plasmid rescue from genomic DNA of the rhy-2 strain, the insertion was localized to the gene white collar-1 ( wc-1). Plasmid DNA was inserted 3' to the sequence encoding the polyglutamine region of the WC-1 gene product, and an mRNA encoding a truncated WC-1 protein must be synthesized under the control of the cmd promoter. The new wc-1 mutant, rhy-2, is still sensitive to light, although only weakly. Since the circadian rhythm of conidiation in continuous darkness is eliminated in the mutant, the polyglutamine region in WC-1 may be essential for both clock function and light-induced carotenogenesis in Neurospora.

Neurospora clock-controlled gene 9 (ccg-9) encodes trehalose synthase: circadian regulation of stress responses and development.

The circadian clock of Neurospora crassa regulates the rhythmic expression of a number of genes encoding diverse functions which, as an ensemble, are adaptive to life in a rhythmic environment of alternating levels of light and dark, warmth and coolness, and dryness and humidity. Previous differential screens have identified a number of such genes based solely on their cycling expression, including clock-controlled gene 9 (ccg-9). Sequence analysis now shows the predicted CCG-9 polypeptide to be homologous to a novel form of trehalose synthase; as such it would catalyze the synthesis of the disaccharide trehalose, which plays an important role in protecting many cells from environmental stresses. Consistent with this, heat, glucose starvation, and osmotic stress induce ccg-9 transcript accumulation. Surprisingly, however, a parallel role in development is suggested by the finding that inactivation of ccg-9 results in altered conidiophore morphology and abolishes the normal circadian rhythm of asexual macroconidial development. Examination of a clock component, FRQ, in the ccg-9-null strain revealed normal cycling, phosphorylation, and light induction, indicating that loss of the conidiation rhythm is not due to changes in either the circadian oscillator or light input into the clock but pointing instead to a defect in circadian output. These data imply an interplay between a role of trehalose in stress protection and an apparent requirement for trehalose in clock regulation of conidiation under constant environmental conditions. This requirement can be bypassed by a daily light signal which drives a light-entrained rhythm in conidiation in the ccg-9-null strain; this bypass suggests that the trehalose requirement is related to clock control of development and not to the developmental process itself. Circadian control of trehalose synthase suggests a link between clock control of stress responses and that of development.