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

Distinct white collar-1 genes control specific lightresponses in Mucor circinelloides

Fátima Silva, Santiago Torres-Martínez andVictoriano Garre*Departamento de Genética y Microbiología, Facultad deBiología, Universidad de Murcia, 30071 Murcia, Spain.

Summary

Light regulates many developmental and physiologi-cal processes in a large number of organisms. Thebest-known light response in the fungus Mucor cir-cinelloides is the biosynthesis of b-carotene. Here, weshow that M. circinelloides sporangiophores alsorespond to light, exhibiting a positive phototropism.Analysis of both responses to different light wave-lengths within the visible spectrum demonstrated thatphototropism is induced by green and blue light,whereas carotenogenesis is only induced by bluelight. The blue regulation of both responses suggeststhe existence of blue-light photoreceptors inM. circinelloides. Three white collar-1 genes (mcwc-1a, mcwc-1b and mcwc-1c) coding for proteinsshowing similarity with the WC-1 photoreceptor ofNeurospora crassa have been identified. All threecontain a LOV (light, oxygen or voltage) domain,similar to that present in fungal and plant blue-lightreceptors. When knockout mutants for each mcwc-1gene were generated to characterize gene functions,only mcwc-1c mutants were defective in lightinduction of carotene biosynthesis, indicating thatmcwc-1c is involved in the light transduction pathwaythat control carotenogenesis. We have also shownthat positive phototropism is controlled by themcwc-1a gene. It seems therefore that mcwc-1a andmcwc-1c genes control different light transductionpathways, although cross-talk between both path-ways probably exists because mcwc-1a is involved inthe light regulation of mcwc-1c expression.

Introduction

Light regulates developmental and physiological pro-cesses in a wide range of organisms, including filamen-tous fungi. In recent years, considerable effort has beendedicated to the study of light perception mechanisms as

well as to the components of the signal transduction path-ways in fungal models, the ascomycete Neurosporacrassa being the best-understood system at molecularlevel (reviewed by Liu et al., 2003). The wc-1 and wc-2genes are the key elements involved in light responses inN. crassa. Mutants in these genes are ‘blind’ to mostphotoresponses, such as mycelial carotenogenesis, regu-lation of the circadian clock, conidiation and phototropismof perithecial beaks (Linden et al., 1997; Liu et al., 2003).The wc-1 and wc-2 genes encode Per-Arnt-Sim (PAS)domain-containing transcription factors with a singleGATA type Zinc-finger DNA binding domain (Zn-fingerdomain) (Ballario et al., 1996; Linden and Macino, 1997).WC-2 protein has only one PAS domain, whereas WC-1shows three PAS domains, the most N-terminal onebelonging to a specialized class of PAS domain known asa LOV (light, oxygen or voltage) domain. This type ofdomain has been implicated in cellular signalling in all lifekingdoms (Taylor and Zhulin, 1999), but were first identi-fied as the domain responsible for blue-light absorption inplant phototropins that control phototropic bending, light-induced stomatal opening and light-induced chloroplastmovement (Crosson et al., 2003). WC-1 and WC-2 formcomplexes through the interaction of the most C-terminalPAS domain of WC-1 and the PAS domain of WC-2, in aprocess that is essential for the functioning of these pro-teins (Cheng et al., 2002; 2003a). These complexes bindto the light response elements found in the promoters oflight-regulated genes (Froehlich et al., 2002). In additionto its role as a transcription factor, WC-1 protein functionsas a blue-light receptor through its LOV domain, whichbinds flavin adenine dinucleotide (FAD) as chromophore(Froehlich et al., 2002; He et al., 2002). WC-1 is not theonly photoreceptor present in N. crassa, because a smallflavoprotein, VIVID, also perceives blue light through aLOV domain, allowing N. crassa to detect and to adapt tochanges in light intensity (Schwerdtfeger and Linden,2003).

Analogous light regulatory systems seem to work inother fungi, because similar genes to wc-1 and wc-2 havebeen cloned or identified in the genome sequences ofseveral ascomycetes and basidiomycetes, although fewhave been characterized in detail (Lombardi and Brody,2005). The best-characterized genes are those of theascomycete Trichoderma atroviride (Casas-Flores et al.,2004) and those of the basidiomycetes Cryptococcus

Accepted 15 June, 2006. *For correspondence. E-mail [email protected]; Tel. (+34) 968367148; Fax (+34) 968363963.

Molecular Microbiology (2006) 61(4), 1023–1037 doi:10.1111/j.1365-2958.2006.05291.xFirst published online 21 July 2006

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd

neoformans and Coprinus cinereus (Idnurm and Heitman,2005; Lu et al., 2005; Terashima et al., 2005). T. atroviridewc homologues are essential for the light induction ofconidiation and expression of the photolyase gene; theyalso regulate the growth rate (Casas-Flores et al., 2004).C. neoformans wc homologues are required for the lightrepression of sexual cell fusion and for filamentation aftercell fusion (Idnurm and Heitman, 2005; Lu et al., 2005),while the C. cinereus wc-1 homologue is involved in light-regulated fruiting-body development (Terashima et al.,2005). Moreover, mutants in the C. neoformans wc homo-logues are hypersensitive to UV light, which indicates thatthese genes are also involved in mechanisms of UV resis-tance (Idnurm and Heitman, 2005).

In zygomycetes, light responses have been extensivelystudied in Phycomyces blakesleeanus (Cerdá-Olmedoand Lipson, 1987; Cerdá-Olmedo, 2001). However, lackof an efficient transformation system in this fungushinders a detailed analysis of the genes involved in theseresponses, most of them identified by mutation (Cerdá-Olmedo, 2001). The zygomycete Mucor circinelloides pro-duces large amounts of b-carotene after illumination,which represents the only response to light described sofar in this fungus (reviewed by Ruiz-Vázquez and Torres-Martínez, 2003). Nevertheless, unlike P. blakesleeanus, anumber of molecular tools have been developed for use inM. circinelloides, including genetic transformation usingreplicative plasmids, the generation of knockout mutants,Agrobacterium-mediated transformation and the use ofRNAi-based procedures to analyse the gene function(Roncero et al., 1989; Navarro et al., 2001; Nicolás et al.,2003; Nyilasi et al., 2005). The isolation of the crgA geneof M. circinelloides (Navarro et al., 2000) has providednew insights into the light transduction pathways involvedin the biosynthesis of carotenoids. The crgA gene acts asa negative regulator of light-inducible carotenogenesis inthis fungus, because null crgA mutants accumulate highlevels of carotenoids in the dark compared with the wild-type strain (Navarro et al., 2001). These high levels ofcarotenoids have been correlated with an increase in themRNA levels of the carotenogenic structural genes carBand carG (Navarro et al., 2001; Lorca-Pascual et al.,2004). Although null crgA mutants accumulate largeamounts of carotenoids and mRNA of the structural caro-tenogenic genes in the dark, they are still able to respondto light, indicating that their light perception is unaffectedand therefore that other genes must be involved in theactivation of gene expression by light (Navarro et al.,2001). In an attempt to further analyse the molecularmechanisms of the light response in M. circinelloides, weidentified three genes in this fungus that show similaritywith the wc-1 gene. The function of these genes in lightregulation has been analysed by producing knockoutmutants for every gene. Phenotypic analysis of these

mutants revealed that one of the genes is involved in thelight regulation of photocarotenogenesis and another inthe sporangiophore phototropism, a light response ofM. circinelloides that is described for the first time in thisreport.

Results

Light regulated responses in M. circinelloides

The induction of carotene biosynthesis by white light is theonly response to light described so far in M. circinelloides.A more detailed analysis of b-carotene accumulation inresponse to different light wavelengths shows that thelevels of b-carotene in blue and white light-illuminatedmycelia were similar, whereas the levels of b-caroteneaccumulated in red or green light-illuminated myceliawere similar to those of dark-grown mycelia (Fig. 1). Thisresult indicates that blue light is basically the only wave-length range within the visible spectrum that induces caro-tenogenesis in M. circinelloides. This is in agreement withthe blue light induction of carB expression (Velayos et al.,2000a). We also found that M. circinelloides sporangio-phores exhibit positive phototropism, a response notdescribed to date. In this response, the tiny sporangio-phores of M. circinelloides bent towards white light whenunilaterally illuminated (Fig. 2). Unilateral illumination withdifferent light wavelengths revealed that M. circinelloidessporangiophores bent towards green and blue light,whereas red light produced a random orientation of thesporangiophores, similar to that observed in dark grownmycelia (Fig. 2). Because green wavelengths were notexpected to induce positive phototropism, the response

Fig. 1. Blue light induces carotene accumulation inM. circinelloides. Carotenes were extracted from theM. circinelloides wild-type strain R7B grown on solid medium (YNBpH 4.5 + leucine) for 84 h in the dark (D), 60 h in the dark and 24 hunder white light (WL), 60 h in the dark and 24 h under blue light(BL), 60 h in the dark and 24 h under green light (GL) or 60 h inthe dark and 24 h under red light (RL). Spectral analysis showedthat in all cases the main carotene accumulated was b-carotene,which was quantified by reference to its absorption coefficients(Davies, 1976). The values are means ± standard errors (bars) offour independent experiments.

1024 F. Silva, S. Torres-Martínez and V. Garre

© 2006 The AuthorsJournal compilation © 2006 Blackwell Publishing Ltd, Molecular Microbiology, 61, 1023–1037

was investigated in more detail using a narrow bandgreen filter (525–540 nm). In this case, too, positive pho-totropism was observed, although the response wasweaker than when broadband filter (500–600 nm) wasused. Taken together, our findings demonstrate thatblue light controls at least two light responses inM. circinelloides, meaning that blue-light receptors mustexist in this fungus.

Cloning of M. circinelloides genes coding forLOV-bearing proteins

As all blue-light receptors described to date in the fungalkingdom contain a LOV domain, a PCR-based strategywas used to clone M. circinelloides genes encoding pro-teins with such a domain, in an attempt to identify Mucorphotoreceptor genes. Four degenerated primers (LOVprimers), corresponding to the amino acid sequence GRN-CRFLQ, which is conserved in several LOV domainsinvolved in light perception (Crosson et al., 2003), weredesigned. A few DNA fragments were PCR-amplified (datanot shown) by using an oriented cDNA library as templateand a pair of primers, a LOV primer and the T7 primer,whose complementary sequence is adjacent to the 3′-endof every cDNA clone. The complete sequence of a 1.8 kbfragment obtained by this approach revealed a truncatedopen reading frame (ORF) with high similarity to the 3′-halfof the wc-1 gene of N. crassa. The corresponding genewas named mcwc-1a for Mucor circinelloides wc-1 gene.To clone the genomic version of this gene, the mcwc-1a

cDNA fragment was used as a probe to screen a genomicLambda GEM-11 library of the M. circinelloides wild-typestrain. Four strongly and nine weakly hybridizing cloneswere isolated, and their DNAs analysed by restriction andSouthern analysis. Strongly hybridizing clones containeddifferent overlapping DNA fragments of the same genomicregion. On the other hand, the weakly hybridizing clonescould be divided into two different groups. Hybridizingfragments from a strongly hybridizing clone and from arepresentative clone of each weakly hybridizing groupwere subcloned in the pUC18 vector and sequenced (seeExperimental procedures). Subsequent sequence analy-sis of the fragment isolated from the strongly hybridizingclone identified the genomic version of the mcwc-1a gene(the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession numberAM040841). The fragments isolated from each group ofweakly hybridizing clones contained ORFs sharing simi-larities to the mcwc-1a gene. However, differencesbetween the ORF sequences indicated that they corre-spond to different genes. One of the genes was namedmcwc-1b (the sequence data have been submitted tothe DDBJ/EMBL/GenBank databases under accessionnumber AM040842) and the other mcwc-1c (the sequencedata have been submitted to the DDBJ/EMBL/GenBankdatabases under accession number AM040843). Com-parison between the 5′-truncated mcwc-1a cDNA and themcwc-1a genomic sequence identified three introns in thegene (Fig. 3A). The intronic sequences of mcwc-1b andmcwc-1c were predicted from the presence of stop codons

Fig. 2. Phototropism in M. circinelloides.Mycelia of the wild-type strain R7B weregrown for 3 days on PDA solid medium withunilateral illumination (indicated by openarrows). Pictures were taken at right angle tothe mycelium surface using a stereomicroscope.

Light regulation in Mucor circinelloides 1025


in the frame and the lack of similarity with the amino acidsequence deduced from mcwc-1a and wc-1. Four intronswere predicted in mcwc-1b and mcwc-1c, three of themconserved in mcwc-1a and the fourth located in a non-conserved position upstream of the other introns (Fig. 3A).

The protein sequences deduced from the three mcwc-1genes showed a high degree of similarity among them-selves and with the sequence of the WC-1 protein(Fig. 3B). Moreover, analysis of the deduced protein

sequence of three mcwc-1 genes revealed the presenceof a putative conserved LOV domain that possesses allthe necessary residues to contact with a flavin chro-mophore (Crosson and Moffat, 2002) and the conservedcysteine that participates in the light-induced formation ofa flavin-cysteinyl adduct (Kasahara et al., 2002) (Fig. 3C).In addition, the deduced MCWC-1A sequence presentstwo putative PAS domains, a putative nuclear localizationsequence (NLS) and a Zn-finger domain, the domain

Fig. 3. A. Schematic representation showing the domain organization of the deduced protein sequences of the mcwc-1 genes (MCWC-1A,MCWC-1B and MCWC-1C) compared with the WC-1 sequence (WC-1). The length of each protein sequence in amino acids (a.a.) is indicatedon the right hand side. AD, putative activation domain; LOV, light-oxygen-voltage domain; PAS, Per-Arnt-Sim domain; NLS, putative nuclearlocalization signal; ZF, GATA type zinc-finger DNA binding domain. Positions of introns in mcwc-1 genes are indicated by arrowheads.B. Similarity and identity (in brackets) between the deduced protein sequences of mcwc-1 genes and WC-1.C. Amino acid sequence alignment of LOV domains of MCWC-1A, MCWC-1B, MCWC-1C, WC-1 (wc-1; Accession No. CAA63964), VIVID(Accession No. AF338412), Adiantum capillus-veneris (Phy3_LOV2; Accession No. BAA36192) and Arabidopsis thaliana phototropin 1(Phot1_LOV1 and Phot1_LOV2; Accession No. AAC01753). Identical residues are indicated by asterisks and similar residues by points andcolons. Secondary structure (open box indicates loop and open arrow indicates b-sheet), according to the crystal structure of LOV2 from thephototropin segment of A. capillus-veneris, is noted above the alignment. The vertical solid arrows mark residues of the phototropin segmentof A. capillus-veneris that interact with FMN chromophore (Crosson and Moffat, 2002). Cysteine residues in bold indicate the residues of LOVdomains of several phototropins that bind the chromophore in response to light.



organization being identical to that of the WC-1. However,the deduced MCWC-1B sequence lacks a NLS and aconsensus Zn-finger domain, whereas the deducedMCWC-1C sequence seems to lack a second PASdomain (Fig. 3A). All tested computer programs (SMART,MotifScan, InterPro Scan and ScanProsite) failed todetect a PAS domain in the MCWC-1C sequence corre-sponding to the second PAS domain of WC-1, which isessential for WC-1 function and the interaction betweenWC-1 and WC-2 (Cheng et al., 2003a). However, as PASdomain similarity can be low and difficult to identify bothby computer programs and manually, the presence of asecond PAS domain in MCWC-1C cannot be completelyruled out.

Generation of knockout mutants for the mcwc-1 genes

To determine the mcwc-1 gene functions, null mutants foreach gene were generated by gene replacement, design-ing three knockout vectors to disrupt each gene. Thesevectors contained the pyrG gene, used as a selectivemarker, flanked by sequences of the correspondingmcwc-1 gene and adjacent regions (see Experimentalprocedures for details). Restriction fragments from eachplasmid containing the pyrG gene and sufficientsequences of the corresponding mcwc-1 gene to allowhomologous recombination (Navarro et al., 2001; Quiles-Rosillo et al., 2003a) were used to transform the MU402strain, which is wild-type for carotenogenesis but aux-otrophic for uracil and leucine. A total of 21 ura+ transfor-mants were obtained using the mcwc-1a replacementDNA fragment, 67 ura+ transformants using the mcwc-1breplacement DNA fragment and 35 ura+ transformantsusing the mcwc-1c replacement DNA fragment. As initialM. circinelloides transformants are heterokaryons due tothe presence of several nuclei in the protoplasts, theywere grown in selective medium for several vegetativecycles to obtain homokaryotic transformants. Thus, ahomokaryotic transformant (MU242) and a heterokaryotictransformant (MU243) showing 80% ura+ spores wereobtained in the experiments to disrupt the mcwc-1a gene.In addition, three homokaryotic transformants (MU244,MU245 and MU246) were obtained using the mcwc-1breplacement DNA fragment and two (MU247 and MU248)using the mcwc-1c replacement DNA fragment.

The disruption of each gene was confirmed by Southernanalysis (Fig. 4). DNA from transformant MU242 wasdigested with SacI and hybridized with a mcwc-1a probethat hybridizes with the wild-type and the disruptedmcwc-1a alleles. The MU242 transformant showed theexpected 4.7 kb fragment and the absence of the 5.9 kbwild-type fragment, indicating that the mcwc-1a wild-typeallele had been replaced (Fig. 4A and B; probe a). Thegene replacement was confirmed by hybridization with an

internal mcwc-1a probe, which only hybridized with twofragments (5.9 kb and 12 kb) of the wild-type allele(Fig. 4A and B; probe b).

DNA from transformants MU244, MU245 and MU246was digested separately with EcoRI and hybridized with amcwc-1b probe that hybridizes with the wild-type and withthe disrupted mcwc-1b alleles. All of the transformantsshowed the expected 5.3 kb fragment and the absence ofthe 2.0 kb and 1.3 kb wild-type fragments, indicating thatthe mcwc-1b wild-type allele had been replaced (Fig. 4Cand D; probe c). The lack of hybridization in the transfor-mant DNA when the same filter was hybridized with aninternal mcwc-1b probe confirmed that the gene had beensuccessfully replaced (Fig. 4C and D; probe d).

DNA from transformants MU247 and MU248 wasdigested separately with SpeI and hybridized with amcwc-1c probe. Both transformants showed the expected9.5 kb fragment and the absence of the 7.0 kb wild-typefragment, indicating that the mcwc-1c wild-type allele hadbeen replaced (Fig. 4E and F; probe e). This was con-firmed by hybridization of the same filter with an internalmcwc-1c probe (Fig. 4E and F; probe f).

Deletion of mcwc-1c and mcwc-1a genes affects thelight induction of carotenogenesis and results in the lossof positive phototropism respectively

The responses of the mcwc-1 mutant strains to light wereexamined and compared with the wild-type strain R7B.The light induction of carotenogenesis in the mcwc-1a nullmutants (MU242 and MU243) and in the mcwc-1b nullmutants (MU244, MU245 and MU246) was phenotypicallysimilar to that observed in the wild-type strain. This wasconfirmed by showing that the light-induced accumulationof b-carotene in MU242 (Dmcwc-1a) and MU244 (Dmcwc-1b) mutants was similar to that seen in the wild-type strain(Fig. 5A), although it was slightly reduced in MU242.However, the mycelia of the mcwc-1c null mutants(MU247 and MU248) remained pale-yellow after lightinduction. The b-carotene levels in dark-grown mycelia ofthe mcwc-1c null mutant MU247 were roughly the sameas those found in dark-grown mycelia of the wild-typestrain. Illumination of the MU247 dark-grown mycelia withwhite or blue light produced around a threefold increase inthe amount of b-carotene compared with the 21-foldincrease seen in the wild-type strain, indicating that light-induced carotene biosynthesis in this mutant is defective,although it is still able to respond to blue light (Fig. 5A).The phenotype shown by the MU247 mutant was exclu-sively due to the absence of the mcwc-1c gene, as wasconfirmed by complementation of the null mutation afterintroducing the plasmid pMAT1131 carrying a mcwc-1cwild-type allele. The presence of the wild-type mcwc-1callele in two independent transformants was confirmed by



Southern blot analysis (data not shown). Light induction ofcarotenogenesis in both MU247 transformants harbouringplasmid pMAT1131 was similar to that seen in the R7Bwild-type strain transformed with control vector pLEU4,while a MU247 transformant carrying control vectorpLEU4 showed a defect in this response similar to thatseen in MU247 (Fig. 5B). Both MU247 transformants car-rying plasmid pMAT1131 showed increased levels ofb-carotene in response to light similar to that observed inthe R7B wild-type strain transformed with control vectorpLEU4. These data indicate that mcwc-1c plays a majorrole in the regulation of carotenogenesis by blue light.

Positive phototropism was also investigated in mutantstrains for each mcwc-1 gene by growing them with unilat-eral illumination. Mutants for mcwc-1b (MU244) andmcwc-1c (MU247) genes showed a wild-type phototropicresponse to white, green and blue light, while the mcwc-1amutant MU242 was unable to sense light of any wave-length, its sporangiophores growing in a random orienta-tion (Fig. 6). The lack of positive phototropism in the nullmcwc-1a strain was exclusively due to the mcwc-1a– muta-tion, as was confirmed by the reintroduction of a mcwc-1awild-type allele in the mutant MU242 (Dmcwc-1a, leuA–).Thus, two transformants carrying plasmid pMAT1133,

Fig. 4. Disruption of the mcwc-1 genes.A. Genomic DNA (0.5 mg) from the recipient strain in transformation (MU402) and mcwc-1a knockout mutant (MU242) was digested with SacIand hybridized with probe a (1.6 kb BamHI-EcoRI fragment purified from plasmid pMAT1110) and with probe b (1.8 kb ApaI fragment purifiedfrom plasmid pMAT1110). The positions and sizes of the fragments of the DNA molecular weight marker (l + HindIII) are indicated in themiddle of both Southern blots.B. Genomic structure of mcwc-1a wild-type locus and after homologous recombination of the replacement fragment. Positions of the usedprobes and expected sizes of SacI fragments detected by the probes are indicated. S, SacI. Black boxes in this part and in parts D and Findicate genomic regions flanking the genes and grey boxes indicate the gene coding sequences of mcwc-1 genes.C. Genomic DNA (0.5 mg) from recipient strain in transformation (MU402) and mcwc-1b knockout mutants (MU244, MU245 and MU246) wasdigested with EcoRI and hybridized with probe c (0.7 kb BamHI fragment purified from plasmid pMAT1118), and subsequently with probe d(1 kb EcoRV-ApaI fragment purified from plasmid pMAT1118). The positions and sizes of the fragments of the DNA molecular weight marker(l + HindIII) are indicated in the middle of both Southern blots.D. Genomic structure of mcwc-1b wild-type locus and after homologous recombination of the replacement fragment. Positions of the usedprobes and expected sizes of EcoRI fragments detected by the probes are indicated. E, EcoRI.E. Genomic DNA (0.5 mg) from recipient strain in transformation (MU402) and mcwc-1c knockout mutants (MU247 and MU248) was digestedwith SpeI and hybridized with probe e (3.2 kb SacI-SphI fragment purified from plasmid pMAT1132), and subsequently with probe f (0.7 kbClaI fragment purified from plasmid pMAT1132). The positions and sizes of the fragments of the DNA molecular weight marker (l + HindIII)are indicated in the middle of both Southern blots.F. Genomic structure of mcwc-1c wild-type locus and after homologous recombination of the replacement fragment. Positions of the usedprobes and expected sizes of SpeI fragments detected by the probes are indicated. S, SpeI.



which contains a wild-type allele of mcwc-1a, and twotransformants carrying empty vector pLEU4 were grownwith unilateral illumination. While sporangiophores of bothtransformants containing plasmid pMAT1133 bent towardsthe light source, the sporangiophores of transformantsbearing the control vector pLEU4 showed a random orien-tation (Fig. 6). The presence of the mcwc-1a wild-typeallele in the transformants carrying plasmid pMAT1133 wasconfirmed by Southern blot analysis (data not shown).These results indicate that the M. circinelloides phototro-pism is controlled by the mcwc-1a gene.

Light regulation of the mcwc-1 genes

The expression of wc-1 gene and its homologue of Tuberborchii (Ambra et al., 2004) is upregulated by light(Ballario et al., 1996), whereas expression of theC. neoformans wc-1 homologue is very low and constitu-tive (Idnurm and Heitman, 2005; Lu et al., 2005). Tounderstand light regulation of the mcwc-1 genes, levels ofthe corresponding mRNAs were ascertained by Northern-blot hybridization using RNA from the wild-type R7B strainand mutant strains for each mcwc-1 gene. Total RNA wasisolated from mycelia grown in the dark for 2 days andthen exposed to light for different periods of time. Hybrid-ization experiments using probes for each of the mcwc-1genes showed that all genes are transcribed in the wild-type strain, but their patterns of expression are clearlydifferent. The mcwc-1a and mcwc-1b mRNA levels did notchange significantly after illumination, although levels ofthe latter were lower, while mcwc-1c mRNA levelsincreased strongly after 5 min of illumination (Fig. 7A).The expression of mcwc-1a in mcwc-1b or mcwc-1cmutant mycelia was similar to that observed in the wild-type strain, indicating that mcwc-1a expression was unaf-fected by the lack of mcwc-1b or mcwc-1c geneexpression. In the same way, the lack of mcwc-1a ormcwc-1c gene expression did not affect the mcwc-1bmRNA accumulation (Fig. 7B). Interestingly, mcwc-1cgene expression was severely affected in the mcwc-1anull mutant, the mcwc-1c mRNA levels in the light-inducedmycelia being much lower than in the wild-type andmcwc-1b null mutant strains (Fig. 7B). This result clearlyindicates that the mcwc-1a gene is involved in the regu-lation of mcwc-1c expression by light.

The mcwc-1c gene controls the light induction ofstructural carotenogenic genes

Light-induced carotenogenesis in M. circinelloides isassociated with light-induced transcription of the carote-nogenic structural genes carRP and carB (Velayos et al.,2000a,b). To analyse the relationship between the lowamount of carotene accumulated by mcwc-1c null

Fig. 5. A. Carotene content of the null mutants of mcwc-1 genes.Carotenes were extracted from mycelia of the indicated straingrown on solid medium (YNB pH 4.5 + leucine) for 84 h in the dark(D), 60 h in the dark and 24 h under white light (WL) or 60 h in thedark and 24 h under blue light (BL). Spectral analysis showed thatin all cases the main carotene accumulated was b-carotene, whichwas quantified by reference to its absorption coefficients (Davies,1976). The values are means ± standard errors (bars) of fourindependent experiments.B. Carotene content of MU247 transformants carrying a wild-typeallele of mcwc-1c. Carotenes were extracted from mycelia of theindicated transformants grown on solid medium (YNB pH 4.5) for3 days in the dark (D) or 2 days in the dark and 1 day in the light(WL). A transformant of R7B strains bearing vector pLEU4(wild-type phenotype), two MU247 transformants carrying plasmidpMAT1131 (1 and 2), and one MU247 transformant carrying vectorpLEU4 (negative control) were analysed. Spectral analysis showedthat in all cases the main carotene accumulated was b-carotene,which was quantified by reference to its absorption coefficients(Davies, 1976). The values are means ± standard errors (bars) offour independent experiments. The different range of accumulatedb-carotene in part A and part B is a consequence of leucineassimilation. Mycelia in part A take the leucine from the medium,whereas mycelia in part B synthesize it.



Fig. 6. Phototropism in mcwc-1 mutants. Mycelia of the indicated strains were grown for 3 days on PDA solid medium unilaterally illuminatedwith white light (part A), blue light (part B) and green light of 500–600 nm (part C). Open arrows indicate the light direction. (D) Mycelia ofMU242 transformants carrying either plasmid pMAT1133 (1 and 2) or control vector pLEU4 (3 and 4) were grown for 3 days on YNB solidmedium unilaterally illuminated with white light (indicated by open arrows). Pictures were taken as described in Fig. 2.



mutants and the light expression of carRP and carBgenes, levels of the corresponding transcripts were analy-sed in null mutants from every mcwc-1 gene. First, theexpression of carB and carRP genes was compared withthe expression of mcwc-1c gene in the wild-type strain(Fig. 7A). Interestingly, the time-course profile of carRPand carB mRNA accumulation in response to lightseemed to be delayed with respect to that of mcwc-1c inthe wild-type strain, which could suggest that light induc-tion of mcwc-1c gene is required for the normal expres-sion of structural genes (Fig. 7A). Consequently, themcwc-1c null mutant showed a very weak and transitoryincrease both in carB and carRP mRNA levels comparedwith the wild-type strain, whereas mcwc-1b null mutantpresented a wild-type carB and carRP mRNA levels (Fig.7B). Surprisingly, carB and carRP expression was alsoaffected in the mcwc-1a null mutants, because the corre-sponding mRNA levels in illuminated mycelia were lowerthan those found in the wild-type strain (Fig. 7B).However, the accumulation patterns of carB and carRP

mRNAs in mcwc-1a and in mcwc-1c null mutants weredifferent. In the former, light-induced mRNA accumulationwas clearly delayed but maintained over time, while in thelatter it dropped significantly in the same illuminationperiod.

Discussion

Light is an important environmental factor in the regulationof a wide range of developmental and physiological pro-cesses from bacteria to humans. A large amount of workhas been carried out to characterize the molecularmechanisms involved in light signal transduction, fromsignal reception to the specific biological response. Infungi, most knowledge comes from studies of lightresponses in N. crassa, where all characterized blue-lightresponses are controlled by the WC-1 protein, which func-tions as both a photoreceptor and transcriptional factor(Liu et al., 2003). The fungal regulatory systems charac-terized in ascomycetes and basidiomycetes seem to work

Fig. 7. Regulation of the expression of mcwc-1 and carotenogenic structural genes.A. Mycelia of the strain R7B grown for 2 days on MMC medium pH 4.5 in the dark (time 0) were illuminated with white light for the indicatedtimes (min). The RNA levels of each gene were examined by successive hybridizations with the cDNA fragment of the mcwc-1a gene, a0.9 kb EcoRI-SacI fragment of the mcwc-1b gene isolated from plasmid pMAT1118, a 1.5 kb NsiI fragment of the mcwc-1c gene isolated fromplasmid pMAT1132, a 2.6 kb PCR-amplified fragment of the carRP gene (Accession No. AJ250827) and a 2.3 kb PCR-amplified fragment ofthe carB gene (Accession No. AJ238028). The membrane was re-probed with a 28S rRNA probe as a control for loading errors. Relativeamount of mcwc-1 transcripts is indicated below each lane and it is the ratio of each mcwc-1 transcript signal in illuminated mycelia versus thecorresponding dark control signal (0), after normalization with the 28S rRNA signal.B. Mycelia of R7B (WT), MU242 (Dmcwc-1a), MU244 (Dmcwc-1b) and MU247 (Dmcwc-1c) were grown for 2 days on YNB solid mediumsupplemented with leucine (pH 4.5) in the dark (time 0) and then illuminated with white light for the periods indicated in each lane. The mRNAlevels of each gene was estimated by successive hybridizations with the probe b of mcwc-1a gene (Fig. 4; 1.8 kb ApaI fragment purified fromplasmid pMAT1110), the probe d of mcwc-1b (Fig. 4; 1 kb EcoRV-ApaI fragment purified from plasmid pMAT1118), the probe f of mcwc-1c(Fig. 4; 0.7 kb ClaI fragment purified from plasmid pMAT1132) and the probes for carB, carRP and 28S rRNA used in part A.



in a similar way, with the sole WC-1 photoreceptor and thesole WC-2 partner controlling the blue-light responses(Casas-Flores et al., 2004; Idnurm and Heitman, 2005; Luet al., 2005). Other blue-light receptors may exist withsupplementary functions to the main photoreceptor, ashappens with VIVID in N. crassa (Schwerdtfeger andLinden, 2003). Zygomycetes, however, seem to havedeveloped a more complex regulatory system to controlthe responses to light. Such is the case forP. blakesleeanus, in which the existence of four transduc-tion pathways that control different light responses hasbeen proposed. According to this model, some elementsof these transduction pathways would be specific andothers would be shared by several pathways (Cerdá-Olmedo and Lipson, 1987; Cerdá-Olmedo, 2001). Duringthe reviewing process of this manuscript, the cloning oftwo P. blakesleeanus genes encoding proteins that showsimilarity with WC-1 was reported. One of them (madA),which was previously identified by mutation, is involved inthe light-induction of carotenogenesis and phototropism,whereas the other (wcoA) is induced by light and itsfunction is unknown (Idnurm et al., 2006). Nevertheless,the molecular characterization of light transduction path-ways in P. Blakesleeanus is a difficult task because of thelack of an efficient transformation system (Obraztsovaet al., 2004). This is not the case with the zygomyceteM. circinelloides, which also responds to light by accumu-lating high amounts of b-carotene (Navarro et al., 1995).Previous results with other fungi (Linden et al., 1997;Cerdá-Olmedo, 2001) and also with M. circinelloides(Velayos et al., 2000a) led us to think that this lightresponse of M. circinelloides would only depend on bluelight within the visible spectrum. The present study con-firms that only the blue light and not green or red light isresponsible for the induction of carotenogenesis inM. circinelloides, which, in turn, suggests the involvementof a blue-light photoreceptor in this response. To best ofour knowledge, we also demonstrate for the first time thatsporangiophores of M. circinelloides respond to light bybending towards the light source, in the same way asP. blakesleeanus sporangiophores (Cerdá-olmedo, 2001)or N. crassa perithecial ‘beaks’ (Harding and Melles,1983). However, unlike carotenogenesis, this response iscontrolled by blue and green light, suggesting either theinvolvement of a different photoreceptor or the intercon-nection between the regulatory pathways that controlcarotenogenesis and phototropism. Phototropism togreen light was previously reported in P. blakesleeanus(Galland and Lipson, 1987).

To identify the putative M. circinelloides blue-lightreceptors, a PCR-based strategy was used to clonegenes coding for LOV domain-containing proteins,bearing in mind that the two photoreceptors (WC-1 andVIVID) described for N. crassa present a LOV domain

(Froehlich et al., 2002; He et al., 2002; Schwerdtfeger andLinden, 2003). Thus, three genes (mcwc-1a, mcwc-1band mcwc-1c) were cloned that code for proteins with aLOV domain and that show similarity with the wc-1 geneof N. crassa. The LOV domains of all three mcwc-1 genesconserve the 11-amino-acid residues present in the well-characterized phototropin segment of the fern Adiantumcapillus-veneris, which interact with the FMN chro-mophore (Crosson and Moffat, 2002) (Fig. 3C), suggest-ing that they may function as chromophore-bindingdomains. In addition, the alignment of LOV domainsof MCWC-1 proteins with LOV domains of well-characterized photoreceptors revealed a segment of11-amino-acid residues inserted between a’A and a’Cloop (Crosson et al., 2003), which is found in all fungalLOV domains, including those of WC-1 and VIVID (Fig.3B) (Cheng et al., 2003b; Casas-Flores et al., 2004). It

has been suggested that this segment might accommo-date the larger terminal adenine moiety of FAD rather thanthe terminal moiety of FMN, which is bound by plantphototropins (Crosson et al., 2003). In fact, WC-1 bindsin vivo FAD but not FMN (He et al., 2002), and FAD isrequired for binding the WC-1/WC-2 complexes to thelight response elements of frq gene (Froehlich et al.,2002). These findings suggest that the LOV domains ofMCWC-1 proteins may share the conserved domain func-tion and be involved in chromophore binding. WhetherFAD is the chromophore binding to MCWC-1 remains tobe determined by fluorescence spectroscopic analysis.

The functional characterization of the mcwc-1 geneswas undertaken by analysis of the light-regulatedresponses in knockout mutants. Knockout mutants formcwc-1a and mcwc-1b genes showed a light-inducedincrease in b-carotene levels similar to that observed inthe wild-type strain, whereas mcwc-1c null mutantsshowed only a slight increase (Fig. 5), suggesting thatlight induction of carotenogenesis is mainly mediated bythe mcwc-1c gene. The lower b-carotene content in illu-minated mycelia of the mcwc-1c mutant was associatedwith an important reduction in the mRNA levels of struc-tural carotenogenic genes (Fig. 7B). Interestingly, thelight induction of mcwc-1c expression precedes theinduction of the carotenogenic genes (Fig. 7A), suggest-ing that MCWC-1C protein must first be synthesized toachieve the light-induced mRNA levels of the structuralcarotenogenic genes. Low levels of carB and carRPtranscripts were also detected in illuminated mycelia ofthe mcwc-1a mutant, although they resulted only in aslight decrease in b-carotene content (Fig. 5A). Differ-ences in light-induced levels of b-carotene betweenmcwc-1a and mcwc-1c mutants could be the result ofdifferent patterns of carB and carRP mRNA accumula-tion, because mcwc-1a showed a low but steadyincrease in mRNA levels during illumination whereas



mcwc-1c mutant showed only a transitory increase(Fig. 7B). Discrepancies between mRNA levels andcarotene content have previously been described inN. crassa (Merrow et al., 2001; Schwerdtfeger andLinden, 2001). Molecular analysis of the response tolight in this fungus reveals that the induction of the caro-tenogenic genes is a relatively rapid and discreteresponse compared with carotene production, which isthe result of a complex, multistep process. This obser-vation alone provides a rationale for the disparitybetween the absolute levels of carotenes and RNAinduction. Alternatively, the differences in light-inducedlevels of b-carotene between mcwc-1a and mcwc-1cmutants can be explained if the presence of MCWC-1Cprotein is required for the activity of the carotenogenicproteins. Although new experiments are required toclarify this point and others, we propose a workingmodel to explain the light regulation of carotene biosyn-thesis and mcwc-1c expression in M. circinelloides,based on our data and the data from N. crassa (Liuet al., 2003; He and Liu, 2005). This model assumesthat MCWC-1A is constitutively present in the mycelium,because mcwc-1a expression is not influenced by light.Light may provoke a post-translational change inMCWC-1A and/or induce the formation of transcriptionalactive complexes with uncharacterized WC-2 homo-logues. M. circinelloides could contain five to six putativewc-2 homologues according to the data from thegenome sequence of the relative zygomycete Rhizopusoryzae (data not shown). Once MCWC-1A protein isactivated and/or its complexes built, they would activatethe mcwc-1c expression. The light induction of mcwc-1cthat is still observed in the absence of MCWC-1A couldbe explained if other light-activated proteins mediate thelight induction of mcwc-1c expression. Subsequently,synthesized MCWC-1C protein would mediate the light-induced expression of structural carotenogenic genes bybinding to their common promoter region (Velayos et al.,2000b). Although MCWC-1C sequence seems to lack aputative PAS domain to interact with M. circinelloidesWC-2 homologues, its presence cannot be completelyruled out because of the low conservation of the PASdomains and, so some of the expected M. circinelloidesWC-2 homologues might interact with MCWC-1C.Whether or not light plays some role in the MCWC-1Cfunction is an open question that requires more experi-ments before it can be resolved. In addition to MCWC-1C, other protein(s), such as MCWC-1A and MCWC-1B,may be involved in the blue light induction of caroteno-genic genes because weak induction is observed in themcwc-1c mutant, which probably provokes the blue lightinduction of small amounts of carotene observed in thismutant. In summary, this model suggests that mcwc-1cand other, as yet, uncharacterized gene(s) are activators

of carotenogenesis and the expression of carotenogenicgenes, in contrast to crgA gene, which represses caro-tenogenesis and mRNA accumulation of carotenogenicgenes (Navarro et al., 2001). A possible functionalredundancy in the function of MCWC-1 proteins cannotbe discarded.

The light phototropic response was also analysed inmcwc-1 mutants. Sporangiophores of knockout mutantsfor mcwc-1b and mcwc-1c showed a phototropicresponse similar to the wild-type strain, but sporangio-phores of the mcwc-1a mutant showed a random orien-tation when grown unilaterally illuminated with white, blueand green light. This result suggests that positive photot-ropism is mediated only by mcwc-1a, whereas mcwc-1band mcwc-1c have no role in this light response. Accord-ing to these data, at least two light transduction pathwaysexist in M. circinelloides. One of them is mainly dependingon mcwc-1c gene and regulates carotenogenesis and theother one depends on mcwc-1a gene and controlsphototropism. This hypothesis is lent weight by the evi-dence that only blue light within the visible spectruminduced carotenogenesis, whereas both blue and greenlight within the visible spectrum induced phototropism.Assuming that interaction with the expectedM. circinelloides WC-2 homologues is required for theMCWC-1 functions, as occurs in N. crassa (Linden et al.,1997; Liu et al., 2003), it is tempting to speculate thatstructural differences in the PAS domains of MCWC-1Aand MCWC-1C would determine the specific WC-2 homo-logue(s) that interact with each protein. The interactionwith different WC-2 homologues would produce differentprotein complexes, which would determine the set ofgenes controlled by each MCWC-1 protein and thereforeby each transduction pathway.

Finally, the function of the third mcwc-1 gene, mcwc-1b,remains unassigned because knockout mutants for thisgene showed no clear defect in any of the light responsesanalysed in this work. However, the conservation of aLOV and two PAS domains in its predicted proteinsequence suggests a putative function of mcwc-1b in lightregulation. The lack of phenotype in mcwc-1b mutantscould be explained by the gene mediating an uncharac-terized light response or if its function can be carried outby any other mcwc-1 gene. The absence of a Zinc-fingerdomain in the predicted MCWC-1B protein does not pre-clude a role in light regulation because this domain isdispensable for light regulation in WC-1 (Cheng et al.,2003a) and it is absent in basidiomycete WC-1 proteins(Idnurm and Heitman, 2005; Lu et al., 2005; Terashimaet al., 2005).

The results reported in this study point to the lightresponses in zygomycetes being more complex than inother taxonomical groups of fungi. Further studies couldidentify other regulatory elements required for light



regulation and provide more insight into the cross-talkbetween the different transduction pathways.

Experimental procedures

Strains, growth and transformation conditions

The leucine auxotroph R7B (Roncero, 1984), derived fromM. circinelloides f. lusitanicus CBS 277.49 (syn. Mucor race-mosus ATCC 1216b) (Schipper, 1976), was used as the wild-type strain. Strain MU402 (kindly provided by F. E. Nicolás-Esteban, Universidad de Murcia), an uracil and leucineauxotroph derived from R7B, was used as the recipient strainin transformation experiments to knock out mcwc-1 genes.Cultures were grown at 26°C in YNB, YPG, potato dextroseagar (PDA; Difco) or in MMC medium (1% casamino acids,0.05% yeast nitrogen base without amino acids andammonium sulphate, 2% glucose; F.E. Nicolás-Esteban,pers. comm.) as described previously (Quiles-Rosillo et al.,2003a). Media were supplemented with uridine (200 mg ml-1)or leucine (20 mg ml-1) when required. The pH was adjustedto 4.5 and 3.2 for mycelial and colonial growth respectively.Transformation was carried out as described previously(Quiles-Rosillo et al., 2003b).

Escherichia coli strain DH5a (Hanahan, 1983) was usedfor all cloning experiments and strain LE392 (Promega,Madison, WI) for the propagation of M. circinelloides genomiclambda clones.

Analysis of carotenes

Carotenes were extracted from mycelia grown on solidmedium with a cellophane sheet to facilitate their harvest.Specific conditions for each experiment are described in thecorresponding figure legend. Mycelia were grown in an incu-bator at 26°C using a battery of fluorescent lamps (Sylvania,standard F18W/154 dlight, Germany) as white light source, whichproduced an energy fluence rate of 4.8 W m-2 (1.4 W m-2 ofblue component). These lamps were always on and darknesswas obtained by wrapping the dishes with aluminium foil.Light of different wavelengths was produced by using thesame battery of fluorescent lamps and interference filters.Blue light (1.44 W m-2) was obtained by using a blue filter(Supergel #83, Rosco, Stamford, CT); red light (1.26 W m-2)was obtained by using a red filter (Supergel #26, Rosco,Stamford, CT) in conjunction with a UV cutoff filter (Rosco,Stamford, CT), and green light (1.28 W m-2) was obtainedusing a green filter (Supergel #91, Rosco, Stamford, CT) inconjunction with a UV cutoff filter (Rosco, Stamford, CT).Energy fluence rates were determined using a 818-SL detec-tor connected to a 1815-C optical power meter (NewportCorporation, CA). Mycelia were dried between paper towels,frozen in liquid nitrogen and ground using mortar and pestle.Before carotene extraction, ground mycelium was lyophilizedand weighed to estimate dry mass. Carotenes were extractedfrom a known amount of lyophilized ground mycelium usingmethanol and light petroleum as described (Govin andCerdá-Olmedo, 1986). The type of carotene accumulatedwas determined by analysis of the absorption spectrumaccording to standards. b-Carotene was quantified spectro-

photometrically using their absorption coefficients (Davies,1976).

Phototropism analysis

To analyse the phototropism of M. circinelloides sporangio-phores, mycelia were grown on solid medium pH 4.5 for3 days. The dishes were placed inside a box at 20 cm froman opening in one side that allowed the passage of light.White light (20 mW m-2) was supplied by a battery of fluo-rescent lamps (Sylvania, standard F18W/154 dlight, Germany).Different light wavelengths were produced using the samebattery of fluorescent lamps and interference filters. Bluelight (4.8 mW m-2) was obtained by using two different bluefilters (Supergel #83 and Supergel #370, Rosco, Stamford,CT), red light (3.2 mW m-2) was obtained by using two redfilters (Supergel #26, Rosco, Stamford, CT) together withtwo UV cut-off filters (Rosco, Stamford, CT), green light of500–600 nm (2.5 mW m-2) was obtained using a 550 nmbroadband filter (Edmun Optics, Barrington, NJ) and greenlight of 525–540 nm (2.0 mW m-2) was obtained using a532 nm narrow band filter (Newport Corporation, CA).Energy fluence rates were determined using a 818-SLdetector connected to a 1815-C optical power meter(Newport, Corporation, CA).

Plasmids

Plasmid pMAT1110 harbours the entire mcwc-1a gene andwas generated by molecular subcloning of a 6.8 kb BamHI-EcoRI genomic fragment, isolated from a hybridizing lambdaclone, into the pUC18 vector. Plasmid pMAT1118 containsthe entire mcwc-1b gene and was generated by molecularsubcloning of a 3.4 kb HindIII-SacI genomic fragment, iso-lated from a hybridizing lambda clone, into the pUC18 vector.Plasmid pMAT1130 contains the entire mcwc-1c gene andwas generated by molecular subcloning of a 5.5 kb KpnIgenomic fragment, isolated from a hybridizing lambda clone,into the pUC18 vector. Plasmid pMAT1132 contains a3′-truncated mcwc-1c version and was generated by molecu-lar subcloning of a 3.2 kb XhoI fragment isolated frompMAT1130.

Plasmid pMAT1113, which contains the M. circinelloidespyrG gene flanked by mcwc-1a sequences, was constructedto disrupt the mcwc-1a gene. In brief, a 1.8 kb ApaI fragmentof the mcwc-1a coding region in plasmid pMAT1110 wasreplaced by the 3.5 kb PuvII fragment from plasmid pEMP1,which contains a wild-type allele of the M. circinelloides pyrGgene (Benito et al., 1995). The 7.5 kb replacement fragmentwas released from plasmid pMAT1113 by SphI and PvuIIdouble digestion and introduced into MU402 protoplasts bytransformation. The construction of plasmids pMAT1120 andpMAT1128 to disrupt mcwc-1b and mcwc-1c, respectively,was based on PCR amplification of part of plasmidspMAT1118 and pMAT1130. In brief, plasmid pMAT1118 wasPCR amplified using primer mcwc-1b-p1 (5′-CCTGAAGATCTATGCATGTCGGCTTGATTGGATGC-3′) and primermcwc-1b-p2 (5′-AAGAAAGATCTATGCATGTGGATCGAATGTAGC-3′), both of which include BamHI restriction sites(underlined) for cloning purpose. These primers amplify the



vector sequence flanked by mcwc-1b sequences, producinga deletion of 1.3 kb of the mcwc-1b coding region. The PCRproduct digested with BamHI was ligated with a 3.2 kb pyrGBamHI fragment from pEMP1 (Benito et al., 1995) to producepMAT1120. As in the case of the disruption of mcwc-1a, a5.6 kb replacement fragment was released from plasmidpMAT1120 by PvuI and SacI double digestion and introducedinto MU402 protoplasts by transformation. For mcwc-1cdisruption, plasmid pMAT1132 was PCR-amplified usingprimers mcwc-1c-p6 (5′-AGCACAGATCTGCAGCCATCCCATCGATTGACAGG-3′) and mcwc-1c-p7 (5′-CGTCGAGATCTTAGCGGTCATGGTCGTGTCC-3′), both of which includeBamHI restriction sites (underlined) for cloning purpose.These primers amplify the vector sequence flanked bymcwc-1c sequences, producing a deletion of 720 bp of themcwc-1c coding region. The PCR product digested withBamHI was ligated with a 3.2 kb pyrG BamHI fragment frompEMP1 (Benito et al., 1995) to produce pMAT1128. As inprevious cases, a 5.7 kb replacement fragment was releasedfrom plasmid pMAT1128 by SphI and SacI double digestionand introduced into MU402 protoplasts by transformation.

Plasmid pMAT1131, which was used in the complementa-tion experiments, contains the complete mcwc-1c gene andthe M. circinelloides leuA to complement the leucine auxotro-phy of strain MU402. To construct pMAT1131, the 4.4 kb PstIfragment from plasmid pLEU4 (Roncero et al., 1989), whichincludes a wild-type allele of leuA gene, was cloned in thePstI site of the multiple cloning site of pMAT1130. Similarly,the plasmid pMAT1133 was constructed to complement themcwc-1a mutant. In this case, the 4.4 kb PstI fragment fromplasmid pLEU4 (Roncero et al., 1989) was cloned in the PstIsite of the multiple cloning site of pMAT1110, which containsthe complete mcwc-1a gene.

Gene expression analysis

For light induction experiments in M. circinelloides, 2.5 ¥ 105

spores were inoculated on solid medium (pH 4.5) with acellophane sheet and incubated for 2 days in the dark.Mycelia were then illuminated with white light (4.8 W m-2) fordifferent periods of time, using a battery of fluorescent lamps(Sylvania, standard F18W/154 dlight, Germany) as light source.RNA isolated from mycelia before illumination was used ascontrol (time 0). Illuminated mycelia were frozen in liquidnitrogen immediately at the respective times. Signal intensi-ties were estimated from autoradiograms using Kodak GelLogic 200 Imaging System and 1D Image Analysis Software(Kodak, Rochester, NY).

Nucleic acid manipulation and analysis

Standard recombinant DNA manipulations were performedas described by Sambrook and Russell (2001). GenomicDNA of M. circinelloides was prepared as reported previ-ously for Phycomyces blakesleeanus (Ruiz-Pérez et al.,1995). For Southern blot analysis, restriction-digestedchromosomal DNA (0.5–2 mg) was blotted onto positivelycharged nylon filters (Hybond-N+, Amersham PharmaciaBiotech, Freiburg, Germany) and hybridized at 65°C toradioactively labelled probes in Denhardt’s hybridization

solution with 0.05 g ml-1 dextran sulphate (Sambrook andRussell, 2001).

For the Northern-blot hybridizations, total RNA was iso-lated using Trizol reagent following supplier-recommendedprotocols (Invitrogen). Fifteen to 25 mg of total RNA from eachsample was electrophoresed in 1.2% agarose formaldehydegels using 1¥ MOPS (Sambrook and Russell, 2001), blottedonto positively charged nylon filters (Hybond-N+), and hybrid-ized in 0.9 M NaCl, 1% SDS and 0.1 g ml-1 dextran sulphate.

Probes were labelled with [a-32P] dCTP using Ready-to-GoDNA labelling beads (Amersham Pharmacia Biotech), follow-ing the instructions of the supplier. The probe for carB gene(Accession No. AJ238028) corresponded to the whole geneand was obtained by PCR amplification of genomic DNAusing primers carb-1 (5′-TTCCCTTACTTTCTATCC) andcarb-2 (5′-AGTTAAGGGAGTTAGTGCTAG-3′). The probe forcarRP gene (Accession No. AJ250827) corresponded to thewhole gene and was obtained by PCR amplification ofgenomic DNA using primer carrp-1 (5′-TTGGGATGTCTGCTGCTAGG-3′) and carrp-2 (5′-AAAAGAGAAAGAGATAGGG-3′).

Computer analysis of the sequence was carried out usingEuropean Bioinformatic Institute Server software (EMBL Out-station, Hinxton, UK), ExPASy Molecular Biology Server(Swiss Institute of Bioinformatics) and Baylor College ofMedicine Search Launcher (Houston, TX).

Cloning of mcwc-1 genes

The cDNA fragment from mcwc-1a gene was cloned using aPCR-based strategy to clone cDNAs coding for LOV domain-containing proteins, from an oriented cDNA library of wild-type strain CBS 277.49, constructed in pAD-GAL4-2.1 vector(Stratagene, La Jolla, CA). Four different PCR reactions wereperformed using 100 ng of library cDNA as template, one ofthe LOV primers (LOV1, 5′-GGTCGAAAUUGYCGNUUYCUNC-3′; LOV2, 5′-GGTCGAAAYUGYCGNUUYUURC-3′; LOV3, 5′-GGTCGAAAYUGYAGRUUYUURC-3′; LOV4,5′-GGTCGAAAYUGYAGRUUYCUNC-3′) and a T7 primer(5′-TAATACGACTCACTATAGGG-3′) in each PCR reaction.T7 primer was complementary to the vector sequence adja-cent to the 3′-end of every cDNA in the library. PCR wascarried out in the presence of 1.2 mM of each primer, 0.2 mMdNTPs, 1.5 mM magnesium chloride, 5% DMSO and 2.5 U ofEcoTaq Plus (Ecogen, Spain). The PCR cycle was 94°C for1 min, 56°C for 1 min, and 72°C for 2 min for 40 cyclesfollowed by 72°C for 10 min. The PCR products were purifiedfrom 1% agarose after size separation and cloned into thepGEM-T vector (Promega, Madison, WI). Subsequentsequencing of both ends of each cloned fragment identified afragment showing similarity with the wc-1 gene. This frag-ment resulted from the PCR amplification using primer LOV2or primer LOV3. The rest of PCR-amplified fragments corre-sponded to cDNAs that code for proteins without LOVdomains.

To clone the genomic version of M. circinelloides mcwc-1genes, 10 000 plaques from a genomic LambdaGEM-11library of wild-type strain CBS 277.49 strain (Quiles-Rosilloet al., 2003a) were transferred to a Colony/Plaque ScreenTM

membrane (NEN, Ma) and screened with the mcwc-1a cDNAas probe.



Acknowledgements

We thank Dr R. M. Ruiz-Vázquez for critically reading themanuscript and J. A. Madrid for technical assistance. Thiswork was funded by the Spanish Dirección General deInvestigación (Ministerio de Ciencia y Tecnología), projectBMC2003-01017, and the Fundación Séneca (ComunidadAutónoma de la Región de Murcia, Spain), project Pb/73/Fs/02.

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