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

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Molecular Microbiology (2006) 61(4), 1023–1037

doi:10.1111/j.1365-2958.2006.05291.x First published online 21 July 2006

Distinct white collar-1 genes control specific light responses in Mucor circinelloides Fátima Silva, Santiago Torres-Martínez and Victoriano Garre* Departamento de Genética y Microbiología, Facultad de Biología, Universidad de Murcia, 30071 Murcia, Spain. Summary Light regulates many developmental and physiological processes in a large number of organisms. The best-known light response in the fungus Mucor circinelloides is the biosynthesis of b-carotene. Here, we show that M. circinelloides sporangiophores also respond to light, exhibiting a positive phototropism. Analysis of both responses to different light wavelengths within the visible spectrum demonstrated that phototropism is induced by green and blue light, whereas carotenogenesis is only induced by blue light. The blue regulation of both responses suggests the existence of blue-light photoreceptors in M. circinelloides. Three white collar-1 genes (mcwc1a, mcwc-1b and mcwc-1c) coding for proteins showing similarity with the WC-1 photoreceptor of Neurospora crassa have been identified. All three contain a LOV (light, oxygen or voltage) domain, similar to that present in fungal and plant blue-light receptors. When knockout mutants for each mcwc-1 gene were generated to characterize gene functions, only mcwc-1c mutants were defective in light induction of carotene biosynthesis, indicating that mcwc-1c is involved in the light transduction pathway that control carotenogenesis. We have also shown that positive phototropism is controlled by the mcwc-1a gene. It seems therefore that mcwc-1a and mcwc-1c genes control different light transduction pathways, although cross-talk between both pathways probably exists because mcwc-1a is involved in the light regulation of mcwc-1c expression. Introduction Light regulates developmental and physiological processes in a wide range of organisms, including filamentous fungi. In recent years, considerable effort has been dedicated to the study of light perception mechanisms as Accepted 15 June, 2006. *For correspondence. E-mail vgarre@ um.es; Tel. (+34) 968367148; Fax (+34) 968363963.

© 2006 The Authors Journal compilation © 2006 Blackwell Publishing Ltd

well as to the components of the signal transduction pathways in fungal models, the ascomycete Neurospora crassa being the best-understood system at molecular level (reviewed by Liu et al., 2003). The wc-1 and wc-2 genes are the key elements involved in light responses in N. crassa. Mutants in these genes are ‘blind’ to most photoresponses, such as mycelial carotenogenesis, regulation of the circadian clock, conidiation and phototropism of 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 single GATA type Zinc-finger DNA binding domain (Zn-finger domain) (Ballario et al., 1996; Linden and Macino, 1997). WC-2 protein has only one PAS domain, whereas WC-1 shows three PAS domains, the most N-terminal one belonging to a specialized class of PAS domain known as a LOV (light, oxygen or voltage) domain. This type of domain has been implicated in cellular signalling in all life kingdoms (Taylor and Zhulin, 1999), but were first identified as the domain responsible for blue-light absorption in plant phototropins that control phototropic bending, lightinduced stomatal opening and light-induced chloroplast movement (Crosson et al., 2003). WC-1 and WC-2 form complexes through the interaction of the most C-terminal PAS domain of WC-1 and the PAS domain of WC-2, in a process that is essential for the functioning of these proteins (Cheng et al., 2002; 2003a). These complexes bind to the light response elements found in the promoters of light-regulated genes (Froehlich et al., 2002). In addition to its role as a transcription factor, WC-1 protein functions as a blue-light receptor through its LOV domain, which binds flavin adenine dinucleotide (FAD) as chromophore (Froehlich et al., 2002; He et al., 2002). WC-1 is not the only photoreceptor present in N. crassa, because a small flavoprotein, VIVID, also perceives blue light through a LOV domain, allowing N. crassa to detect and to adapt to changes in light intensity (Schwerdtfeger and Linden, 2003). Analogous light regulatory systems seem to work in other fungi, because similar genes to wc-1 and wc-2 have been cloned or identified in the genome sequences of several ascomycetes and basidiomycetes, although few have been characterized in detail (Lombardi and Brody, 2005). The best-characterized genes are those of the ascomycete Trichoderma atroviride (Casas-Flores et al., 2004) and those of the basidiomycetes Cryptococcus

1024 F. Silva, S. Torres-Martínez and V. Garre neoformans and Coprinus cinereus (Idnurm and Heitman, 2005; Lu et al., 2005; Terashima et al., 2005). T. atroviride wc homologues are essential for the light induction of conidiation and expression of the photolyase gene; they also regulate the growth rate (Casas-Flores et al., 2004). C. neoformans wc homologues are required for the light repression of sexual cell fusion and for filamentation after cell fusion (Idnurm and Heitman, 2005; Lu et al., 2005), while the C. cinereus wc-1 homologue is involved in lightregulated fruiting-body development (Terashima et al., 2005). Moreover, mutants in the C. neoformans wc homologues are hypersensitive to UV light, which indicates that these genes are also involved in mechanisms of UV resistance (Idnurm and Heitman, 2005). In zygomycetes, light responses have been extensively studied in Phycomyces blakesleeanus (Cerdá-Olmedo and Lipson, 1987; Cerdá-Olmedo, 2001). However, lack of an efficient transformation system in this fungus hinders a detailed analysis of the genes involved in these responses, most of them identified by mutation (CerdáOlmedo, 2001). The zygomycete Mucor circinelloides produces large amounts of b-carotene after illumination, which represents the only response to light described so far in this fungus (reviewed by Ruiz-Vázquez and TorresMartínez, 2003). Nevertheless, unlike P. blakesleeanus, a number of molecular tools have been developed for use in M. circinelloides, including genetic transformation using replicative plasmids, the generation of knockout mutants, Agrobacterium-mediated transformation and the use of RNAi-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 gene of M. circinelloides (Navarro et al., 2000) has provided new insights into the light transduction pathways involved in the biosynthesis of carotenoids. The crgA gene acts as a negative regulator of light-inducible carotenogenesis in this fungus, because null crgA mutants accumulate high levels of carotenoids in the dark compared with the wildtype strain (Navarro et al., 2001). These high levels of carotenoids have been correlated with an increase in the mRNA levels of the carotenogenic structural genes carB and carG (Navarro et al., 2001; Lorca-Pascual et al., 2004). Although null crgA mutants accumulate large amounts of carotenoids and mRNA of the structural carotenogenic genes in the dark, they are still able to respond to light, indicating that their light perception is unaffected and therefore that other genes must be involved in the activation of gene expression by light (Navarro et al., 2001). In an attempt to further analyse the molecular mechanisms of the light response in M. circinelloides, we identified three genes in this fungus that show similarity with the wc-1 gene. The function of these genes in light regulation has been analysed by producing knockout mutants for every gene. Phenotypic analysis of these

Fig. 1. Blue light induces carotene accumulation in M. circinelloides. Carotenes were extracted from the M. circinelloides wild-type strain R7B grown 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), 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 in the dark and 24 h under red light (RL). Spectral analysis showed that 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) of four independent experiments.

mutants revealed that one of the genes is involved in the light regulation of photocarotenogenesis and another in the sporangiophore phototropism, a light response of M. circinelloides that is described for the first time in this report. Results Light regulated responses in M. circinelloides The induction of carotene biosynthesis by white light is the only response to light described so far in M. circinelloides. A more detailed analysis of b-carotene accumulation in response to different light wavelengths shows that the levels of b-carotene in blue and white light-illuminated mycelia were similar, whereas the levels of b-carotene accumulated in red or green light-illuminated mycelia were similar to those of dark-grown mycelia (Fig. 1). This result indicates that blue light is basically the only wavelength range within the visible spectrum that induces carotenogenesis in M. circinelloides. This is in agreement with the blue light induction of carB expression (Velayos et al., 2000a). We also found that M. circinelloides sporangiophores exhibit positive phototropism, a response not described to date. In this response, the tiny sporangiophores of M. circinelloides bent towards white light when unilaterally illuminated (Fig. 2). Unilateral illumination with different light wavelengths revealed that M. circinelloides sporangiophores bent towards green and blue light, whereas red light produced a random orientation of the sporangiophores, similar to that observed in dark grown mycelia (Fig. 2). Because green wavelengths were not expected to induce positive phototropism, the response

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Fig. 2. Phototropism in M. circinelloides. Mycelia of the wild-type strain R7B were grown for 3 days on PDA solid medium with unilateral illumination (indicated by open arrows). Pictures were taken at right angle to the mycelium surface using a stereo microscope.

was investigated in more detail using a narrow band green filter (525–540 nm). In this case, too, positive phototropism was observed, although the response was weaker than when broadband filter (500–600 nm) was used. Taken together, our findings demonstrate that blue light controls at least two light responses in M. circinelloides, meaning that blue-light receptors must exist in this fungus. Cloning of M. circinelloides genes coding for LOV-bearing proteins As all blue-light receptors described to date in the fungal kingdom contain a LOV domain, a PCR-based strategy was used to clone M. circinelloides genes encoding proteins with such a domain, in an attempt to identify Mucor photoreceptor genes. Four degenerated primers (LOV primers), corresponding to the amino acid sequence GRNCRFLQ, which is conserved in several LOV domains involved in light perception (Crosson et al., 2003), were designed. A few DNA fragments were PCR-amplified (data not shown) by using an oriented cDNA library as template and a pair of primers, a LOV primer and the T7 primer, whose complementary sequence is adjacent to the 3′-end of every cDNA clone. The complete sequence of a 1.8 kb fragment obtained by this approach revealed a truncated open reading frame (ORF) with high similarity to the 3′-half of the wc-1 gene of N. crassa. The corresponding gene was 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 genomic Lambda GEM-11 library of the M. circinelloides wild-type strain. Four strongly and nine weakly hybridizing clones were isolated, and their DNAs analysed by restriction and Southern analysis. Strongly hybridizing clones contained different overlapping DNA fragments of the same genomic region. On the other hand, the weakly hybridizing clones could be divided into two different groups. Hybridizing fragments from a strongly hybridizing clone and from a representative clone of each weakly hybridizing group were subcloned in the pUC18 vector and sequenced (see Experimental procedures). Subsequent sequence analysis of the fragment isolated from the strongly hybridizing clone identified the genomic version of the mcwc-1a gene (the sequence data have been submitted to the DDBJ/ EMBL/GenBank databases under accession number AM040841). The fragments isolated from each group of weakly hybridizing clones contained ORFs sharing similarities to the mcwc-1a gene. However, differences between the ORF sequences indicated that they correspond to different genes. One of the genes was named mcwc-1b (the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number AM040842) and the other mcwc-1c (the sequence data have been submitted to the DDBJ/EMBL/GenBank databases under accession number AM040843). Comparison between the 5′-truncated mcwc-1a cDNA and the mcwc-1a genomic sequence identified three introns in the gene (Fig. 3A). The intronic sequences of mcwc-1b and mcwc-1c were predicted from the presence of stop codons

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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 indicated on the right hand side. AD, putative activation domain; LOV, light-oxygen-voltage domain; PAS, Per-Arnt-Sim domain; NLS, putative nuclear localization 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 and colons. Secondary structure (open box indicates loop and open arrow indicates b-sheet), according to the crystal structure of LOV2 from the phototropin segment of A. capillus-veneris, is noted above the alignment. The vertical solid arrows mark residues of the phototropin segment of A. capillus-veneris that interact with FMN chromophore (Crosson and Moffat, 2002). Cysteine residues in bold indicate the residues of LOV domains of several phototropins that bind the chromophore in response to light.

in the frame and the lack of similarity with the amino acid sequence deduced from mcwc-1a and wc-1. Four introns were predicted in mcwc-1b and mcwc-1c, three of them conserved in mcwc-1a and the fourth located in a nonconserved position upstream of the other introns (Fig. 3A). The protein sequences deduced from the three mcwc-1 genes showed a high degree of similarity among themselves 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 presence of a putative conserved LOV domain that possesses all the necessary residues to contact with a flavin chromophore (Crosson and Moffat, 2002) and the conserved cysteine that participates in the light-induced formation of a flavin-cysteinyl adduct (Kasahara et al., 2002) (Fig. 3C). In addition, the deduced MCWC-1A sequence presents two putative PAS domains, a putative nuclear localization sequence (NLS) and a Zn-finger domain, the domain

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

Light regulation in Mucor circinelloides organization being identical to that of the WC-1. However, the deduced MCWC-1B sequence lacks a NLS and a consensus Zn-finger domain, whereas the deduced MCWC-1C sequence seems to lack a second PAS domain (Fig. 3A). All tested computer programs (SMART, MotifScan, InterPro Scan and ScanProsite) failed to detect a PAS domain in the MCWC-1C sequence corresponding to the second PAS domain of WC-1, which is essential for WC-1 function and the interaction between WC-1 and WC-2 (Cheng et al., 2003a). However, as PAS domain similarity can be low and difficult to identify both by computer programs and manually, the presence of a second PAS domain in MCWC-1C cannot be completely ruled out. Generation of knockout mutants for the mcwc-1 genes To determine the mcwc-1 gene functions, null mutants for each gene were generated by gene replacement, designing three knockout vectors to disrupt each gene. These vectors contained the pyrG gene, used as a selective marker, flanked by sequences of the corresponding mcwc-1 gene and adjacent regions (see Experimental procedures for details). Restriction fragments from each plasmid containing the pyrG gene and sufficient sequences of the corresponding mcwc-1 gene to allow homologous recombination (Navarro et al., 2001; QuilesRosillo et al., 2003a) were used to transform the MU402 strain, which is wild-type for carotenogenesis but auxotrophic for uracil and leucine. A total of 21 ura+ transformants were obtained using the mcwc-1a replacement DNA fragment, 67 ura+ transformants using the mcwc-1b replacement DNA fragment and 35 ura+ transformants using the mcwc-1c replacement DNA fragment. As initial M. circinelloides transformants are heterokaryons due to the presence of several nuclei in the protoplasts, they were grown in selective medium for several vegetative cycles to obtain homokaryotic transformants. Thus, a homokaryotic transformant (MU242) and a heterokaryotic transformant (MU243) showing 80% ura+ spores were obtained in the experiments to disrupt the mcwc-1a gene. In addition, three homokaryotic transformants (MU244, MU245 and MU246) were obtained using the mcwc-1b replacement DNA fragment and two (MU247 and MU248) using the mcwc-1c replacement DNA fragment. The disruption of each gene was confirmed by Southern analysis (Fig. 4). DNA from transformant MU242 was digested with SacI and hybridized with a mcwc-1a probe that hybridizes with the wild-type and the disrupted mcwc-1a alleles. The MU242 transformant showed the expected 4.7 kb fragment and the absence of the 5.9 kb wild-type fragment, indicating that the mcwc-1a wild-type allele had been replaced (Fig. 4A and B; probe a). The gene replacement was confirmed by hybridization with an

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internal mcwc-1a probe, which only hybridized with two fragments (5.9 kb and 12 kb) of the wild-type allele (Fig. 4A and B; probe b). DNA from transformants MU244, MU245 and MU246 was digested separately with EcoRI and hybridized with a mcwc-1b probe that hybridizes with the wild-type and with the disrupted mcwc-1b alleles. All of the transformants showed the expected 5.3 kb fragment and the absence of the 2.0 kb and 1.3 kb wild-type fragments, indicating that the mcwc-1b wild-type allele had been replaced (Fig. 4C and D; probe c). The lack of hybridization in the transformant DNA when the same filter was hybridized with an internal mcwc-1b probe confirmed that the gene had been successfully replaced (Fig. 4C and D; probe d). DNA from transformants MU247 and MU248 was digested separately with SpeI and hybridized with a mcwc-1c probe. Both transformants showed the expected 9.5 kb fragment and the absence of the 7.0 kb wild-type fragment, indicating that the mcwc-1c wild-type allele had been replaced (Fig. 4E and F; probe e). This was confirmed by hybridization of the same filter with an internal mcwc-1c probe (Fig. 4E and F; probe f). Deletion of mcwc-1c and mcwc-1a genes affects the light induction of carotenogenesis and results in the loss of positive phototropism respectively The responses of the mcwc-1 mutant strains to light were examined and compared with the wild-type strain R7B. The light induction of carotenogenesis in the mcwc-1a null mutants (MU242 and MU243) and in the mcwc-1b null mutants (MU244, MU245 and MU246) was phenotypically similar to that observed in the wild-type strain. This was confirmed by showing that the light-induced accumulation of b-carotene in MU242 (Dmcwc-1a) and MU244 (Dmcwc1b) 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 light induction. The b-carotene levels in dark-grown mycelia of the mcwc-1c null mutant MU247 were roughly the same as those found in dark-grown mycelia of the wild-type strain. Illumination of the MU247 dark-grown mycelia with white or blue light produced around a threefold increase in the amount of b-carotene compared with the 21-fold increase seen in the wild-type strain, indicating that lightinduced 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 exclusively due to the absence of the mcwc-1c gene, as was confirmed by complementation of the null mutation after introducing the plasmid pMAT1131 carrying a mcwc-1c wild-type allele. The presence of the wild-type mcwc-1c allele in two independent transformants was confirmed by

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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 SacI and hybridized with probe a (1.6 kb BamHI-EcoRI fragment purified from plasmid pMAT1110) and with probe b (1.8 kb ApaI fragment purified from plasmid pMAT1110). The positions and sizes of the fragments of the DNA molecular weight marker (l + HindIII) are indicated in the middle of both Southern blots. B. Genomic structure of mcwc-1a wild-type locus and after homologous recombination of the replacement fragment. Positions of the used probes and expected sizes of SacI fragments detected by the probes are indicated. S, SacI. Black boxes in this part and in parts D and F indicate 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) was digested 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 used probes 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 digested with SpeI and hybridized with probe e (3.2 kb SacI-SphI fragment purified from plasmid pMAT1132), and subsequently with probe f (0.7 kb ClaI 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 used probes and expected sizes of SpeI fragments detected by the probes are indicated. S, SpeI.

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

Positive phototropism was also investigated in mutant strains for each mcwc-1 gene by growing them with unilateral illumination. Mutants for mcwc-1b (MU244) and mcwc-1c (MU247) genes showed a wild-type phototropic response to white, green and blue light, while the mcwc-1a mutant MU242 was unable to sense light of any wavelength, its sporangiophores growing in a random orientation (Fig. 6). The lack of positive phototropism in the null mcwc-1a strain was exclusively due to the mcwc-1a– mutation, as was confirmed by the reintroduction of a mcwc-1a wild-type allele in the mutant MU242 (Dmcwc-1a, leuA–). Thus, two transformants carrying plasmid pMAT1133,

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which contains a wild-type allele of mcwc-1a, and two transformants carrying empty vector pLEU4 were grown with unilateral illumination. While sporangiophores of both transformants containing plasmid pMAT1133 bent towards the light source, the sporangiophores of transformants bearing the control vector pLEU4 showed a random orientation (Fig. 6). The presence of the mcwc-1a wild-type allele in the transformants carrying plasmid pMAT1133 was confirmed by Southern blot analysis (data not shown). These results indicate that the M. circinelloides phototropism is controlled by the mcwc-1a gene. Light regulation of the mcwc-1 genes

Fig. 5. A. Carotene content of the null mutants of mcwc-1 genes. Carotenes were extracted from mycelia of the indicated strain grown 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 the dark and 24 h under blue light (BL). Spectral analysis showed that 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) of four independent experiments. B. Carotene content of MU247 transformants carrying a wild-type allele of mcwc-1c. Carotenes were extracted from mycelia of the indicated transformants grown on solid medium (YNB pH 4.5) for 3 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 plasmid pMAT1131 (1 and 2), and one MU247 transformant carrying vector pLEU4 (negative control) were analysed. Spectral analysis showed that 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) of four independent experiments. The different range of accumulated b-carotene in part A and part B is a consequence of leucine assimilation. Mycelia in part A take the leucine from the medium, whereas mycelia in part B synthesize it.

The expression of wc-1 gene and its homologue of Tuber borchii (Ambra et al., 2004) is upregulated by light (Ballario et al., 1996), whereas expression of the C. neoformans wc-1 homologue is very low and constitutive (Idnurm and Heitman, 2005; Lu et al., 2005). To understand light regulation of the mcwc-1 genes, levels of the corresponding mRNAs were ascertained by Northernblot hybridization using RNA from the wild-type R7B strain and mutant strains for each mcwc-1 gene. Total RNA was isolated from mycelia grown in the dark for 2 days and then exposed to light for different periods of time. Hybridization experiments using probes for each of the mcwc-1 genes showed that all genes are transcribed in the wildtype strain, but their patterns of expression are clearly different. The mcwc-1a and mcwc-1b mRNA levels did not change significantly after illumination, although levels of the latter were lower, while mcwc-1c mRNA levels increased strongly after 5 min of illumination (Fig. 7A). The expression of mcwc-1a in mcwc-1b or mcwc-1c mutant mycelia was similar to that observed in the wildtype strain, indicating that mcwc-1a expression was unaffected by the lack of mcwc-1b or mcwc-1c gene expression. In the same way, the lack of mcwc-1a or mcwc-1c gene expression did not affect the mcwc-1b mRNA accumulation (Fig. 7B). Interestingly, mcwc-1c gene expression was severely affected in the mcwc-1a null mutant, the mcwc-1c mRNA levels in the light-induced mycelia being much lower than in the wild-type and mcwc-1b null mutant strains (Fig. 7B). This result clearly indicates that the mcwc-1a gene is involved in the regulation of mcwc-1c expression by light. The mcwc-1c gene controls the light induction of structural carotenogenic genes Light-induced carotenogenesis in M. circinelloides is associated with light-induced transcription of the carotenogenic structural genes carRP and carB (Velayos et al., 2000a,b). To analyse the relationship between the low amount of carotene accumulated by mcwc-1c null

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Fig. 6. Phototropism in mcwc-1 mutants. Mycelia of the indicated strains were grown for 3 days on PDA solid medium unilaterally illuminated with 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 of MU242 transformants carrying either plasmid pMAT1133 (1 and 2) or control vector pLEU4 (3 and 4) were grown for 3 days on YNB solid medium unilaterally illuminated with white light (indicated by open arrows). Pictures were taken as described in Fig. 2.

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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 indicated times (min). The RNA levels of each gene were examined by successive hybridizations with the cDNA fragment of the mcwc-1a gene, a 0.9 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 from plasmid pMAT1132, a 2.6 kb PCR-amplified fragment of the carRP gene (Accession No. AJ250827) and a 2.3 kb PCR-amplified fragment of the carB gene (Accession No. AJ238028). The membrane was re-probed with a 28S rRNA probe as a control for loading errors. Relative amount of mcwc-1 transcripts is indicated below each lane and it is the ratio of each mcwc-1 transcript signal in illuminated mycelia versus the corresponding 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 medium supplemented with leucine (pH 4.5) in the dark (time 0) and then illuminated with white light for the periods indicated in each lane. The mRNA levels of each gene was estimated by successive hybridizations with the probe b of mcwc-1a gene (Fig. 4; 1.8 kb ApaI fragment purified from plasmid 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.

mutants and the light expression of carRP and carB genes, levels of the corresponding transcripts were analysed in null mutants from every mcwc-1 gene. First, the expression of carB and carRP genes was compared with the expression of mcwc-1c gene in the wild-type strain (Fig. 7A). Interestingly, the time-course profile of carRP and carB mRNA accumulation in response to light seemed to be delayed with respect to that of mcwc-1c in the wild-type strain, which could suggest that light induction of mcwc-1c gene is required for the normal expression of structural genes (Fig. 7A). Consequently, the mcwc-1c null mutant showed a very weak and transitory increase both in carB and carRP mRNA levels compared with the wild-type strain, whereas mcwc-1b null mutant presented a wild-type carB and carRP mRNA levels (Fig. 7B). Surprisingly, carB and carRP expression was also affected in the mcwc-1a null mutants, because the corresponding mRNA levels in illuminated mycelia were lower than 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 were different. In the former, light-induced mRNA accumulation was clearly delayed but maintained over time, while in the latter it dropped significantly in the same illumination period. Discussion Light is an important environmental factor in the regulation of a wide range of developmental and physiological processes from bacteria to humans. A large amount of work has been carried out to characterize the molecular mechanisms involved in light signal transduction, from signal reception to the specific biological response. In fungi, most knowledge comes from studies of light responses in N. crassa, where all characterized blue-light responses are controlled by the WC-1 protein, which functions as both a photoreceptor and transcriptional factor (Liu et al., 2003). The fungal regulatory systems characterized in ascomycetes and basidiomycetes seem to work

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

1032 F. Silva, S. Torres-Martínez and V. Garre in a similar way, with the sole WC-1 photoreceptor and the sole WC-2 partner controlling the blue-light responses (Casas-Flores et al., 2004; Idnurm and Heitman, 2005; Lu et al., 2005). Other blue-light receptors may exist with supplementary functions to the main photoreceptor, as happens with VIVID in N. crassa (Schwerdtfeger and Linden, 2003). Zygomycetes, however, seem to have developed a more complex regulatory system to control the responses to light. Such is the case for P. blakesleeanus, in which the existence of four transduction pathways that control different light responses has been proposed. According to this model, some elements of these transduction pathways would be specific and others would be shared by several pathways (CerdáOlmedo and Lipson, 1987; Cerdá-Olmedo, 2001). During the reviewing process of this manuscript, the cloning of two P. blakesleeanus genes encoding proteins that show similarity with WC-1 was reported. One of them (madA), which was previously identified by mutation, is involved in the light-induction of carotenogenesis and phototropism, whereas the other (wcoA) is induced by light and its function is unknown (Idnurm et al., 2006). Nevertheless, the molecular characterization of light transduction pathways in P. Blakesleeanus is a difficult task because of the lack of an efficient transformation system (Obraztsova et al., 2004). This is not the case with the zygomycete M. circinelloides, which also responds to light by accumulating 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 light response of M. circinelloides would only depend on blue light within the visible spectrum. The present study confirms that only the blue light and not green or red light is responsible for the induction of carotenogenesis in M. circinelloides, which, in turn, suggests the involvement of a blue-light photoreceptor in this response. To best of our knowledge, we also demonstrate for the first time that sporangiophores of M. circinelloides respond to light by bending towards the light source, in the same way as P. blakesleeanus sporangiophores (Cerdá-olmedo, 2001) or N. crassa perithecial ‘beaks’ (Harding and Melles, 1983). However, unlike carotenogenesis, this response is controlled by blue and green light, suggesting either the involvement of a different photoreceptor or the interconnection between the regulatory pathways that control carotenogenesis and phototropism. Phototropism to green light was previously reported in P. blakesleeanus (Galland and Lipson, 1987). To identify the putative M. circinelloides blue-light receptors, a PCR-based strategy was used to clone genes coding for LOV domain-containing proteins, bearing in mind that the two photoreceptors (WC-1 and VIVID) described for N. crassa present a LOV domain

(Froehlich et al., 2002; He et al., 2002; Schwerdtfeger and Linden, 2003). Thus, three genes (mcwc-1a, mcwc-1b and mcwc-1c) were cloned that code for proteins with a LOV domain and that show similarity with the wc-1 gene of N. crassa. The LOV domains of all three mcwc-1 genes conserve the 11-amino-acid residues present in the wellcharacterized phototropin segment of the fern Adiantum capillus-veneris, which interact with the FMN chromophore (Crosson and Moffat, 2002) (Fig. 3C), suggesting that they may function as chromophore-binding domains. In addition, the alignment of LOV domains of MCWC-1 proteins with LOV domains of wellcharacterized photoreceptors revealed a segment of 11-amino-acid residues inserted between a’A and a’C loop (Crosson et al., 2003), which is found in all fungal LOV 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 accommodate the larger terminal adenine moiety of FAD rather than the terminal moiety of FMN, which is bound by plant phototropins (Crosson et al., 2003). In fact, WC-1 binds in vivo FAD but not FMN (He et al., 2002), and FAD is required for binding the WC-1/WC-2 complexes to the light response elements of frq gene (Froehlich et al., 2002). These findings suggest that the LOV domains of MCWC-1 proteins may share the conserved domain function and be involved in chromophore binding. Whether FAD is the chromophore binding to MCWC-1 remains to be determined by fluorescence spectroscopic analysis. The functional characterization of the mcwc-1 genes was undertaken by analysis of the light-regulated responses in knockout mutants. Knockout mutants for mcwc-1a and mcwc-1b genes showed a light-induced increase in b-carotene levels similar to that observed in the wild-type strain, whereas mcwc-1c null mutants showed only a slight increase (Fig. 5), suggesting that light induction of carotenogenesis is mainly mediated by the mcwc-1c gene. The lower b-carotene content in illuminated mycelia of the mcwc-1c mutant was associated with an important reduction in the mRNA levels of structural carotenogenic genes (Fig. 7B). Interestingly, the light induction of mcwc-1c expression precedes the induction of the carotenogenic genes (Fig. 7A), suggesting that MCWC-1C protein must first be synthesized to achieve the light-induced mRNA levels of the structural carotenogenic genes. Low levels of carB and carRP transcripts were also detected in illuminated mycelia of the mcwc-1a mutant, although they resulted only in a slight decrease in b-carotene content (Fig. 5A). Differences in light-induced levels of b-carotene between mcwc-1a and mcwc-1c mutants could be the result of different patterns of carB and carRP mRNA accumulation, because mcwc-1a showed a low but steady increase in mRNA levels during illumination whereas

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

Light regulation in Mucor circinelloides mcwc-1c mutant showed only a transitory increase (Fig. 7B). Discrepancies between mRNA levels and carotene content have previously been described in N. crassa (Merrow et al., 2001; Schwerdtfeger and Linden, 2001). Molecular analysis of the response to light in this fungus reveals that the induction of the carotenogenic genes is a relatively rapid and discrete response compared with carotene production, which is the result of a complex, multistep process. This observation alone provides a rationale for the disparity between the absolute levels of carotenes and RNA induction. Alternatively, the differences in light-induced levels of b-carotene between mcwc-1a and mcwc-1c mutants can be explained if the presence of MCWC-1C protein is required for the activity of the carotenogenic proteins. Although new experiments are required to clarify this point and others, we propose a working model to explain the light regulation of carotene biosynthesis and mcwc-1c expression in M. circinelloides, based on our data and the data from N. crassa (Liu et al., 2003; He and Liu, 2005). This model assumes that MCWC-1A is constitutively present in the mycelium, because mcwc-1a expression is not influenced by light. Light may provoke a post-translational change in MCWC-1A and/or induce the formation of transcriptional active complexes with uncharacterized WC-2 homologues. M. circinelloides could contain five to six putative wc-2 homologues according to the data from the genome sequence of the relative zygomycete Rhizopus oryzae (data not shown). Once MCWC-1A protein is activated and/or its complexes built, they would activate the mcwc-1c expression. The light induction of mcwc-1c that is still observed in the absence of MCWC-1A could be explained if other light-activated proteins mediate the light induction of mcwc-1c expression. Subsequently, synthesized MCWC-1C protein would mediate the lightinduced expression of structural carotenogenic genes by binding to their common promoter region (Velayos et al., 2000b). Although MCWC-1C sequence seems to lack a putative PAS domain to interact with M. circinelloides WC-2 homologues, its presence cannot be completely ruled out because of the low conservation of the PAS domains and, so some of the expected M. circinelloides WC-2 homologues might interact with MCWC-1C. Whether or not light plays some role in the MCWC-1C function is an open question that requires more experiments before it can be resolved. In addition to MCWC1C, other protein(s), such as MCWC-1A and MCWC-1B, may be involved in the blue light induction of carotenogenic genes because weak induction is observed in the mcwc-1c mutant, which probably provokes the blue light induction of small amounts of carotene observed in this mutant. In summary, this model suggests that mcwc-1c and other, as yet, uncharacterized gene(s) are activators

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of carotenogenesis and the expression of carotenogenic genes, in contrast to crgA gene, which represses carotenogenesis and mRNA accumulation of carotenogenic genes (Navarro et al., 2001). A possible functional redundancy in the function of MCWC-1 proteins cannot be discarded. The light phototropic response was also analysed in mcwc-1 mutants. Sporangiophores of knockout mutants for mcwc-1b and mcwc-1c showed a phototropic response similar to the wild-type strain, but sporangiophores of the mcwc-1a mutant showed a random orientation when grown unilaterally illuminated with white, blue and green light. This result suggests that positive phototropism is mediated only by mcwc-1a, whereas mcwc-1b and mcwc-1c have no role in this light response. According to these data, at least two light transduction pathways exist in M. circinelloides. One of them is mainly depending on mcwc-1c gene and regulates carotenogenesis and the other one depends on mcwc-1a gene and controls phototropism. This hypothesis is lent weight by the evidence that only blue light within the visible spectrum induced carotenogenesis, whereas both blue and green light within the visible spectrum induced phototropism. Assuming that interaction with the expected M. circinelloides WC-2 homologues is required for the MCWC-1 functions, as occurs in N. crassa (Linden et al., 1997; Liu et al., 2003), it is tempting to speculate that structural differences in the PAS domains of MCWC-1A and MCWC-1C would determine the specific WC-2 homologue(s) that interact with each protein. The interaction with different WC-2 homologues would produce different protein complexes, which would determine the set of genes controlled by each MCWC-1 protein and therefore by each transduction pathway. Finally, the function of the third mcwc-1 gene, mcwc-1b, remains unassigned because knockout mutants for this gene showed no clear defect in any of the light responses analysed in this work. However, the conservation of a LOV and two PAS domains in its predicted protein sequence suggests a putative function of mcwc-1b in light regulation. The lack of phenotype in mcwc-1b mutants could be explained by the gene mediating an uncharacterized light response or if its function can be carried out by any other mcwc-1 gene. The absence of a Zinc-finger domain in the predicted MCWC-1B protein does not preclude a role in light regulation because this domain is dispensable 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; Terashima et al., 2005). The results reported in this study point to the light responses in zygomycetes being more complex than in other taxonomical groups of fungi. Further studies could identify other regulatory elements required for light

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

1034 F. Silva, S. Torres-Martínez and V. Garre regulation and provide more insight into the cross-talk between the different transduction pathways.

photometrically using their absorption coefficients (Davies, 1976).

Experimental procedures

Phototropism analysis

Strains, growth and transformation conditions

To analyse the phototropism of M. circinelloides sporangiophores, mycelia were grown on solid medium pH 4.5 for 3 days. The dishes were placed inside a box at 20 cm from an opening in one side that allowed the passage of light. White light (20 mW m-2) was supplied by a battery of fluorescent lamps (Sylvania, standard F18W/154 dlight, Germany). Different light wavelengths were produced using the same battery of fluorescent lamps and interference filters. Blue light (4.8 mW m-2) was obtained by using two different blue filters (Supergel #83 and Supergel #370, Rosco, Stamford, CT), red light (3.2 mW m-2) was obtained by using two red filters (Supergel #26, Rosco, Stamford, CT) together with two UV cut-off filters (Rosco, Stamford, CT), green light of 500–600 nm (2.5 mW m-2) was obtained using a 550 nm broadband filter (Edmun Optics, Barrington, NJ) and green light of 525–540 nm (2.0 mW m-2) was obtained using a 532 nm narrow band filter (Newport Corporation, CA). Energy fluence rates were determined using a 818-SL detector connected to a 1815-C optical power meter (Newport, Corporation, CA).

The leucine auxotroph R7B (Roncero, 1984), derived from M. circinelloides f. lusitanicus CBS 277.49 (syn. Mucor racemosus ATCC 1216b) (Schipper, 1976), was used as the wildtype strain. Strain MU402 (kindly provided by F. E. NicolásEsteban, Universidad de Murcia), an uracil and leucine auxotroph derived from R7B, was used as the recipient strain in transformation experiments to knock out mcwc-1 genes. Cultures were grown at 26°C in YNB, YPG, potato dextrose agar (PDA; Difco) or in MMC medium (1% casamino acids, 0.05% yeast nitrogen base without amino acids and ammonium 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 adjusted to 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 used for all cloning experiments and strain LE392 (Promega, Madison, WI) for the propagation of M. circinelloides genomic lambda clones.

Analysis of carotenes Carotenes were extracted from mycelia grown on solid medium with a cellophane sheet to facilitate their harvest. Specific conditions for each experiment are described in the corresponding figure legend. Mycelia were grown in an incubator at 26°C using a battery of fluorescent lamps (Sylvania, standard F18W/154 dlight, Germany) as white light source, which produced an energy fluence rate of 4.8 W m-2 (1.4 W m-2 of blue component). These lamps were always on and darkness was obtained by wrapping the dishes with aluminium foil. Light of different wavelengths was produced by using the same 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 obtained using a green filter (Supergel #91, Rosco, Stamford, CT) in conjunction with a UV cutoff filter (Rosco, Stamford, CT). Energy fluence rates were determined using a 818-SL detector connected to a 1815-C optical power meter (Newport Corporation, CA). Mycelia were dried between paper towels, frozen in liquid nitrogen and ground using mortar and pestle. Before carotene extraction, ground mycelium was lyophilized and weighed to estimate dry mass. Carotenes were extracted from a known amount of lyophilized ground mycelium using methanol and light petroleum as described (Govin and Cerdá-Olmedo, 1986). The type of carotene accumulated was determined by analysis of the absorption spectrum according to standards. b-Carotene was quantified spectro-

Plasmids Plasmid pMAT1110 harbours the entire mcwc-1a gene and was generated by molecular subcloning of a 6.8 kb BamHIEcoRI genomic fragment, isolated from a hybridizing lambda clone, into the pUC18 vector. Plasmid pMAT1118 contains the entire mcwc-1b gene and was generated by molecular subcloning of a 3.4 kb HindIII-SacI genomic fragment, isolated from a hybridizing lambda clone, into the pUC18 vector. Plasmid pMAT1130 contains the entire mcwc-1c gene and was generated by molecular subcloning of a 5.5 kb KpnI genomic fragment, isolated from a hybridizing lambda clone, into the pUC18 vector. Plasmid pMAT1132 contains a 3′-truncated mcwc-1c version and was generated by molecular subcloning of a 3.2 kb XhoI fragment isolated from pMAT1130. Plasmid pMAT1113, which contains the M. circinelloides pyrG gene flanked by mcwc-1a sequences, was constructed to disrupt the mcwc-1a gene. In brief, a 1.8 kb ApaI fragment of the mcwc-1a coding region in plasmid pMAT1110 was replaced by the 3.5 kb PuvII fragment from plasmid pEMP1, which contains a wild-type allele of the M. circinelloides pyrG gene (Benito et al., 1995). The 7.5 kb replacement fragment was released from plasmid pMAT1113 by SphI and PvuII double digestion and introduced into MU402 protoplasts by transformation. The construction of plasmids pMAT1120 and pMAT1128 to disrupt mcwc-1b and mcwc-1c, respectively, was based on PCR amplification of part of plasmids pMAT1118 and pMAT1130. In brief, plasmid pMAT1118 was PCR amplified using primer mcwc-1b-p1 (5′-CCTGAA GATCTATGCATGTCGGCTTGATTGGATGC-3′) and primer mcwc-1b-p2 (5′-AAGAAAGATCTATGCATGTGGATCGAAT GTAGC-3′), both of which include BamHI restriction sites (underlined) for cloning purpose. These primers amplify the

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

Light regulation in Mucor circinelloides vector sequence flanked by mcwc-1b sequences, producing a deletion of 1.3 kb of the mcwc-1b coding region. The PCR product digested with BamHI was ligated with a 3.2 kb pyrG BamHI fragment from pEMP1 (Benito et al., 1995) to produce pMAT1120. As in the case of the disruption of mcwc-1a, a 5.6 kb replacement fragment was released from plasmid pMAT1120 by PvuI and SacI double digestion and introduced into MU402 protoplasts by transformation. For mcwc-1c disruption, plasmid pMAT1132 was PCR-amplified using primers mcwc-1c-p6 (5′-AGCACAGATCTGCAGCCATCCC ATCGATTGACAGG-3′) and mcwc-1c-p7 (5′-CGTCGAGATC TTAGCGGTCATGGTCGTGTCC-3′), both of which include BamHI restriction sites (underlined) for cloning purpose. These primers amplify the vector sequence flanked by mcwc-1c sequences, producing a deletion of 720 bp of the mcwc-1c coding region. The PCR product digested with BamHI was ligated with a 3.2 kb pyrG BamHI fragment from pEMP1 (Benito et al., 1995) to produce pMAT1128. As in previous cases, a 5.7 kb replacement fragment was released from plasmid pMAT1128 by SphI and SacI double digestion and introduced into MU402 protoplasts by transformation. Plasmid pMAT1131, which was used in the complementation experiments, contains the complete mcwc-1c gene and the M. circinelloides leuA to complement the leucine auxotrophy of strain MU402. To construct pMAT1131, the 4.4 kb PstI fragment from plasmid pLEU4 (Roncero et al., 1989), which includes a wild-type allele of leuA gene, was cloned in the PstI site of the multiple cloning site of pMAT1130. Similarly, the plasmid pMAT1133 was constructed to complement the mcwc-1a mutant. In this case, the 4.4 kb PstI fragment from plasmid pLEU4 (Roncero et al., 1989) was cloned in the PstI site of the multiple cloning site of pMAT1110, which contains the 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 a cellophane sheet and incubated for 2 days in the dark. Mycelia were then illuminated with white light (4.8 W m-2) for different 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 as control (time 0). Illuminated mycelia were frozen in liquid nitrogen immediately at the respective times. Signal intensities were estimated from autoradiograms using Kodak Gel Logic 200 Imaging System and 1D Image Analysis Software (Kodak, Rochester, NY).

Nucleic acid manipulation and analysis Standard recombinant DNA manipulations were performed as described by Sambrook and Russell (2001). Genomic DNA of M. circinelloides was prepared as reported previously for Phycomyces blakesleeanus (Ruiz-Pérez et al., 1995). For Southern blot analysis, restriction-digested chromosomal DNA (0.5–2 mg) was blotted onto positively charged nylon filters (Hybond-N+, Amersham Pharmacia Biotech, Freiburg, Germany) and hybridized at 65°C to radioactively labelled probes in Denhardt’s hybridization

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solution with 0.05 g ml dextran sulphate (Sambrook and Russell, 2001). For the Northern-blot hybridizations, total RNA was isolated using Trizol reagent following supplier-recommended protocols (Invitrogen). Fifteen to 25 mg of total RNA from each sample was electrophoresed in 1.2% agarose formaldehyde gels using 1¥ MOPS (Sambrook and Russell, 2001), blotted onto positively charged nylon filters (Hybond-N+), and hybridized 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-Go DNA labelling beads (Amersham Pharmacia Biotech), following the instructions of the supplier. The probe for carB gene (Accession No. AJ238028) corresponded to the whole gene and was obtained by PCR amplification of genomic DNA using primers carb-1 (5′-TTCCCTTACTTTCTATCC) and carb-2 (5′-AGTTAAGGGAGTTAGTGCTAG-3′). The probe for carRP gene (Accession No. AJ250827) corresponded to the whole gene and was obtained by PCR amplification of genomic DNA using primer carrp-1 (5′-TTGGGATGTC TGCTGCTAGG-3′) and carrp-2 (5′-AAAAGAGAAAGAGA TAGGG-3′). Computer analysis of the sequence was carried out using European Bioinformatic Institute Server software (EMBL Outstation, Hinxton, UK), ExPASy Molecular Biology Server (Swiss Institute of Bioinformatics) and Baylor College of Medicine Search Launcher (Houston, TX).

Cloning of mcwc-1 genes The cDNA fragment from mcwc-1a gene was cloned using a PCR-based strategy to clone cDNAs coding for LOV domaincontaining proteins, from an oriented cDNA library of wildtype strain CBS 277.49, constructed in pAD-GAL4-2.1 vector (Stratagene, La Jolla, CA). Four different PCR reactions were performed using 100 ng of library cDNA as template, one of the LOV primers (LOV1, 5′-GGTCGAAAUUGYCGNUUY CUNC-3′; LOV2, 5′-GGTCGAAAYUGYCGNUUYUURC3′; 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 adjacent to the 3′-end of every cDNA in the library. PCR was carried out in the presence of 1.2 mM of each primer, 0.2 mM dNTPs, 1.5 mM magnesium chloride, 5% DMSO and 2.5 U of EcoTaq Plus (Ecogen, Spain). The PCR cycle was 94°C for 1 min, 56°C for 1 min, and 72°C for 2 min for 40 cycles followed by 72°C for 10 min. The PCR products were purified from 1% agarose after size separation and cloned into the pGEM-T vector (Promega, Madison, WI). Subsequent sequencing of both ends of each cloned fragment identified a fragment showing similarity with the wc-1 gene. This fragment resulted from the PCR amplification using primer LOV2 or primer LOV3. The rest of PCR-amplified fragments corresponded to cDNAs that code for proteins without LOV domains. To clone the genomic version of M. circinelloides mcwc-1 genes, 10 000 plaques from a genomic LambdaGEM-11 library of wild-type strain CBS 277.49 strain (Quiles-Rosillo et al., 2003a) were transferred to a Colony/Plaque ScreenTM membrane (NEN, Ma) and screened with the mcwc-1a cDNA as probe.

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

1036 F. Silva, S. Torres-Martínez and V. Garre Acknowledgements We thank Dr R. M. Ruiz-Vázquez for critically reading the manuscript and J. A. Madrid for technical assistance. This work was funded by the Spanish Dirección General de Investigación (Ministerio de Ciencia y Tecnología), project BMC2003-01017, and the Fundación Séneca (Comunidad Autónoma de la Región de Murcia, Spain), project Pb/73/Fs/ 02.

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