Light reception and circadian behavior in 'blind' and 'clock-less' mutants of Neurospora crassa

The filamentous fungus Neurospora crassa is a model organism for the genetic dissection of blue light photoreception and circadian rhythms. WHITE COLLAR-1 (WC-1) and WC-2 are considered necessary for all light responses, while FREQUENCY (FRQ) is required for light-regulated asexual development (conidia formation); without any of the three, self-sustained (circadian) rhythmicity in constant conditions fails. Here we show that light-regulated and self-sustained development occur in the individual or mutant white collar strains. These strains resemble wild type in their organization of the daily bout of light-regulated conidiation. Molecular profiles of light- induced genes indicate that the individual white collar-1 and white collar-2 mutants utilize distinct pathways, despite their similar appearance in all aspects. Titration of fluence rate also demonstrates different light sensitivities between the two strains. The data require the existence of an as-yet-unidentified photoreceptor. Furthermore, the extant circadian clock machinery in these mutant strains supports the notion that the circadian system in Neurospora involves components outside the WC-FRQ loop.     

Protein kinase C modulates light responses in Neurospora by regulating the blue light photoreceptor WC-1

The Neurospora protein kinase C (NPKC) is a regulator of light responsive genes. We have studied the function of NPKC in light response by investigating its biochemical and functional interaction with the blue light photoreceptor white-collar 1 (WC-1), showing that activation of NPKC leads to a significant decrease in WC-1 protein levels. Furthermore, we show that WC-1 and NPKC interact in a light-regulated manner in vivo, and that protein kinase C (PKC) phosphorylates WC-1 in vitro. We designed dominant negative and constitutively active forms of PKC which are able to induce either a large increase of WC-1 protein level or a strong reduction respectively. Moreover, these changes in PKC activity result in an altered light response. As WC-1 is a key component of Neurospora circadian clock and regulates the clock oscillator component FRQ we investigated the effect of NPKC-mutated forms on FRQ levels. We show that changes in PKC activity affect FRQ levels and the robustness of the circadian clock. Together these data identify NPKC as a novel component of the Neurospora light signal transduction pathway that modulates the circadian clock.     

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.     

Distinct white collar-1 genes control specific light responses in Mucor circinelloides

Light regulates many developmental and physiological processes in a large number of organisms. The best-known light response in the fungus Mucor circinelloides is the biosynthesis of beta-carotene. Here, we show that M. circinelloides sporangiophores also respond to light, exhibiting a positive phototropism. Analysis of both responses to different light wavelengths within the visible spectrum demonstrated that phototropism is induced by green and blue light, whereas carotenogenesis is only induced by blue light. The blue regulation of both responses suggests the existence of blue-light photoreceptors in M. circinelloides. Three white collar-1 genes (mcwc-1a, mcwc-1b and mcwc-1c) coding for proteins showing similarity with the WC-1 photoreceptor of Neurospora crassa have been identified. All three contain a LOV (light, oxygen or voltage) domain, similar to that present in fungal and plant blue-light receptors. When knockout mutants for each mcwc-1 gene were generated to characterize gene functions, only mcwc-1c mutants were defective in light induction of carotene biosynthesis, indicating that mcwc-1c is involved in the light transduction pathway that control carotenogenesis. We have also shown that positive phototropism is controlled by the mcwc-1a gene. It seems therefore that mcwc-1a and mcwc-1c genes control different light transduction pathways, although cross-talk between both pathways probably exists because mcwc-1a is involved in the light regulation of mcwc-1c expression.     

The Neurospora circadian clock: simple or complex?

The fungus Neurospora crassa is being used by a number of research groups as a model organism to investigate circadian (daily) rhythmicity. In this review we concentrate on recent work relating to the complexity of the circadian system in this organism. We discuss: the advantages of Neurospora as a model system for clock studies; the frequency (frq), white collar-1 and white collar-2 genes and their roles in rhythmicity; the phenomenon of rhythmicity in null frq mutants and its implications for clock mechanisms; the study of output pathways using clock-controlled genes; other rhythms in fungi; mathematical modelling of the Neurospora circadian system; and the application of new technologies to the study of Neurospora rhythmicity. We conclude that there may be many gene products involved in the clock mechanism, there may be multiple interacting oscillators comprising the clock mechanism, there may be feedback from output pathways onto the oscillator(s) and from the oscillator(s) onto input pathways, and there may be several independent clocks coexisting in one organism. Thus even a relatively simple lower eukaryote can be used to address questions about a complex, networked circadian system.     

Temperature-sensitive and circadian oscillators of Neurospora crassa share components

In Neurospora crassa, the interactions between products of the frequency (frq), frequency-interacting RNA helicase (frh), white collar-1 (wc-1), and white collar-2 (wc-2) genes establish a molecular circadian clockwork, called the FRQ-WC-Oscillator (FWO), which is required for the generation of molecular and overt circadian rhythmicity. In strains carrying nonfunctional frq alleles, circadian rhythms in asexual spore development (conidiation) are abolished in constant conditions, yet conidiation remains rhythmic in temperature cycles. Certain characteristics of these temperature-synchronized rhythms have been attributed to the activity of a FRQ-less oscillator (FLO). The molecular components of this FLO are as yet unknown. To test whether the FLO depends on other circadian clock components, we created a strain that carries deletions in the frq, wc-1, wc-2, and vivid (vvd) genes. Conidiation in this ΔFWO strain was still synchronized to cyclic temperature programs, but temperature-induced rhythmicity was distinct from that seen in single frq knockout strains. These results and other evidence presented indicate that components of the FWO are part of the temperature-induced FLO.     

Stress factor-induced changes in the activity of antioxidant protective mechanisms in the wild type strain of Neurospora crassa and in its photoreceptor complex mutants

The effect of stress factors (changes in oxygen content, temperature, and illumination) on superoxide dismutase (SOD) and catalase activity, as well as on the content of thiol and disulfide groups in low-molecular-weight compounds and proteins of Neurospora crassa mycelium was studied in the wild type strain and white collar-1 (wc-1) and white collar-2 (wc-2) mutants. Environmental stress factors induced the activation of both SOD and catalase, as well as an increase in the thiol level in the wild type strain of Neurospora crassa. In the wc-1 and wc-2 mutants, an increase in catalase activity and in the total thiol level was revealed; however, activation of superoxide dismutase was not observed. A decrease in the formation of disulfide bonds in the proteins of wc-1 and wc-2 mutants (as compared with the wild type strain) was recorded. These results indicate disrupted transduction in the WCC mutants of stress factor signals that promote ROS (reactive oxygen species) formation.     

A comparative study of the components of the antioxidant defense system during growth of the mycelium of a wild-type Neurospora crassa strain and mutants, white collar-1 and white collar-2

A comparative study of the changes in the components of the antioxidant defense system (ADS), the activity of superoxide dismutase (SOD) and catalase and the level of extractable SH-groups, during the growth of wild-type and mutant (white collar-1 and white colar-2) Neurospora crassa strains was performed. Oxidative stress developing during spore germination and upon the transition to a stationary growth phase was accompanied in all strains by an increase in the level of extractable SH-groups and SOD activity, whereas the total catalase activity decreased during growth. However, in contrast to the wild-type strain, the activity of the catalase in the mutant strains wc-1 and wc-2 slightly increased upon the transition to the stationary phase. In the wc-2 mutant, SOD activity and the level of extractable SH-groups in the exponential growth phase were always lower than in the wild-type and wc-2 strains. The role of wc-1 and wc-2 genes in the level regulation of reactive oxygen species is discussed.     

The White Collar protein WcoA of Fusarium fujikuroi is not essential for photocarotenogenesis, but is involved in the regulation of secondary metabolism and conidiation

The fungal proteins of the White Collar photoreceptor family, represented by WC-1 from Neurospora crassa, mediate the control by light of different biochemical and developmental processes, such as carotenogenesis or sporulation. Carotenoid biosynthesis is induced by light in the gibberellin-producing fungus Fusarium fujikuroi. In an attempt to identify the photoreceptor for this response, we cloned the only WC-1-like gene present in the available Fusarium genomes, that we called wcoA. The predicted WcoA polypeptide is highly similar to WC-1 and contains the relevant functional domains of this protein. In contrast to the Neurospora counterpart, wcoA expression is not affected by light. Unexpectedly, targeted wcoA disruptant strains maintain the light-induced carotenogenesis. Furthermore, the wcoA mutants show a drastic reduction of fusarin production in the light, and produce less gibberellins and more bikaverins than the parental strain under nitrogen-limiting conditions. The changes in the production of the different products indicate a key regulatory role for WcoA in secondary metabolism of this fungus. Additionally, the mutants are severely affected in conidiation rates under different culture conditions, indicating a more general regulatory role for this protein.     

The White Collar protein WcoA of Fusarium fujikuroi is not essential for photocarotenogenesis, but is involved in the regulation of secondary metabolism and conidiation.

The fungal proteins of the White Collar photoreceptor family, represented by WC-1 from Neurospora crassa, mediate the control by light of different biochemical and developmental processes, such as carotenogenesis or sporulation. Carotenoid biosynthesis is induced by light in the gibberellin-producing fungus Fusarium fujikuroi. In an attempt to identify the photoreceptor for this response, we cloned the only WC-1-like gene present in the available Fusarium genomes, that we called wcoA. The predicted WcoA polypeptide is highly similar to WC-1 and contains the relevant functional domains of this protein. In contrast to the Neurospora counterpart, wcoA expression is not affected by light. Unexpectedly, targeted wcoA disruptant strains maintain the light-induced carotenogenesis. Furthermore, the wcoA mutants show a drastic reduction of fusarin production in the light, and produce less gibberellins and more bikaverins than the parental strain under nitrogen-limiting conditions. The changes in the production of the different products indicate a key regulatory role for WcoA in secondary metabolism of this fungus. Additionally, the mutants are severely affected in conidiation rates under different culture conditions, indicating a more general regulatory role for this protein.     

Circadian clock-specific roles for the light response protein WHITE COLLAR-2

To understand the role of white collar-2 in the Neurospora circadian clock, we examined alleles of wc-2 thought to encode partially functional proteins. We found that wc-2 allele ER24 contained a conservative mutation in the zinc finger. This mutation results in reduced levels of circadian rhythm-critical clock gene products, frq mRNA and FRQ protein, and in a lengthened period of the circadian clock. In addition, this mutation altered a second canonical property of the clock, temperature compensation: as temperature increased, period length decreased substantially. This temperature compensation defect correlated with a temperature-dependent increase in overall FRQ protein levels, with the relative increase being greater in wc-2 (ER24) than in wild type, while overall frq mRNA levels were largely unaltered by temperature. We suggest that this temperature-dependent increase in FRQ levels partially rescues the lowered levels of FRQ resulting from the wc-2 (ER24) defect, yielding a shorter period at higher temperatures. Thus, normal activity of the essential clock component WC-2, a positive regulator of frq, is critical for establishing period length and temperature compensation in this circadian system.     

Genetic Analysis of Phototropism of Neurospora crassa Perithecial Beaks Using White Collar and Albino Mutants

Positive phototropism of perithecial beaks in the fungus Neurospora crassa has been demonstrated. The effect was shown to be mediated by blue light. When mutants (white collar-1 and white collar-2) which are blocked in the light induction of enzymes in the carotenoid biosynthetic pathway were used as the protoperithecial parent in crosses, the resulting perithecial beaks did not show a phototropic response. However, when wild type, albino-1, albino-2, or albino-3 strains were used as the protoperithecial parent, phototropism occurred.

The results show that both photoinduced carotenogenesis and phototropism in N. crassa are controlled by the white collar-1 and white collar-2 loci. Thus, the sensory transduction pathways for the two photoresponses must have some steps in common. The results further support the proposal that the white collar strains are regulatory mutants blocked in the light induction process, whereas the albino-1, albino-2, and albino-3 strains can carry out light induction but have the albino phenotype because they are each defective for a different enzyme in the carotenoid biosynthetic pathway.