The band mutation in Neurospora crassa is a dominant allele of ras-1 implicating RAS signaling in circadian output

Abstract

band, an allele enabling clear visualization of circadianly regulated spore formation (conidial banding), has remained an integral tool in the study of circadian rhythms for 40 years. bd was mapped using single-nucleotide polymorphisms (SNPs), cloned, and determined to be a T79I point mutation in ras-1. Alterations in light-regulated gene expression in the ras-1(bd) mutant suggests that the Neurospora photoreceptor WHITE COLLAR-1 is a target of RAS signaling, and increases in transcription of both wc-1 and fluffy show that regulators of conidiation are elevated in ras-1(bd). Comparison of ras-1(bd) with dominant active and dominant-negative ras-1 mutants and biochemical assays of RAS function indicate that RAS-1(bd) displays a modest enhancement of GDP/GTP exchange and no change in GTPase activity. Because the circadian clock in ras-1(bd) appears to be normal, ras-1(bd) apparently acts to amplify a subtle endogenous clock output signal under standard assay conditions. Reactive oxygen species (ROS), which can affect and be affected by RAS signaling, increase conidiation, suggesting a link between generation of ROS and RAS-1 signaling; surprisingly, however, ROS levels are not elevated in ras-1(bd). The data suggest that interconnected RAS- and ROS-responsive signaling pathways regulate the amplitude of circadian- and light-regulated gene expression in Neurospora.

Figure 1.

Cloning and identification of the bd mutation. (A) Germinated spores isolated from cross 402 between wild-type Mauriceville and Oak Ridge bd strains were scored on race tubes. The panel shows representative strains with and without the bd mutation grown on standard race tube media. (B) Forced heterokaryon analysis was used to establish the dominance of ras-1^bd. Heterokaryons were generated and banding was scored on race tubes. The corresponding genotype of each heterokaryon is Het A, 901-64 (inl, bd, A) and 418-11 (pan-2, A); Het B, 418-11 (pan-2, A) and 901-64 (inl, bd, A); Het C, 418-11 (pan-2, A) and 418-24 (inl, A); Het D, 901-62 (pan-2, bd, A) and 418-18 (inl, A); Het E, 418-18 (inl, A) and 901-62 (pan-2, bd, A); and Het F, 901-62 (pan-2, bd, A) and 418-16 (inl, bd, A). (C) Structure and sequence alignment indicating the location of the bd mutation in RAS-1. The structure of RAS and SOS (Boriack-Sjodin et al. 1998) was modified using PDB (PDB:1BKD) (http://www.rcsb.org/pdb/explore/pubmed.do?structureid=1BKD). The number sign (#) marks the region comprising the GEF-binding domain, and the asterisk (*) marks the switch II loop. The homology alignment compares RAS-1 between Neurospora (N.c.), Homo sapien (H.s.), Trametes hirsuta (T.h.), and Schizosaccharomyces pombe (S.p.). The T mutated to I in RAS-1^bd is highlighted with an arrow. (D) Northern blots showing the regulation of ras-1 in a wild-type strain where RNA was extracted at the times shown after standard circadian entrainment.

Figure 2.

Analysis of RAS-1^bd activity. (A) Comparison of wild type and ras-1^bd with dominant-negative GDP-bound (K21N) and dominant active GTP-bound (G17V) forms of RAS-1. Strains were grown on Vogel’s media supplemented with 0.1% glucose, 0.17% arginine, yeast extract, and malt extract. (B) The amount of activated RAS-1 was measured in wild-type and ras-1^bd strains in the dark and following exposure to light (LP) for 30, 60, or 180 min. GTP-bound RAS was assessed by its ability to bind GST–RAF–RBD and was detected with a monoclonal antibody that specifically recognizes RAS-1. Lanes marked GTP represent lysate preincubated with GTPγS, and lanes marked GDP represent those incubated with GDP. (C) Densitometric analysis of GTP-bound RAS-1 obtained from five independent experiments; error bars represent ±SEM. (D) GDP/GTP exchange was measured by incubating lysates in the presence of GTPγS, and then GST–RBD-bound RAS-1 was detected by immunoblot analysis. TOT represents 10% of the total lysate used, and lanes marked GDP show lysate incubated with GDP; see Materials and Methods. (E). Graphical representation of GTP exchange in wild type compared with ras-1^bd: Error bars represent ±SEM; n = 6.

Figure 3.

Light-activated gene expression is elevated in ras-1^bd. (A) Following treatment with saturating amounts of light for the indicated times, total RNA from wild-type and ras-1^bd strains was isolated and analyzed by Northern blotting using probes specific for wc-1, fluffy, and vvd. rRNA represents the loading control. (B) Protein lysates were isolated from the same cultures used in A, and immunoblot analysis was performed using antibodies specific for FRQ and WC-1. Blots are representative of three replicates. (See also Supplementary Fig. 2 for densitometric analyses.)

Figure 4.

Circadian-regulated transcription in wild type versus ras-1^bd. Following growth in liquid culture after standard circadian entrainment, total RNA from wild-type and ras-1^bd strains was isolated at the indicated times in constant darkness (or after a 15-min light pulse given at DD24) and analyzed by Northern blotting with probes specific for frq (A), wc-1 (B), or vvd (C). Blots are representative of three to four replicates. (See also Supplementary Fig. 3 for densitometric analyses.)

Figure 5.

Increased ROS causes circadianly regulated banding. (A) The wild-type strain exhibits circadian banding when evaluated in race tube media containing the ROS-generating compound menadione (ME). The effect can be reverted by the addition of the antixodant NAC, while higher concentrations of NAC abrogate banding in the ras-1^bd strain. Race tube assays were conducted under conditions of constant darkness (DD) and temperature (25°C). Standard race tube media was supplemented with the indicated compounds at the specified concentrations. NAG was used as a control for NAC addition. Two out of six replicate tubes are shown for each treatment, and period lengths are reported as mean ± SD for n = 6 race tubes. (B) To confirm the circadian nature of the rhythm observed for wild type, the long-period mutant frq⁷ and the arrhythmic frq¹⁰ were also evaluated in menadione-race tubes and compared with ras-1^bd, frq⁷ and ras-1^bd, frq¹⁰. (C) The Δsod-1 strain displays circadian banding when assayed in race tubes, and the banding phenotype can be reverted by addition of 10 mM NAC. Where possible, circadian period lengths were measured and are reported as mean ± SD (n = 6 race tubes).

Figure 6.

Measurement of ROS levels in wild type, ras-1^bd, and Δsod-1. (A) Superoxide was detected by lucigenin-enhanced chemiluminescence in mycelial layers grown for 20 h at 30°C in 0.1% liquid culture medium as described in Materials and Methods. (RLU) Relative light units. (B) ROS levels were measured following H₂DCFDA diacetate oxidation in mycelia (top panel) or extracellular media (bottom panel) as indicated in Material and Methods. Values are averages of six samples and corrected by protein (A) or dry weight (B). Error bars represent the standard deviation of the mean.

Figure 7.

RAS-1^bd and ROS increase fluffy trancription. Following growth in liquid culture after standard circadian entrainment, total RNA from wild-type, ras-1^bd, and Δsod-1 strains was isolated at the indicated times in constant darkness (or after a 15-min light pulse given at DD24) and analyzed by Northern blotting with a probes specific for fluffy. Blots are representative of three to four replicates. (See also Supplementary Fig. 3D for densitometric analyses.)