A Novel Cryptochrome-Dependent Oscillator in Neurospora Crassa

Genetics: Early Online, published on October 30, 2014 as 10.1534/genetics.114.169441

A Novel Cryptochrome-Dependent Oscillator in Neurospora crassa

Imade Y. Nsa, Nirmala Karunarathna, Xiaoguang Liu1, Howard Huang, Brittni Boetteger, and Deborah Bell-Pedersen

Center for Biological Clocks Research, Program for the Biology of Filamentous Fungi, and Department of Biology, Texas A&M University, College Station, TX 77843, U.S.A. 1 Current address: Department of Microbial Engineering, College of Bioengineering Tianjin University of Science and Technology, 29, 13th Avenue, TEDA, Tianjin, P.O. 300457

Running Title: CRY-dependent oscillator Key Words: Circadian clock, oscillator, cryptochrome, FRQ-less oscillator, FRQ-WCC oscillator. Correspondence to: Deborah Bell-Pedersen; 3258 TAMU Dept of Biology, College Station, TX 77843 USA; Tel.: 979-847-9237; Fax: 979-845-2891; Email: [email protected]

ABSTRACT Several lines of evidence suggest that the circadian clock is constructed of multiple molecular feedback oscillators that function to generate robust rhythms in organisms. However, while core oscillator mechanisms driving specific behaviors are well described in several model systems, the nature of other potential circadian oscillators is not understood. Using genetic approaches in the fungus Neurospora crassa, we uncovered an oscillator mechanism that drives rhythmic spore development in the absence of the well-characterized FRQ/WCC oscillator, and in constant light, conditions in which the FRQ/WCC oscillator is not functional. While this novel oscillator does not require the FWO for activity, it does require the blue light photoreceptor CRYPTOCHROME, thus we call it the CDO (CRY-dependent oscillator). The CDO was uncovered in a strain carrying a mutation in cog-1 (cry-dependent oscillator gate -1), has a period of about a day in LL, and is temperature compensated. In addition, cog-1 cells lacking the circadian blue-light photoreceptor WC-1 respond to blue-light, suggesting that alternate light inputs function in cog-1 mutant cells. We show that the blue-light photoreceptors, VIVID and CRY compensate for each other, and for WC-1, in CRY-dependent oscillator light responses, but that WC-1 is necessary for circadian light entrainment.

Circadian clocks, composed of generate daily rhythms in gene expression, physiology, and behavior, in molecular transcription/translation- all kingdoms of life. The circadian clock based feedback loop (TTFL) oscillators,

Copyright 2014. provides a mechanism for organisms to (FRH), the blue-light photoreceptor anticipate cyclic changes in the WHITE COLLAR 1 (WC-1), and WHITE environment in order to carry out COLLAR 2 (WC-2). WC-1 and WC-2 specific tasks at advantageous times of form a complex, called the WCC that the day. During investigations of clock functions as a positive element in the mechanisms, several studies have oscillator loop. WCC binds the frq revealed evidence for the existence of promoter and directly activates multiple autonomous oscillators in cells transcription of the frq gene (FROEHLICH and/or tissues. First, there exist free- et al. 2003). As FRQ protein running rhythms of different periods in accumulates it interacts with itself and the same organism (MORSE et al. 1994; FRH (CHENG et al. 2001; CHENG et al. SAI and JOHNSON 1999; CAMBRAS et al. 2005), and then binds to, and promotes 2007). Second, residual rhythmicity is the phosphorylation and inactivation of found in some strains defective in the WCC (SCHAFMEIER et al. 2005; HE et known oscillator components (LOROS al. 2006). Inhibition of WCC activity and FELDMAN 1986; STANEWSKY et al. results in reduced frq transcription and 1998; EMERY et al. 2000; COLLINS et al. FRQ protein levels. Once FRQ protein 2005). Third, some tissue-specific levels are sufficiently decreased, oscillators are constructed differently FRQ/FRH-directed inhibition of the from core oscillators located in the activity of the WCC is released, and the brains of insects and animals cycle reactivates the next day. The (STANEWSKY et al. 1998; EMERY et al. FWO controls daily rhythms in 2000; IVANCHENKO et al. 2001; KRISHNAN expression of about 20% of the genome, et al. 2001; COLLINS et al. 2005). Thus, and overt rhythms in asexual spore multiple oscillators may exist both development (conidiation) (VITALINI et al. within, and among, cells in organisms 2006). The conidiation rhythm, typically with differentiated tissues. Furthermore, measured using the race tube assay, recent data revealed circadian rhythms has a period in constant dark (DD) in the oxidation state of highly conditions of about 22 h in wild type conserved peroxiredoxin in the absence strains (LOROS and DUNLAP 2001). of transcription and thus a functional TTFL, adding additional levels of Several studies revealed that rhythms complexity to the circadian clock system can persist in the absence of a (EDGAR et al. 2012). functional FWO under certain growth conditions, and/or in specific genetic The fungus Neurospora crassa is a backgrounds, providing evidence to leading model for studying the clock suggest the existence of additional (BELL-PEDERSEN 2000; HEINTZEN and LIU oscillators in N. crassa cells (LOROS and 2007; LAKIN-THOMAS et al. 2011; BAKER FELDMAN 1986; ARONSON et al. 1994; et al. 2012) and light signaling (LINDEN MERROW et al. 1999; RAMSDALE and et al. 1997; BELL-PEDERSEN et al. 2001; LAKIN-THOMAS 2000; DRAGOVIC et al. LIU 2003; MERROW et al. 2006; CHEN 2002; CORREA et al. 2003; GRANSHAW et and LOROS 2009). In N. crassa, the core al. 2003; CHRISTENSEN et al. 2004; HE et FRQ/WCC oscillator (FWO) contains the al. 2005; DE PAULA et al. 2006; BRODY et negative elements FREQUENCY (FRQ) al. 2010; HUNT et al. 2012). The term and FRQ-interacting RNA helicase FLO (frq-less-oscillator) was coined to

2 collectively describe these putative regarding their nature and role in the circadian and/or non-circadian circadian system. oscillators (IWASAKI and DUNLAP 2000). Indeed, in most cases, rhythms In an attempt to identify key components attributed to FLOs were shown to lack of the FLO(s), we carried out a genetic one or more of the three canonical clock screen for mutations that enhance properties, including the generation of a rhythmicity in strains that are deficient in free-running rhythm of about 24 h in the both positive and negative components absence of environmental cues, of the FWO. We identified a mutation entrainment of the free-running rhythm called cry-dependent oscillator gate-1 to 24 h by environmental cues, and (cog-1) that displayed robust rhythms in temperature compensation of the clock conidiation in constant light (LL), (LAKIN-THOMAS 2000; BAKER et al. independent of the FWO. The oscillator 2012). In entrainment of the clock, the controlling these rhythms fulfills two of period of the rhythm becomes equal, on the three criteria for a circadian average, to an imposed environmental oscillator; it free runs in constant cycle, and a unique stable phase conditions with a period close to a day, relationship is established between the and is temperature compensated. imposed environmental cycle and the However, while cog-1 mutant strains entrained oscillator (JOHNSON et al. were synchronized by LD cycles 2003). Synchronization is distinguished independent of the photoreceptor and from entrainment in that the cycle output clock component WC-1, circadian occurs in response to the stimulus in a entrainment in LD required WC-1. set time frame, and does not depend on Finally, we show that the blue-light the length of the imposed environmental photoreceptor CRYPTOCHROME cycle. Temperature compensation of the (CRY), a core component of the clock means that the rate is relatively mammalian circadian oscillator (GRIFFIN independent of temperature within the et al. 1999), is necessary for the novel physiological range, with a temperature oscillator activity in Neurospora cells in coefficient (Q10) near 1. The lack of full LL. We therefore call the oscillator the circadian properties of the FLOs led to CRY-dependent Oscillator (CDO), suggestions that the FWO serves as a pacemaker in N. crassa cells, driving MATERIALS AND METHODS rhythms in downstream, so-called slave, Strains FLOs (DUNLAP and LOROS 2005). In this The N. crassa strains used in this study model, the FLOs are intrinsically are listed in Table 1, their periods under rhythmic, but require the FWO for full different growth conditions in Table 2, circadian properties. Furthermore, the and growth rates of representative number of FLOs in Neurospora cells is strains in Table 3. The vvd knockout not known. While studies have been (KO) strain was obtained from Dr. undertaken to identify molecular Christian Heintzen (HEINTZEN et al. components of the FLO(s) (LOMBARDI et 2001). The Δwc-1::hph strain was al. 2007; SHI et al. 2007; YOSHIDA et al. obtained from Dr. Jay Dunlap 2008; SCHNEIDER et al. 2009; LAKIN- (Dartmouth Medical School), and Δwc- THOMAS et al. 2011; LI et al. 2011; HUNT 1::bar was generated in our lab et al. 2012), little is understood (BENNETT et al. 2013). The cry KO

3 (FGSC 12981) was generated by the N. Growth Conditions crassa KO project (COLOT et al. 2006), and obtained from the Fungal Genetics All vegetative cultures were maintained Stock Center (FGSC). All strains used in on 1X Vogel’s, 2% glucose, minimal this study carry the ras-1bd mutation, medium with the appropriate which clarifies the conidiation rhythm on supplements as required (VOGEL 1956; race tubes (SARGENT et al. 1966; DAVIS and DE SERRES 1970). Sexual bd BELDEN et al. 2007). The ras-1 strain crosses were performed on serves as the clock wild type (WT) Westergaard’s crossing agar plates control strain. To generate the cog-1 (WESTERGAARD and MITCHELL 1947). KO mutation, a strain defective in the strains containing the hph marker were positive and negative arm of the FWO maintained on Vogel’s minimal medium (wc-2234W, ras-1bd, Δfrq) was supplemented with 200 µg/ml mutagenized by ultraviolet (UV) light hygromycin B (Sigma Aldrich, St. Louis, according to standard procedures MO). KO strains containing the bar gene (DAVIS and DE SERRES 1970). The were maintained on nitrate free Vogel’s construction of double, and triple medium supplemented with 250 µg/ml photoreceptor deletion strains are glufosinate (Sigma Aldrich, St. Louis, described in Figure S1 A&B. To MO). The composition of race tube complement Δcry, cog-1 cells, a 4.9 kb media was 11.5 ml of 1X Vogel’s, 0.1% PCR fragment containing the cry gene glucose, 0.17% arginine, and 1.5% was cloned into pCR-BluntBar that agar. After autoclaving, race tubes were contains the bar gene for selection and allowed to dry for 7 days. The dried race transformed to Δcry, cog-1 cells using tubes were inoculated with mycelia or standard Neurospora transformation conidia from 7-day old slants, and grown techniques (PALL and BRUNELLI 1993). for one day at 25°C, and then PCR was used to verify the genotypes transferred to the indicated conditions in of double and triple photoreceptor Percival growth chambers (Perry, IA). mutant strains from genomic DNA The growth front was marked at the time isolated from 7-day old slant cultures as of transfer. Light was from cool white described (JIN et al. 2007) (Figure S1 fluorescent bulbs, unless otherwise C). The PCR primers used to verify the indicated. Light intensity was measured KO strains are listed in Table S1. with a VWR Scientific Dual Range Light Meter (Radnor, PA), and maintained at To determine if the cog-1 mutation is 1200 lux in all light experiments, unless dominant or recessive, a heterokaryon otherwise indicated. To examine the of strain matA, arg-5, ras-1bd and matA, effects of different wavelengths of light trp-3, ras-1bd, cog-1 was generated. on the CDO, four different light sources Heterokaryons were selected by growth (red: 60 lux, blue: 80 lux, green: 64 lux, on minimal media as described (Davis white: 72 lux) were used in Percival and de Serres, 1970). Heterokayotic incubators. Temperature recording in strains with nuclear ratios of 1:1, as the incubators was accomplished using determined by plating on selective an EasyLog EL-USB thermometer media, were examined for rhythmicity on (Lascar Electronics, Erie, PA). The race tubes. growth fronts of the race tubes were marked at 24h intervals using a red light for cultures in DD, and at lights on for

4 cultures in light/dark (LD) cycles. Race strains that were defective in both the tubes were scanned with an EPSON positive and negative arms of the FWO. scanner (Long Beach, CA), and growth This approach ruled out any possible rates and periods were calculated. n = residual activity from the FWO. The wc- the number of conidial bands measured 2234W, Δfrq strain was mutagenized by on replicate race tubes. The percent UV light to 50% survival, and a mutant rhythmic was calculated as the number strain, originally identified as Light of individual race tubes displaying at Mutant 1, and renamed cry-dependent least 5 clear conidial bands for which we oscillator gate (cog-1), was identified could measure period, out of the total that had robust conidiation rhythms in number of race tubes examined for each LL (Figure 1, Table 2). The cog-1 strain under a given growth condition. mutant strain was outcrossed multiple times to the clock WT strain ras-1bd to isolate the cog-1 mutation from all other Western blots mutations. The cog-1 mutant phenotype Protein was extracted from ground segregated with a 1:1 ratio in crosses, tissue (GARCEAU et al. 1997), and 100 suggesting that the rhythmic phenotype µg of total protein was run on 10% SDS is due to a single gene mutation. The PAGE gels, and transferred to resulting ras-1bd, cog-1 strain (called polyvinylidene difluoride membranes cog-1) was used for all subsequent (IPVH00010; Millipore, Billarica, MA). analyses. CRY protein was detected by immunoblotting with primary anti-CRY To confirm that the cog-1 mutation antibody (polyclonal, 1:1000 v/v, a gift rescued developmental rhythms in from Dr. Jay Dunlap, Dartmouth Medical strains that lack a functional FWO, the School), and secondary goat anti-rabbit cog-1 strain was crossed to frq-null horseradish peroxide 2 conjugated (Δfrq) and wc-1-null (Δwc-1) strains. antibody (diluted 1:20,000 v/v, 170- Progeny that were Δfrq, cog-1 and Δwc- 6515, Bio Rad, Hercules, CA). Protein 1, cog-1 were isolated and examined, bands were visualized using a along with control sibling strains, for chemiluminescence kit (SuperSignal rhythmicity in LL (Figure 2 and Table West Femto, ThermoFischer Scientific, 2). Consistent with previous Waltham, MA) according to the observations, the WT strain was manufacturer’s instructions. The arrhythmic in LL (CROSTHWAITE et al. membranes were subsequently stained 1995; ELVIN et al. 2005). Strains that with amido black (0.1% amido black lacked FWO components were also [Sigma-Aldrich, St. Louis MO], 10% arrhythmic in LL. However, rhythms in acetic acid, 25% isopropanol) for protein conidial development within the level normalization. circadian range were observed in all strains that contained the cog-1 RESULTS mutation. In the absence of the FWO The CDO functions independently of components, the period of the cog-1 the FWO rhythm was near 24 h, whereas cog-1 To identify components of N. crassa strains with a functional FWO had a FLO’s, we carried out a screen for shorter period. These data suggested mutations that would enhance that the FWO and the CDO genetically developmental rhythms on race tubes in

5 interact in LL. While all cog-1 and Δwc- CDO rhythm on race tubes is 1, cog-1 replicate race tube cultures pronounced. While most biochemical displayed rhythms in LL (100%), the reactions are temperature-dependent, number of rhythmic race tubes of Δfrq, with Q10 temperature coefficient values cog-1 was reduced to 75% (Table 2). of 2-3, temperature compensated clocks have Q10 values of between 0.8-1.4 The CDO free runs in constant (SWEENEY and HASTINGS 1960). The conditions and is temperature period of the rhythm differed between compensated. the cog-1 and ∆wc-1, cog-1 strains as Using heterokaryon analyses, we expected; however, the Q10 values determined that the cog-1 mutation was measured for both strains between 17°C recessive (Figure S2), suggesting cog-1 and 27°C was 1.2, confirming that the is a loss of function mutation that CDO is temperature compensated uncovers an autonomous CDO. To test (Figure 3B). if the CDO has properties of a circadian oscillator, cog-1 strains were further The third defining property of a circadian examined for free-running rhythms in clock is entrainment. The best way to DD, light entrainment, and temperature demonstrate entrainment is to examine compensation. Consistent with the LL environmental cycles with periods that data, independent of the FWO, strains are not equal to 24 h (JOHNSON et al. containing the cog-1 mutation are 2003). If the rhythm entrains to rhythmic in DD (Figure 3A). However, environmental cycles it will display a the robustness of the rhythms (% period that equals the length of the rhythmic) in cog-1 strains that also environmental cycle, and have stable lacked components of the FWO was phase angles that differ in different decreased in DD, as compared to LL environmental cycle lengths. If instead, (Table 2), and robustness of the the rhythms display periods equal to the rhythms in cog-1 strains was cycle length, but have similar phase independent of whether the clock was angles in different cycles, then the initially synchronized in these cells by a rhythms are synchronized (driven), not light and/or a temperature transition entrained, by the imposing cycle. Thus, (data not shown). Together, these data to investigate entrainment of the CDO, suggest that in DD, the FWO overrides, we examined strains that display the and/or enhances, the CDO rhythms. CDO in different light/dark (LD) cycle Consistent with the FWO overriding the lengths (Figure 4A). For WT, cog-1, and CDO in DD, the cog-1 period in DD was ∆wc-1, cog-1 strains, the period of the similar to the WT strain (22 h), whereas rhythm matched the imposed LD cycle when only the CDO is expressed (cog-1 length. Strains with functional WC-1 in LL), the period is about 17 h (Figure displayed conidiation in different phases 2). in LD cycles of different duration, consistent with entrainment. However, To determine if the CDO rhythm is the phase angles in ∆wc-1, cog-1 strains temperature compensated, we assayed stayed relatively constant, with the developmental rhythm in cog-1 and conidiation occurring just after lights on ∆wc-1, cog-1 strains in LL, conditions in in the different LD cycles, indicative of which the FWO is not functional, but the an LD-driven rhythm (Figure 4B).

6 period of the rhythm was reduced, and To rule out the possibility that the more variable, as compared to cog-1 synchronization of ∆wc-1, cog-1 strains (compare Figure 5A to Figure 2). The in LD cycles was due to changes in ∆vvd, cog-1 strains also entrained to LD temperature, rather than changes in cycles (Figure 5B & 5C). Surprisingly, light, we carefully monitored the in ∆cry, cog-1 strains grown in LL, the temperature of the incubators with a developmental rhythms were abolished. temperature-recording device. The The rhythm was rescued by ectopic maximum temperature variance transformation of the cry gene into the recorded was an increase in ∆cry, cog-1 strain (∆cry::cry, cog-1) temperature in the light of 0.5°C in each (Figure 5A). In addition, the ∆cry, cog-1 cycle. A 1°C temperature change was strain entrained normally to LD cycles not sufficient to drive the developmental (Figure 5B & 5C). Entrainment of ∆vvd, rhythm in ∆wc-1 cells (Figure S3). cog-1 and ∆cry, cog-1 strains to LD Therefore, we can rule out the possibility cycles is consistent with the presence of that the rhythms observed in ∆wc-1, a functional FWO in these cells. cog-1 strains in LD cycles was due to Together, these data demonstrated that 0.5°C cycles in the incubators. CRY is necessary for function of the Together, these data demonstrate that CDO in LL, and that both CRY and VVD WC-1 is required for stable entrainment are dispensable for light responses in of the circadian clock to light in N. cog-1 cells. However, the WC-1 crassa, and that photoreceptors other photoreceptor likely compensated for than WC-1 are capable of responding to the loss of individual VVD or CRY light in ∆wc-1, cog-1 cells. photoreceptors in these strains.

CRY is essential for CDO rhythms To determine if light responses in the The N. crassa clock is only known to be ∆vvd, cog-1 and ∆cry, cog-1 strains are responsive to blue light (SARGENT and due to the presence of WC-1, we BRIGGS 1967). Consistent with this, the generated double photoreceptor response of the CDO to light is mutants in the cog-1 mutant restricted to the blue light range (Figure background, and examined rhythms in S4). Therefore, to identify the LL and responses to light in LD cycles at photoreceptors responsible for light 25°C. Consistent with a requirement for responses in the ∆wc-1, cog-1 strain, we CRY in CDO function, all of the cog-1 crossed the cog-1 mutation to strains double photoreceptor mutants that carrying deletions of the other known harbor cry deletions were arrhythmic in blue light photoreceptors in N. crassa, LL (Figure 6A and Table 2). Of the VVD (ZOLTOWSKI et al. 2007) and CRY double photoreceptor mutants, only the (FROEHLICH et al. 2010) to generate ∆vvd, ∆wc-1, cog-1 strain displayed ∆vvd, cog-1, and ∆cry, cog-1 strains. rhythms in LL. The period of the rhythm These strains, and the control siblings, of ∆vvd, ∆wc-1, cog-1 cells was similar were assayed for rhythms in LL and for to ∆wc-1, cog-1 (Figure 2), but was entrainment in LD cycles at 25°C significantly longer than ∆vvd, cog-1 (Figure 5). In the ∆vvd, cog-1 strain in (Figure 5A and Table 2), suggesting LL, rhythms in development were that while VVD may modify the period of observed (Figure 5A); however, the the CDO, VVD is not necessary for CDO

7 rhythms. In LD cycles, light responses and Table 2). In wc-1 deletion strains, were observed in all of the double development is arrhythmic in DD as photoreceptor mutant strains that expected, but the cog-1 mutation harbored the cog-1 mutation (Figure rescued rhythmicity (Figure 3). 6B), and consistent with our earlier Consistent with a central role for CRY in results, entrainment to light required the CDO, rescue of rhythmicity in ∆wc-1 WC-1, but not VVD and CRY (Figure cells by cog-1 depended upon CRY, as 6C). evidenced by arrhythmic development in the ∆cry, ∆wc-1, cog-1 strain (Figure We next generated strains that lacked 7A). Together, these data suggest that all 3 blue light photoreceptors, with and CRY is necessary for the function of the without the cog-1 mutation, and assayed CDO in LL (Figure 5A) and in DD the developmental rhythm in LL and LD (Figure 7A). cycles at 25°C (Figure 6 A&B and Table 2). Independent of the cog-1 The cry promoter is bound by the WCC mutations, the triple photoreceptor after a light pulse (SMITH et al. 2010), deletion mutant strains were arrhythmic providing a direct link between the FWO under both lighting conditions. and the CDO. To examine this link Arrhythmicity in the triple photoreceptor further, we assayed the levels of CRY mutant cells may be due to a lack of protein in WT, and ∆wc-1 strains with or synchronization of the clock by light in without the cog-1 mutation in DD and LL the culture, or to loss of function of the (Figure 7B). In cells grown for 24 h in oscillators. To distinguish these LL, CRY levels were increased relative possibilities, we used a temperature to the DD samples in WT, and cog-1 shift from 30°C to 25°C to synchronize cells, but not in ∆wc-1, and ∆wc-1, cog-1 the cells (GOOCH et al. 1994; LIU et al. cells, consistent with previous data 1998). In support of loss of clock demonstrating a role for WC-1 in CRY function in the triple photoreceptor light responses (FROEHLICH et al. 2010). mutant, the strain was arrhythmic Low levels of CRY protein were also following a temperature shift (Figure observed in ∆wc-1 and ∆wc-1, cog-1 S5). Together, these data indicated that cells grown in LL and harvested at any of the three blue light different times of the day (Figure S6). photoreceptors are able to substitute for Developmental rhythms persist in cog-1 each other to drive rhythms in LD cycles and ∆wc-1, cog-1 cells in LL, but the in strains containing the cog-1 mutation; levels of CRY protein varied widely however, in all cases, WC-1 was between these strains, with very low required for normal circadian levels in ∆wc-1, cog-1 cells and high entrainment to light. levels in cog-1 cells (Figures 7B & S6). Thus, the levels of CRY protein do not To further examine the requirement for appear to correlate with rhythmicity of CRY in the function of the CDO, we the strains in LL. We also examined assayed the photoreceptor mutant WC-1 steady-state levels in the same strains in DD at 25°C. In strains where cells. WC-1 levels were increased in the FWO was functional, rhythms with cells grown in LL for 24 h as compared periods close to WT were observed to the 24 h DD samples in WT, cog-1, independent of cry or cog-1 (Figure 7A and ∆cry cells, and slightly elevated

8 ∆cry, cog-1 cells (Figure 7C). Similar and is temperature compensated in LL, results were observed in cells grown for development in strains expressing the 12, 15, and 18 h in DD and LL (Figure CDO, but lacking WC-1, were driven by S7). Consistent with previous data, we light, rather than entrained by LD cycles. observed that WC-1 levels were Thus, the CDO, similar to other reported elevated in ∆cry cells as compared to FLO’s (DUNLAP and LOROS 2004), lacks WT cells in DD (OLMEDO et al. 2010). full circadian properties. In addition, Surprisingly, this increase in WC-1 these data revealed that blue light levels was not observed in ∆cry, cog-1 photoreceptors besides WC-1 are cells in DD (Figures 7 and S6), capable of perceiving light information to suggesting the possibility that COG-1 ultimately drive development in the functions as a CRY-dependent activator fungus. Surprisingly, any of the three of the WCC. However, similar to CRY blue light photoreceptors, VVD, CRY, or protein levels, no correlations between WC-1, can compensate for each other in the levels of WC-1 protein and cog-1 mutant light responses. These rhythmicity were observed. data support the notion that the photoreceptors function in multi-protein CDO activity reduces growth rate complexes, providing opportunities for To determine if activity of the CDO has crosstalk in response to light (BAYRAM et an affect on Neurospora cells, we al. 2010) compared the growth rates of representative strains with variations in The cog-1 mutation is recessive (Figure FWO and CDO function in DD and LL S2), suggesting that cog-1 is most likely (Table 3). Under both conditions, CDO a loss of function mutation. While we activity reduced the growth rate as have not yet identified the defective compared to WT cells. Cells with a cry gene product in the cog-1 mutant strain, deletion had a slightly slower growth these data indicate that the gene rate in DD. In cells that lack both FWO specified by the cog-1 mutation encodes and CDO activity in LL and DD, the a protein that either directly or indirectly growth rate was increased as compared reduces the activity the CDO when it is to WT cells, suggesting that while the present, rather than as a component of clock provides an overall adaptive the CDO that is necessary for its advantage to organisms in natural LD function. There are several possible cycles, its activity negatively affects reasons for why the CDO might be growth of the fungus in DD or LL, similar repressed via cog-1. For example, the to results obtained in cyanobacteria (MA CDO may only be required under certain et al. 2013). growth conditions, and shutting off the CDO when those conditions are not met DISCUSSION may provide a growth advantage to the To investigate the organization of the N. organism. Consistent with this crassa circadian system, we identified hypothesis, we observed that strains the cog-1 mutation that restores rhythms with a functional CDO had a reduced to strains that lack a functional FWO in growth rate (Table 3). Alternatively, the DD and LL. While the oscillator that CDO may be an ancient oscillator that drives this rhythm free-runs with a was shut down to allow a more efficient circadian period in constant conditions, oscillator, the FWO, which fully entrains to environmental conditions, to take over

9 and provide a mechanism for surprising (CHEN and LOROS 2009; anticipation of environmental cycles. FROEHLICH et al. 2010; OLMEDO et al. 2010). While our data strongly supports Our data are consistent with a role for a central role for CRY in the CDO, we CRY in the function of the CDO, as CRY were surprised to find that steady-state deletion strains are arrhythmic under levels of CRY protein do not correlate conditions in which the CDO is normally with the rhythmicity of the strains. For expressed (Figure 5, 6 &7). In plants example, the levels of CRY protein are and insects, CRY is necessary for light low in Δwc-1, cog-1 and Δwc-1 cells in entrainment of the circadian clock LL, but only the Δwc-1, cog-1 displays (EMERY et al. 1998; STANEWSKY et al. rhythms in development (Figures 2, 7 & 1998; EMERY et al. 2000), and in S6). The data also pointed to the animals, CRY 1 and CRY 2 function as possibility that cog-1 functions both as a negative components of the core CRY-dependent activator of WC-1 (as circadian oscillator (REPPERT and evidenced by the low levels of WC-1 WEAVER 2002). Some insects, such as protein in Δcry, cog-1 cells as compared the monarch butterfly, have both Δcry cells in LL) and as a negative Drosophila and mammalian versions of regulator of CDO activity. Taken CRY, supporting an ancestral-like clock together, CDO rhythms appear in cells mechanism that involves both light that lack a functional FWO, have low sensing and transcriptional repressor levels (or activity) of CRY protein, and roles for CRY (ZHU et al. 2008). CRY in carry the cog-1 mutation. Furthermore, N. crassa is a member of the CRY- in cog-1 strains in LL, the period is 17 h DASH family of proteins, and while it and the levels of CRY are high, whereas can bind chromophores, it does not in Δwc-1, cog-1, cells the period is 25 h appear to have photolyase activity and the levels of CRY are low. These typical of CRY-DASH proteins data suggest that low levels (or activity) (FROEHLICH et al. 2010). Both cry of CRY correlate with a longer CDO mRNA and protein are induced by light, period. These data, together with our and light induction requires WC-1, results showing no detectable consistent with direct binding of the consequences in rhythms in DD in WCC to the cry promoter (SMITH et al. strains completely lacking CRY (and 2010). Furthermore, cry mRNA thus a functional CDO), support the idea accumulates with a low amplitude that the CDO functions downstream of circadian rhythm, peaking in the the FWO and is not necessary for nighttime (FROEHLICH et al. 2010). The developmental rhythms. cry deletion strains show a small decrease in amplitude of a few light Similar to circadian oscillators, we induced genes, and a slight phase delay predict that the CDO functions as an in LD entrainment in otherwise WT autonomous molecular feedback loop, strains (CHEN et al. 2009). Given the although this still needs to be central role of CRY in the insect and determined. Because the CDO does not animal clockworks, the lack of have full circadian properties, requiring pronounced circadian or light WC-1 for LD entrainment, we surmise phenotypes in CRY deletion mutants in that it lies downstream of the FWO, and Neurospora has, until now, been may function as a slave oscillator to

10 enhance rhythmic outputs, such as the known. Light signals may directly affect development rhythm. This observation is CRY activity to synchronize the CDO reminiscent of previous studies rhythm in LD cycles. In LL, some demonstrating that WCC plays a central mechanism would be expected to exist role in generating FLO-like oscillatory to desensitize CRY activity during behavior in development in FWO- chronic light treatment. It has been deficient strains in temperature cycles shown that VVD functions in N. crassa (HUNT et al. 2012). In LL and naturally to desensitize photo-transduction occurring LD cycles, the CDO may take pathways during chronic light treatment, on a more prominent role, to maintain and plays a role in establishing the rhythms during long periods of light phase of the clock in LD cycles (ELVIN et when the FWO would normally break al. 2005). Thus, VVD is a good down. This idea is congruent with candidate for desensitizing CRY to previous suggestions characterizing the chronic light to promote CDO function. role of VVD in LD entrainment (ELVIN et Consistent with a role for VVD in LL, the al. 2005), in which the FWO oscillator is period of the CDO rhythm is significantly predicted to fully function in the night, reduced in Δvvd, cog-1 strains (Figure and whereas a slave oscillator, such as 5). This model leads to several testable the CDO, may function during periods of predictions; 1) that the activity of CRY light to complete the downstream would be increased in cog-1 mutant rhythmic events. Our observation that strains, and reduced in WC-1 deletions the CDO is less robust in DD (Figure 3) in LL, 2) the activity of CRY would cycle also fits with this hypothesis. Additional in LL and LD in the absence of WC-1, 3) evidence points to an interaction that Δvvd will double the cycling of the between the FWO and the CDO, CDO due to increased CRY activation in including differences in the period of the LL, which would be dependent on WC- rhythm in cog-1 strains FWO-deficient 1, 4) mutations in cry, or artificially versus FWO-sufficient strains grown in increasing the levels of CRY in Δwc-1, LL (Figure 2), and the demonstration cog-1 strains would alter the period of that the cry promoter is a direct target of the CDO rhythm, and 5) identification of the WCC (SMITH et al. 2010). However, CRY interacting proteins would uncover because the phase of the rhythm in cry additional components of the CDO. In (nighttime peak) does not match the any case, the clock system is likely to be morning activity of the WCC (FROEHLICH even more complex than depicted here. et al. 2010), it is possible that other For example, under specialized growth components of the CDO control cry conditions, developmental rhythms with rhythmicity. periods ranging between 6-21 h were observed in vvd mutant alleles in LL that We propose a simple model to provide a are dependent on WC-1, but not FRQ framework for future tests of the (SCHNEIDER et al. 2009). In addition, complexity of the clock (Figure 8). In rhythms in the expression of the ccg-16 cog-1 deficient strains, and independent gene in N. crassa are controlled by a of the FWO, CRY is active and the CDO FLO that requires WC-1, but not FRQ, feedback loop oscillates. How cog-1 for activity. This FLO, called the WC- represses the CDO under conditions in FLO, that appears to be both which the FWO is not operative (such as temperature compensated and in mutants of the FWO, or in LL) is not

11 entrained to environmental cycles by a single open reading frame independent of WCC and FRQ (DE defines period length and PAULA et al. 2006; DE PAULA et al. 2007). temperature compensation. Proc Natl Acad Sci U S A 91: 7683- In summary, this work provides new 7687. insights into the complexity of the oscillator system in N. crassa and light BAKER, C. L., J. J. LOROS and J. C. responses. The identification of CRY as DUNLAP, 2012 The circadian clock an CDO component, and the discovery of Neurospora crassa. FEMS of the cog-1 mutation that uncovered the Microbiol Rev 36: 95-110. CDO, will undoubtedly aid in testing models of the CDO, its connections to BAYRAM, O., G. H. BRAUS, R. FISCHER the environment, and to the FWO. As and J. RODRIGUEZ-ROMERO, 2010 CRY is considered to be an ancient Spotlight on Aspergillus nidulans photoreceptor that has different photosensory systems. Fungal activities in diverse organisms, including Genet Biol 47: 900-908. DNA repair, light perception, and running of the circadian clock (SOMERS BELDEN, W. J., L. F. LARRONDO, A. C. et al. 1998; STANEWSKY et al. 1998; FROEHLICH, M. SHI, C. H. CHEN et REPPERT and WEAVER 2002; DAIYASU et al., 2007 The band mutation in al. 2004; FROEHLICH et al. 2010), our Neurospora crassa is a dominant finding of the CRY-dependent CDO may allele of ras-1 implicating RAS provide key insights into the evolution of signaling in circadian output. the clock. Genes Dev 21: 1494-1505.

ACKNOWLEDGEMENTS BELL-PEDERSEN, D., 2000 Understanding circadian rhythmicity in We thank the Neurospora Program Neurospora crassa: from Project (P01GM68087), the Fungal behavior to genes and back Genetics Stock Center, Dr. Jay Dunlap, again. Fungal Genet Biol 29: 1- and Dr. Christian Heintzen for strains, 18. Dr. Jay Dunlap for CRY antibodies, Drs. Kyung Suk Seo and Louis Morgan for BELL-PEDERSEN, D., S. K. CROSTHWAITE, the initial identification of cog-1, and P. L. LAKIN-THOMAS, M. MERROW Salem Hamiani and Dr. Xiuyun Tian for and M. OKLAND, 2001 The assistance with data collection. We also Neurospora circadian clock: thank Dr. Teresa Lamb for insightful simple or complex? Philos Trans comments on the manuscript and advice R Soc Lond B Biol Sci 356: 1697- throughout the course of this study. This 1709. work was supported by a grant from the National Institutes of Health to DBP BENNETT, L. D., P. BEREMAND, T. L. (P01 NS39546). THOMAS and D. BELL-PEDERSEN, 2013 Circadian activation of the REFERENCES mitogen-activated protein kinase MAK-1 facilitates rhythms in ARONSON, B. D., K. A. JOHNSON and J. C. clock-controlled genes in DUNLAP, 1994 Circadian clock locus frequency: protein encoded

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Figure 1. Identification of the cog-1 mutant strain that uncovers the CDO. A FWO mutant strain (wc-2234W, ∆frq) was mutagenized by ultraviolet (UV) light, and the resulting strains were assayed in constant light (LL) (1200 lux) at 25°C for rescue of developmental rhythms on race tubes. The cog-1 mutation displayed rhythmic development under these conditions, whereas the parental strain was arrhythmic. The direction of growth is from left to right, and the solid black lines correspond to 24 h of growth.

Figure 2. The cog-1 mutation restores rhythms in strains that lack a functional FWO in LL. Representative photographs of race tubes of the indicated strains in LL (1200 lux) at 25°C. Black lines on the tubes represent 24 h growth fronts. The period of the rhythmic strains are shown on the right (± SD, n≥60). AR indicates that the strain was arrhythmic.

Figure 3. The CDO cycles in DD and is temperature compensated. (A) The cog-1 mutation restores circadian rhythms in development to frq-null and wc-1-null strains in DD. Representative race tube pictures of the indicated strains are shown from cultures grown in DD at 25° C and labeled as in Figure 2. (B) cog-1 rhythms are temperature compensated. Plots of the period (h) versus temperature (°C) are shown for the indicated strains (± SD, n≥60).

Figure 4. WC-1 is required for light entrainment of the circadian clock. (A) Race tube cultures were exposed to different light:dark (LD) photoperiods (h). For example, LD 6:6 indicates a 12 hour photoperiod with cycles of 6 h light and 6 h dark. Representative race tube pictures are shown for the indicated strains. Black lines on the race tubes denote when the light was turned on. (B) Plot of the phase of the conidiation band in relation to lights on in strains that showed light responses in each of the photoperiods from (A) (± SEM, n≥5). A positive number indicates that the conidiation band occurred after lights on, and a negative number indicates that the band occurred prior to lights on. In some cases, the error bar is smaller than the symbol.

Figure 5. The blue-light photoreceptor CRY, but not VVD, is necessary for CDO function, and other photoreceptors can compensate for rhythms in LD cycles. Representative race tube photographs of the indicated photoreceptor mutant strains are shown in LL at 25°C (A) and in LD 12:12 cycles at 25°C in (B), and labeled as in Figure 2. (C) Plot of the phase of the conidiation bands in relation to lights on for the indicated strains (± SEM, n≥5).

Figure 6. CRY and VVD are not required for light entrainment of the circadian clock. (A) Race tube cultures of the indicated photoreceptor mutant strains were grown in LL, and (B) were exposed to different LD photoperiods (h), and labeled as in Figure 2. (C) The phase of the conidiation band in relation to lights on were plotted (± SEM, n≥5) as in Figure 4B.

Figure 7. CRY, but not VVD or WC-1, is required for CDO-generated rhythms in DD. (A) Representative race tubes of the indicated strains, and labeled as in Figure 2. (B & C) Representative western blots of CRY protein (B) and WC-1 protein (C) in the indicated strains harvested following 24 h of growth in DD or LL, and plotted above (± SEM, n=3). The amido stained membrane is shown below, and was used to normalize protein loading.

Figure 8. Model of the circadian clock composed of the FWO and CDO. See the text for a description of the model.

27 Table 1. List of strains used in this study. STRAIN STRAIN NAME GENOTYPE NUMBER SOURCE/REFERENCE WT matA, ras-1bd DBP369 FGSC 1858 WT mata, ras-1bd DBP368 FGSC 1859 cog-1 matA, ras-1bd, cog-1 DBP694 this study cog-1 mata, ras-1bd, cog-1 DBP695 this study ∆frq mata, ras-1bd, frq10 DBP287 Aronson et al., 1994 ∆frq matA, ras-1bd, frq10 DBP776 FGSC 7490 ∆frq, cog-1 matA, ras-1bd, frq10, cog-1 DBP831 this study ∆wc-1 matA, ras-1bd, ∆wc-1hyg DBP580 Lee at al., 2003 ∆wc-1, cog-1 matA, ras-1bd, ∆wc-1hyg, cog-1 DBP696 this study ∆wc-1 bar matA, ras-1bd; ∆wc-1bar DBP1223 Bennett et al., 2013 ∆wc-1 bar, cog-1 mata, ras-1bd, ∆wc-1bar, cog-1 DBP1369 this study (DBP 1223 x DBP 695)a ∆vvd mata, ras-1bd, ∆vvd hyg DBP693 Heintzen et al., 2001 ∆vvd matA, ras-1bd, ∆vvd hyg DBP1634 this study (DBP 369 x DBP 693) ∆vvd, cog-1 matA, ras-1bd, ∆vvd hyg , cog-1 DBP1335 this study (DBP 693 x DBP 694) ∆vvd, cog-1 mata, ras-1bd, ∆vvd hyg , cog-1 DBP1638 this study (DBP 695 x DBP 1335) ∆cry ∆cry hyg, mata, ras-1bd DBP963 Laboratory stock ∆cry, cog-1 ∆cry hyg, matA, ras-1bd, cog-1 DBP1022 this study (DBP 963 x DBP 694) ∆cry, ∆wc-1 ∆cry hyg, matA, ras-1bd, ∆wc-1bar DBP1598 this study (DBP 963 x DBP 1223) ∆cry, ∆wc-1 ∆cry hyg, mata, ras-1bd, ∆wc-1bar DBP1599 this study (DBP 963 x DBP 1223) this study (DBP 1022 x DBP ∆cry, ∆wc-1, cog-1 ∆cry hyg, matA, ras-1bd, ∆wc-1bar, cog-1 DBP1645 1369) ∆cry, ∆vvd ∆cry hyg, mata, ras-1bd, ∆vvd hyg DBP1640 this study (DBP 693 x DBP 963) ∆cry, ∆vvd ∆cry hyg, matA, ras-1bd, ∆vvd hyg DBP1600 this study (DBP 693 x DBP 963) this study (DBP 1022 x DBP ∆cry, ∆vvd, cog-1 ∆cry hyg, matA, ras-1bd, ∆vvd hyg, cog-1 DBP1601 1638) ∆vvd, ∆wc-1 matA, ras-1bd, ∆vvd hyg, ∆wc-1bar DBP1639 this study (DBP 693 x DBP 1223) ∆vvd. ∆wc-1 mata, ras-1bd, ∆vvd hyg, ∆wc-1bar DBP1637 this study (DBP 693 x DBP 1223) this study (DBP 1335 x DBP ∆vvd, ∆wc-1, cog-1 matA, ras-1bd, ∆vvd hyg, ∆wc-1bar, cog-1 DBP1635 1369) this study (DBP 1335 x DBP ∆vvd, ∆wc-1, cog-1 mata, ras-1bd, ∆vvd hyg, ∆wc-1bar, cog-1 DBP1636 1369) mata, ∆cry hyg, ras-1bd, ∆vvd hyg, ∆wc- this study (DBP 1599 x DBP ∆cry, ∆vvd, ∆wc-1 1bar DBP1593 1634) matA, ∆cry hyg, ras-1bd, ∆vvd hyg, ∆wc- this study (DBP 1599 x DBP ∆cry, ∆vvd, ∆wc-1 1bar DBP1592 1634) matA, ∆cry hyg, ras-1bd, ∆vvd hyg, ∆wc- ∆cry, ∆vvd, ∆wc-1, cog-1 1bar , cog-1 DBP1597 this study (DBP1022 x DBP 1636) mata, ∆cry hyg, ras-1bd, ∆vvd hyg, ∆wc- ∆cry, ∆vvd, ∆wc-1, cog-1 1bar , cog-1 DBP1596 this study (DBP1022 x DBP 1636) a Derived from the indicated cross (X) between lab strains.

28 Table 2. Period of strains

DD LL GENOTYPE cog-1 + cog-1 - cog-1 + cog-1 -

Period % Period % Period % Period % (h) Rhythmic (h) Rhythmic (h) Rhythmic (h) Rhythmic

WT 22.3±0.8a 100 22.7±0.5, 100 AR 0 17.1±1.3, 100 n=60b n=77 n=194

Δwc-1 ARc 0 23.9±1.3, 40 AR 0 25.1±0.8, 100 n=77 n=191

Δfrq AR 0 21.7±2.7, 50 AR 0 23.0±2.0, 75 n=34 n=80

Δcry 22.2±0.5, 100 22.4±0.5, 100 AR 0 AR 0 n=60 n=60

Δvvd 21.7±0.4, 100 23.2±0.9, 100 AR 0 12.9±1.7, 100 n=60 n=77 n=47

Δvvd, Δwc-1 AR 0 21.3±1.8, 60 AR 0 23.0±1.3, 100 n=44 n=62

Δcry, Δwc-1 AR 0 AR 0 AR 0 AR 0

Δcry, Δvvd 22.2±0.5, 100 22.2±0.5, 100 AR 0 AR 0 n=60 n=60

Δcry, Δvvd, AR 0 AR 0 AR 0 AR 0 Δwc-1

LD6:6 LD 9:9 LD12:12 LD14:14

GENOTYPE cog-1 + cog-1 - cog-1 + cog-1 - cog-1 + cog-1 - cog-1 + cog-1 -

Period Period Period Period Period Period Period Period (h) (h) (h) (h) (h) (h) (h) (h)

WT 12.1±0.1, 11.9±0.1, 18.1±0.1, 17.8±0.3, 23.9±0. 23.9±0.2, 27.3±0.5, 27.6±0.4, n=172 n=179 n=90 n=96 2, n=84 n=84 n=68 n=74

Δwc-1 AR 11.9±0.1, AR 17.9±0.2, AR 23.8±0.2, AR 27.8±0.2, n=126 n=74 n=61 n=61

Δfrq NDd ND ND ND ND ND ND ND

Δcry 12.0±0.1, 12.0±0.2, 17.8±0.3, 17.9±0.1, 24.2±0. 23.8±0.3, 27.9±0.4, 27.8±0.3, n=162 n=150 n=78 n=83 2, n=69 n=70 n=53 n=70

Δvvd 12.0±0.1, 11.9±0.2, 17.8±0.2, 17.8±0.2, 23.6±0. 23.5±0.4, 27.8±0.2, 27.8±0.2, n=168 n=190 n=84 n=108 2, n=72 n=99 n=62 n=72

Δvvd, Δwc-1 AR 12.0±0.5, AR 17.8±0.3, AR 23.9±0.4, AR 27.7±0.5, n=74 n=91 n=60 n=60

Δcry, Δwc-1 AR 12.0±0.5, AR 18.1±0.5, AR 24.0±0.2, AR 27.4±1.7, n=68 n=88 n=63 n=63

Δcry, Δvvd 12.0±0.5, 12.1±0.1, 17.8±0.2, 17.8±0.2, 23.6±0. 23.5±0.2, 27.9±0.2, 28.0±0.2, n=155 n=132 n=101 n=88 2, n=60 n=60 n=60 n=60

Δcry, Δvvd, AR AR AR AR AR AR AR AR Δwc-1

a Values represent mean ± SD b n=number of bands per strain used to calculate period c AR=arrhythmic d ND=not determined

30 Table 3. Growth rates of strains with various oscillator function.

DD LL GENOTYPE GROWTH OSCILLATOR GROWTH OSCILLATOR RATEA FUNCTION RATE FUNCTION

WT 4.0±0.2b FWO 4.0±0.2 none cog-1 3.3±0.3 FWO, CDO 3.5±0.2 CDO

Δwc-1 4.3±0.4 none 4.3±0.3 none

Δwc-1, cog-1 3.7±0.2 CDO 3.2±0.2 CDO

Δcry 3.8±0.4 FWO 4.5±0.4 none

Δcry, cog-1 3.8±0.4 FWO 4.8±0.4 none

a cm/day b values are mean±SD, n≥12