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

Densitometry of <i>chol⁺</i>; <i>frq⁺</i> progeny.

<p>Race tube cultures of progeny from the cross of <i>csp-1</i>; <i>ras^bd</i>; UV90 to Mauriceville were analyzed by densitometry. Six <i>csp-1</i> progeny from each class were randomly chosen for analysis. Densitometry traces were averaged for each class and plotted ± one S.E.M. A. <i>ras^bd</i> progeny. B. <i>ras⁺</i> progeny. Thick black lines: UV90⁺ progeny. Thin blue lines: UV90 progeny. Gray lines: ± one S.E.M. All cultures were grown without choline. Density is in arbitrary units. Time is in hours after transfer from LL to DD.</p

Effects of UV90 mutation on phase-resetting.

<p>All strains carry the <i>ras-1^bd</i> and <i>csp-1</i> mutations. Cultures were grown in DD at 22°C. Solid triangles: UV90⁺. Open triangles: UV90. Each point represents an individual race tube. Top row: Resetting by 2 min light pulses. Bottom row: Resetting by 15 min heat pulses. Left column: Phase response curves, plotting the change in phase (phase shift in circadian hours) against the circadian time (CT) of the pulse. Average phase shifts are connected by lines. Right column: Phase transition curves, plotting the new phase after the pulse in circadian hours against the circadian time of the pulse (“old phase”). Diagonal lines indicate the locus of “no effect” where new phase equals old phase. Note that the UV90⁺ data for old phase 22.5 in the heat PTC have been double-plotted to show the continuity of the data.</p

Effects of UV90 mutation on entrainment to high temperature pulses.

<p>All strains carry the <i>ras-1^bd</i>, <i>csp-1</i> and <i>chol-1</i> mutations and were grown on high choline to repair the <i>chol-1</i> defect. Cultures were grown in DD at 22°C and were subjected to 2-h 32°C pulses at repeated intervals of T hours (16, 20, 24 or 28 h), in which T is the length of one cycle from one pulse start to the next pulse start. Culture density was determined and the last two complete cycles on each race tube were averaged for 6 race tubes. Density traces were normalized and are plotted ± one S.E.M., N = 12, against hours after the beginning of the 2-h heat pulse. Thick black lines: wild-type for UV90; thin black lines: UV90 mutant; grey lines: ± S.E.M. A–D. Comparison of UV90 wild-type with UV90 mutant in the <i>frq⁺</i> background. E–H. Comparison of UV90 wild-type with UV90 mutant in the <i>frq¹⁰</i> background.</p

Densitometry of <i>chol-1</i>; <i>frq¹⁰</i> progeny.

<p>Race tube cultures of <i>csp-1</i>; <i>chol-1 ras^bd</i>; <i>frq¹⁰</i> progeny from the backcross of <i>csp-1</i>; <i>chol-1 ras^bd</i>; <i>frq¹⁰</i>; UV90 to <i>ras^bd</i> were analyzed by densitometry. Six progeny from each class were randomly chosen for analysis. Left column: UV90⁺ progeny. Right column: UV90 progeny. All cultures were grown without choline. Density is in arbitrary units. Time is in hours after transfer from LL (constant light) to DD.</p

Effects of UV90 mutation on expression of FRQ protein.

<p>Both strains carry the <i>ras-1^bd</i>, <i>csp-1</i> and <i>chol-1</i> mutations and were grown on high choline to repair the <i>chol-1</i> defect. Cultures were grown in DD for varying lengths of time and protein extracts were assayed for FRQ protein by immunoblotting. A. Relative level of FRQ, normalized to total protein by Coomassie staining. Solid circles: UV90 wild-type; open circles: UV90 mutant. Points are from a single series of experiments; similar results were obtained in a replicate experiment. B. Upper panel: Immunoblotting of FRQ protein for one cycle, 3–27 h in DD, corresponding to the time-series in A. UV90 wild-type samples are on the left half and UV90 mutant samples on the right half of the blot. Lower panel: Coomassie stained membrane of samples in upper panel. C. Ratios of relative FRQ levels in UV90 vs. UV90⁺. Ratios were calculated at three peak time points (21, 42 and 63 hours in DD) in three independent experiments and mean ratios are plotted ± one S.E.M. *: significantly less than 1.00, p<0.05. **: significantly less than 1.00, p<0.01.</p

Segregation of growth rates.

<p>Progeny from the backcross of <i>csp-1</i>; <i>chol-1 ras^bd</i>; <i>frq¹⁰</i>; UV90 to <i>ras^bd</i> were grown at 22°C in DD in race tubes on MA medium with 100 µM choline (high choline) or without added choline (low choline) and growth rates were determined. All progeny carry the <i>csp-1</i>, <i>frq⁺</i> and <i>ras^bd</i> alleles. Note the change in scales between the X and Y axes.</p

Densitometry of <i>chol-1</i>; <i>frq⁺</i> progeny.

<p>Race tube cultures of <i>csp-1</i>; <i>chol-1 ras^bd</i> progeny from the backcross of <i>csp-1</i>; <i>chol-1 ras^bd</i>; <i>frq¹⁰</i>; UV90 to <i>ras^bd</i> were analyzed by densitometry. All other conditions as in <a href="http://www.plosgenetics.org/article/info:doi/10.1371/journal.pgen.1002151#pgen-1002151-g002" target="_blank">Figure 2</a>.</p