Chen, C., Loros, J. J. (2009) Neurospora Sees the Light. Journal of Communicative and Integrative Biology 2, 448-451
Brunner, M., Káldi, K. (2008) Interlocked Feedback Loops of the Circadian Clock of Neurospora crassa. Molecular Microbiology 68, 255-262
WHITE COLLAR COMPLEX
WHITE COLLAR-1 and WC-2 are GATA-type transcription factors forming heteromeric complexes via their PAS domains (Talora et al., 1999; Cheng et al., 2002). WC-1 contains three PAS domains, PAS-A, -B and C (Fig. 1). PAS-A belongs to a specialized class of flavin-binding PAS photo-sensors, the so-called LOV (light-oxygenvoltage) domains, which are well characterized in phototropins, specific blue-light receptors of plants (Crosson et al., 2003). The LOV domain of WC-1 also serves as a blue-light photoreceptor and regulates the activity of the WHITE COLLAR COMPLEX (WCC). Purified WC-1 is associated with flavin adenine dinucleotide (FAD) and it is likely that photo-activation of WC-1 involves the formation of a photo-adduct between a conserved cysteine in the LOV domain and the bound FAD cofactor (He et al., 2002). The apparent photocycle of the WCC in vivo seems to be in the range of hours (He and Liu, 2005), in contrast to recombinant LOV domains of phototropins, which have a photocycle in the range of a few minutes. The PAS-B domain is essential for WC-1 function but its molecular role is not clear (Cheng et al., 2003). PAS-C is required for the interaction of WC-1 with WC-2 (Cheng et al., 2002; 2003). A putative nuclear localization signal (NLS) and a GATA-type zinc-finger DNA binding domain are located downstream of the PAS domains. The DNA binding domain of WC-1 is required for the function of WCC in the circadian clock in constant darkness, but it is not required for induction of genes by light (Cheng et al., 2003). WC-1 homologues in several fungi lack the DNA binding domain (Idnurm and Heitman, 2005). The N- and
C-terminal regions of WC-1 carry polyglutamine-rich stretches that are not required to maintain FRQ expression in light or dark (Cheng et al., 2003; Káldi, unpubl. obs.).
WHITE COLLAR-2 (Fig. 1) contains one PAS domain, which is crucial for complex formation with WC-1, a coiled-coil domain of unknown function, a putative NLS and a GATA-type zinc-finger domain, which is essential to maintain circadian rhythm (Crosthwaite et al., 1997; Linden and Macino, 1997; Cheng et al., 2002). WHITE COLLAR-2 is more abundant than WC-1 (Denault et al., 2001). WC-1 does not accumulate in the absence of its assembly partner. The WCC is the major transcriptional activator of frq, vvd and a large number of light-induced genes (Lewis et al., 2002; Froehlich et al., 2003). The activity of the WCC is enhanced by light (Froehlich et al., 2002; He and Liu, 2005) and inhibited by phosphorylation (He et al., 2005b; Schafmeier et al., 2005). WCC binds in a light-dependent fashion to two sites at the frq promoter, a proximal and a distal light-responsive element (pLRE and dLRE) (Froehlich et al., 2002). The dLRE also functions as clock-box, promoting rhythmic transcription of frq in the dark (Froehlich et al., 2003). The WCC activates frq transcription by inducing histone remodelling, a process that is assisted by CLOCKSWITCH-1 (Belden et al., 2007).
Purified WCC contains only WC-1 and WC-2 polypeptides, but the precise subunit composition is not known. Its apparent molecular mass, estimated by gel filtration, is about 450 kDa, suggesting that it contains more than one WC-1 (110 kDa) and one WC-2 (55 kDa) subunit (Schafmeier et al., 2005). When purified WCC is exposed to light, it undergoes a conformational change or a subunit shuffling, which results in a reduced electrophoretic mobility in electrophoretic mobility shift assay (EMSA) (Froehlich et al., 2002). WCC binds to tandem GATX repeats. Although the number of characterized binding sites is low, WCC appears to display an extraordinary flexibility with respect to the sequence requirement of its binding sites. A, C and G have been found in the variable position of the GATX motifs and the spacing of the two motifs varies between 5 and 16 bp (Froehlich et al., 2002).
VIVID is a small flavin-binding blue-light photoreceptor consisting of a single LOV domain (Fig. 1) preceded by an a-helix and a short N-terminal extension that is not required for function (Schwerdtfeger and Linden, 2003; Zoltowski et al., 2007). Transcription of vvd is induced by the light-activated WCC (Heintzen et al., 2001). VVD is considered to be a negative regulator of light responses, and is required to the adaptation of light-activated transcription of vvd, frq, wc-1 and many other immediate light-induced genes (Heitzen et al., 2001;Schwerdtfeger and Linden, 2001; Shrode et al., 2001; Schwerdtfeger and Linden, 2003; Liu and Bell-Pedersen, 2006). VVD does not contain a NLS and it is localized in the cytosol (Schwerdtfeger and Linden, 2003). Interaction partners of VVD have not been reported and the mechanism is still obscure how the dominantly cytosolic VVD may inhibit the nuclear WCC. VVD is constitutively expressed in constant light. When Neurospora is transferred to the dark, vvd RNA levels still oscillate during the first day showing a peak in the first subjective morning. After extended periods in darkness, vvd transcript is not detected (Heintzen et al., 2001). By muting acute light-induced transcription at dawn, VVD sustains a circadian clock running during the day (Elvin et al., 2005).
The crystal structure of a functional fragment of VVD lacking 36 N-terminal amino acid residues has been solved in the light and dark form (Zoltowski et al., 2007). In the dark form, the N-terminal helix packs tightly onto the LOV domain. When exposed to light, the conserved cysteine residue of the LOV domain forms a photo-adduct with the bound FAD, leading to a conformational change that promotes repacking of the N-terminal helix. Scission of the photo-adduct and return to the dark state are slow (t1/2~3 h) (Zoltowski et al., 2007).
FREQUENCY is a phosphoprotein that regulates WCC activity and abundance. FRQ is progressively hyperphosphorylated at numerous sites and is then degraded in the course of a circadian period. An essential coiled-coil domain that mediates homo-dimerization is located close to its N-terminus (Fig. 1). An NLS is located next to the coiled-coil and, although only a minor fraction of FRQ is localized in the nucleus (Schafmeier et al., 2005), the NLS is essential for clock function (Luo et al., 1998). The central and C-terminal portions of FRQ do not share any similarity with known protein domains and are predicted to be largely unstructured. Binding sites for CKI (CK-1a) and FRH have been identified in this region (Cheng et al., 2005; He et al., 2006), which may serve as a scaffold for recruitment of accessory factors required for clock function. FRQ also contains two Pro/Glu,(Asp)/Ser/ Thr-rich sequence (PEST)-like sequences, PEST-1 and
PEST-2, in the central and C-terminal portion of the protein, respectively, which are targets for phosphorylation by CK1-a in vitro. PEST-1 and a putative phosphorylation site close by (Ser513) are critical for regulated turnover of FRQ and are major determinants of period length (Liu et al., 2000; Görl et al., 2001). Phosphorylation of PEST-2, presumably by CK-1a, is required for the positive feedback of FRQ-supporting WC-1 accumulation (Schafmeier et al., 2006).
Corrochano, L.M. (2007) Fungal Photoreceptors: Sensory Molecules for Fungal Development and Behaviour. The Royal Society of Chemistry 6, 725-736
Genes similar to Neurospora wc-1 and wc-2 in other fungi: is the white collar complex a model for fungal photoreception?
Genes similar to Neurospora wc-1 and wc-2 have been isolated and characterized in ascomycete, basidiomycete, and zygomycete fungi. In many instances, strains with mutations in these genes have reduced photosensitivity, suggesting that the corresponding gene products should play roles similar to WC-1 and WC-2 in Neurospora. The widespread presence of genes similar to wc-1 and wc-2 in fungi suggests that the white collar complex and its homologous counterparts represent an ancient photoreceptor system for blue-light vision in fungi.
Blue light inhibits mating and haploid fruiting in the basidiomycete Cryptococcus neoformans. A mutant strain with an inactivation of a gene similar to wc-1 showed a blind phenotype, with equal mating reactions in dark and light.12,62 A gene with similarities to the Neurospora wc-2 gene was identified by two independent approaches: by sequence similarities62 and by the isolation of an insertional mutant with a blind-mating phenotype.12 Mutations in any of the Cryptococcus wc genes resulted in a blind mating phenotype,12,62 and their overexpression resulted in a stronger light-dependent inhibition of mating.62 The Cryptococcus WC-1 protein contains a putative chromophore binding domain but does not contain a Zn finger domain, unlike Neurospora WC-1 (Fig. 3). However, a putative Zn finger is present in the Cryptococcus WC-2 protein.12,62 The Cryptococcus wc-1 and wc-2 genes are expressed at very low levels in the dark but the wc-2 gene is induced by light.12,62 In addition, the twoWC proteins were shown to physically interact in a yeast two-hybrid assay.12 These results suggested that the Cryptococcus WC proteins will form a complex that will bind the promoters of light-regulated genes through the WC-2 Zn finger in a mode of action similar to the Neurospora WC complex.12,62
Idnurm, A., Heitman, J. (2005) Light Controls Growth and Development via a Conserved Pathway in the Fungal Kingdom. Public Library of Science Biology 3, 0615-0626
Light inhibits mating and haploid fruiting of the human fungal pathogen Cryptococcus neoformans, but the mechanisms involved were unknown. Two genes controlling light responses were discovered through candidate gene and insertional mutagenesis approaches. Deletion of candidate genes encoding a predicted opsin or phytochrome had no effect on mating, while strains mutated in the white collar 1 homolog gene BWC1 mated equally well in the light or the dark. The predicted Bwc1 protein shares identity with Neurospora crassa WC-1, but lacks the zinc finger DNA binding domain. BWC1 regulates cell fusion and repression of hyphal development after fusion in response to blue light. In addition, bwc1 mutant strains are hypersensitive to ultraviolet light. To identify other components required for responses to light, a novel self-fertile haploid strain was created and subjected to Agrobacterium-mediated insertional mutagenesis. One UV-sensitive mutant that filaments equally well in the light and the dark was identified and found to have an insertion in the BWC2 gene, whose product is structurally similar to N. crassa WC-2. The C. neoformans Bwc1 and Bwc2 proteins interact in the yeast two-hybrid assay. Deletion of BWC1 or BWC2 reduces the virulence of C. neoformans in a murine model of infection; the Bwc1-Bwc2 system thus represents a novel protein complex that influences both development and virulence in a pathogenic fungus. These results demonstrate that a role for blue/UV light in controlling development is an ancient process that predates the divergence of the fungi into the ascomycete and basidiomycete phyla.
Light is the fundamental energy source for life on earth and as such is a major environmental signal for organisms from all kingdoms of life. In the fungal kingdom, light can regulate growth, the direction of growth, asexual and sexual reproduction, and pigment formation, all of which are important aspects for the survival and dissemination of fungal species. These processes have negative implications to many aspects of human life, as the uncontrolled proliferation of fungi can lead to devastating plant disease, mold, and human disease. On the other hand, fungi are essential for recycling nutrients in the environment, in mycorrhizal interactions with plants, and as a source of food and pharmaceutical metabolites for humans. Understanding the role of environmental signals in fungal development is vital to increase the benefits and decrease the costs that fungi present. Despite the importance of light to fungal development, much has yet to be determined to illuminate the mechanisms fungi use to perceive and respond to light.
The effects of light have been investigated in model fungal species. While spectral analyses and the morphological effects of light have been well characterized in genera such as Coprinus (a basidiomycete) or Phycomyces (a zygomycete), at the molecular level Neurospora crassa (an ascomycete) is best understood based on the functions of the white collar (wc-1 and wc-2) genes in light sensing [1,2,3]. In N. crassa, blue light regulates induction of carotenoid pigment production, protoperithecia (sexual fruiting body) formation and phototropism of perithecial beaks, and circadian rhythm, all of which are abolished by mutations in wc-1 or wc-2 . These two genes encode proteins with several conserved domains, including a zinc finger DNA binding domain found in both proteins [5,6,7]. The two proteins physically interact through PAS (conserved in Per, Arnt, Sim proteins) domains [8,9,10]. The WC-1 protein functions as the blue light receptor through a specialized PAS domain responsible for sensing light, oxygen, and voltage in other proteins (LOV domain), and, together with WC-2, acts as a transcription factor. The WC-1 protein interacts with a flavin chromophore [flavin adenine dinucleotide (FAD)] to function as the blue light receptor [11,12]. A small protein, VIVID, also perceives blue light through a LOV domain and modulates N. crassa sensitivity to light . N. crassa has an additional four candidate photoactive protein homologs whose functions in photoperception remain elusive [14,15].
We set out to identify genes involved in the process by which light inhibits mating of the basidiomycete Cryptococcus neoformans. In nature, cryptococcal varieties are associated with bird excreta, soil, tree hollows, and even caves [16,17]. Thus, the light stimuli studied under laboratory conditions are highly relevant to the varying light signals the fungus experiences in the wild. C. neoformans exists as a haploid yeast with two bipolar mating types (a and a). MATa and MATa cells fuse to form a dikaryotic hypha that terminates in a basidium in which nuclear fusion and meiosis occur, producing four long chains of haploid basidiospores by mitosis and budding. A similar process, known as haploid or monokaryotic fruiting, can occur with only one mating partner that also gives rise to filaments that terminate in basidium-like structures and produce short spore chains. Spores have been implicated as an infectious propagule, further underscoring the importance of understanding the regulatory processes governing basidiospore production [18,19]. Mating and fruiting are controlled in the laboratory by stimuli such as the presence of potential mating partners (via pheromone signaling), nutrient limitation, desiccation, temperature, and light . Many aspects of the transduction pathways for these signals have been elucidated, particularly in response to pheromones and nutrient limitation , but no components of light signaling had been reported to date for this important human pathogen. We identify here two genes required for C. neoformans responses to light, and demonstrate their role in blue light regulation of development and sensitivity to UV light, and their requirement for full virulence of this pathogen in a mammalian host.