Rhythmic conidiation in the blue-light fungus Trichoderma pleuroticola

Trichoderma species conidiate in response to blue light, however, unlike in the blue-light model fungus Neurospora crassa, conidiation in Trichoderma spp. has been considered to be non-circadian. In this study we uncovered evidence for circadian conidiation in Trichoderma pleuroticola and identified orthologues of the key N. crassa clock components, wc-1 (blr-1) and frq.     

OS-2 mitogen activated protein kinase regulates the clock-controlled gene ccg-1 in Neurospora crassa

OS-2 MAP kinase is involved in osmoadaptation in Neurospora crassa. Clock-controlled genes ccg-1, bli-3, and con-10 were induced by osmotic stress in an OS-2 dependent manner. In contrast, osmotic stress did not affect the expression of clock genes frq, wc-1, and wc-2 or of clock-controlled genes ccg-2 and bli-4. These results suggest that OS-2 participates in the regulation of certain circadian-clock output genes.     

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 genetic selection for circadian output pathway mutations in Neurospora crassa

In most organisms, circadian oscillators regulate the daily rhythmic expression of clock-controlled genes (ccgs). However, little is known about the pathways between the circadian oscillator(s) and the ccgs. In Neurospora crassa, the frq, wc-1, and wc-2 genes encode components of the frq-oscillator. A functional frq-oscillator is required for rhythmic expression of the morning-specific ccg-1 and ccg-2 genes. In frq-null or wc-1 mutant strains, ccg-1 mRNA levels fluctuate near peak levels over the course of the day, whereas ccg-2 mRNA remains at trough levels. The simplest model that fits the above observations is that the frq-oscillator regulates a repressor of ccg-1 and an activator of ccg-2. We utilized a genetic selection for mutations that affect the regulation of ccg-1 and ccg-2 by the frq-oscillator. We find that there is at least one mutant strain, COP1-1 (circadian output pathway derived from ccg-1), that has altered expression of ccg-1 mRNA, but normal ccg-2 expression levels. However, the clock does not appear to simply regulate a repressor of ccg-1 and an activator of ccg-2 in two independent pathways, since in our selection we identified three mutant strains, COP1-2, COP1-3, and COP1-4, in which a single mutation in each strain affects the expression levels and rhythmicity of both ccg-1 and ccg-2.     

The frequency gene is required for temperature-dependent regulation of many clock-controlled genes in Neurospora crassa

The circadian clock of Neurospora broadly regulates gene expression and is synchronized with the environment through molecular responses to changes in ambient light and temperature. It is generally understood that light entrainment of the clock depends on a functional circadian oscillator comprising the products of the wc-1 and wc-2 genes as well as those of the frq gene (the FRQ/WCC oscillator). However, various models have been advanced to explain temperature regulation. In nature, light and temperature cues reinforce one another such that transitions from dark to light and/or cold to warm set the clock to subjective morning. In some models, the FRQ/WCC circadian oscillator is seen as essential for temperature-entrained clock-controlled output; alternatively, this oscillator is seen exclusively as part of the light pathway mediating entrainment of a cryptic "driving oscillator" that mediates all temperature-entrained rhythmicity, in addition to providing the impetus for circadian oscillations in general. To identify novel clock-controlled genes and to examine these models, we have analyzed gene expression on a broad scale using cDNA microarrays. Between 2.7 and 5.9% of genes were rhythmically expressed with peak expression in the subjective morning. A total of 1.4-1.8% of genes responded consistently to temperature entrainment; all are clock controlled and all required the frq gene for this clock-regulated expression even under temperature-entrainment conditions. These data are consistent with a role for frq in the control of temperature-regulated gene expression in N. crassa and suggest that the circadian feedback loop may also serve as a sensor for small changes in ambient temperature.     

Circadian rhythms in Neurospora crassa: clock mutant effects in the absence of a frq-based oscillator

In Neurospora, the circadian rhythm is expressed as rhythmic conidiation driven by a feedback loop involving the protein products of frq (frequency), wc-1 (white collar-1), and wc-2, known as the frq/wc (FWC) oscillator. Although strains carrying null mutations such as frq(10) or wc-2Delta lack a functional FWC oscillator and do not show a rhythm under most conditions, a rhythm can be observed in them by the addition of geraniol or farnesol to the media. Employing this altered media as an assay, the effect of other clock mutations in a frq(10)- or wc-2Delta-null background can be measured. It was found that the existing clock mutations fall into three classes: (1) those, such as prd-3 or prd-4 or frq(1), that showed no effect in a clock null background; (2) those, such as prd-1 or prd-2 or prd-6, that did have a measurable effect in the frq(10) background; and (3) those, such as the new mutation ult, that suppressed the frq(10) or wc-2Delta effect, i.e., geraniol/farnesol was not required for a visible rhythm. This classification suggests that some of the known clock mutations are part of a broader multioscillator 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.     

The interplay of light and the circadian clock. Independent dual regulation of clock-controlled gene ccg-2(eas).

Ambient light is the major agent mediating entrainment of circadian rhythms and is also a major factor influencing development and morphogenesis. We show that in Neurospora crassa the expression of clock-controlled gene 2 (ccg-2), a gene under the control of the circadian clock and allelic to the developmental gene easy wettable (eas), is regulated by light in wild-type strains. Light elicits a direct and important physiological effect on ccg-2(eas) expression as demonstrated using several mutant Neurospora strains. In white collar mutants (wc-1 and wc-2) that are "blind" to blue light, ccg-2(eas) mRNA shows no variation following illumination with saturating light. By contrast, ccg-2(eas) mRNA is photoinduced in clock-null strains such as frequency (bd;frq). The results in the clock mutants show that an intact circadian oscillator is not required for light induction of ccg-2(eas). Thus, ccg-2(eas) is subject to a dual regulation that involves separable regulation by light and circadian rhythm.     

Effect of 5-azacytidine on the light-sensitive formation of sexual and asexual reproductive structures in wc-1 and wc-2 mutants of Neurospora crassa

Under the conditions of nitrogen starvation, illumination by blue light of wc-1 and wc-2 mutants of the ascomycete Neurospora crassa failed to stimulate the formation of protoperithecia and inhibit conidiation (contrary to what was observed in the mycelium of the wild-type fungus). The data obtained indicate that wc-1 and wc-2 genes of N. crassa are involved in light-dependent formation of protoperithecia and conidia. The effects of 5-azacytidine (an inhibitor of DNA methylation) under the same experimental conditions suggest that the balance between the formation of sexual and asexual reproductive structures, maintained in N. crassa, depends on genome methylation processes sensitive to the action of light, which is mediated by the photoreceptor complex of WC proteins.     

Phylogenetic Analysis of Heterothallic Neurospora Species

We examined the phylogenetic relationships among five heterothallic species of Neurospora using restriction fragment polymorphisms derived from cosmid probes and sequence data from the upstream regions of two genes, al-1 and frq. Distance, maximum likelihood, and parsimony trees derived from the data support the hypothesis that strains assigned to N. sitophila, N. discreta, and N. tetrasperma form respective monophyletic groups. Strains assigned to N. intermedia and N. crassa, however, did not form two respective monophyletic groups, consistent with a previous suggestion based on analysis of mitochondrial DNAs that N. crassa and N. intermedia may be incompletely resolved sister taxa. Trees derived from restriction fragments and the al-1 sequence position N. tetrasperma as the sister species of N. sitophila. None of the trees produced by our data supported a previous analysis of sequences in the region of the mating type idiomorph that grouped N. crassa and N. sitophila as sister taxa, as well as N. intermedia and N. tetrasperma as sister taxa. Moreover, sequences from al-1, frq, and the mating-type region produced different trees when analyzed separately. The lack of consensus obtained with different sequences could result from the sorting of ancestral polymorphism during speciation or gene flow across species boundaries, or both.     

Isolation of new white collar mutants of Neurospora crassa and studies on their behavior in the blue light-induced formation of protoperithecia

White collar (wc) mutants of Neurospora crassa are thought to be regulatory mutants blocked in the photoinduction of carotenogenesis. Eight new wc mutants have been isolated after UV mutagenesis; their morphology and linear growth rate are not altered, although blue light-induced carotenogenesis is completely blocked. All of the wc mutations fall into two complementation groups corresponding to the already-known wc-1 and wc-2 loci. It is shown that the wc mutations impair another blue light effect, the photoinduction of protoperithecia formation, as well as the low constitutive production of protoperithecia in the dark. These effects are not due to the lack of carotenoids since the albino mutants show a normal sexual development. The pleiotropic effects of the mutations in the wc genes indicate that they play a key role in the mechanisms of regulation of the blue light-induced responses of N. crassa.     

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.     

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.     

Structural variations and optional introns in the mitochondrial DNAs of Neurospora strains isolated from nature

Mitochondrial DNAs from ten wild-type Neurospora crassa, Neurospora intermedia, and Neurospora sitophila strains collected from different geographical areas were screened for structural variations by restriction enzyme analysis. The different mtDNAs show much greater structural diversity, both within and among species, than had been apparent from previous studies of mtDNA from laboratory N. crassa strains. The mtDNAs range in size from 60 to 73 kb, and both the smallest and largest mtDNAs are found in N. crassa strains. In addition, four strains contain intramitochondrial plasmid DNAs that do not hybridize with the standard mtDNA. All of the mtDNA species have a basically similar organization. A 25-kb region that includes the rRNA genes and most tRNA genes shows very strong conservation of restriction sites in all strains. The 2.3-kb intron found in the large rRNA gene in standard N. crassa mtDNAs is present in all strains examined, including N. intermedia and N. sitophila strains. The size differences between the different mtDNAs are due to insertions or deletions that occur outside of the rRNA-tRNA region. Restriction enzyme and heteroduplex mapping suggest that four of these insertions are optional introns in the gene encoding cytochrome oxidase subunit I. Mitochondrial DNAs from different wild-type strains contain zero, one, three, or four of these introns.