Phototropism

In response to light, plants can reorient organ growth toward (positive phototropism) or away (negative phototropism) from a directional light source. Not only are etiolate seedling first exposed to light but also light grown seedling, petioles and inflorescence stems exhibit asymmetric growth, called phototropism, in response to unilateral light illumination. Historically, asymmetrical growth caused by differential distribution of auxin across the organ including hypocotyl has been reported. Auxin is a plant hormone, mainly synthesized in the shoot apex and young leaves (Fankhauser and Christie, 2015; Liscum et al., 2014).

Phototropism regulation and signaling pathway in plants

Through the light perception mechanism, both root and hypocotyl phototropisms are mainly mediated by blue light phototropins called as PHOT1 and PHOT2. In Arabidopsis, the phot1 mediates the phototropic response over a broad range of blue light intensities, therefore phot1 is the primary receptor to regulate the phototropism.

Both phot2 and phot1 genes are also responsible for hypocotyl phototropism under high blue light intensity. Beside phototropism, phot1 and phot2 also play important roles in the regulation of chloroplast distribution and movement along edges of the cell. PHOT1 and PHOT2 photoreceptors are associated with plasma membrane (in darkness) and translocated to cytoplasm and Golgi apparatus, respectively in response to light illumination. In the presence of light, blue light induces the formations of the LOV2 cysteinyl-FMN adduct and results in autophosphorylation of phototropin, that is essential to initiate phototropin-mediated active signaling. The activated PHOTs directly phosphorylate the signaling components including ATP binding cassette B (ABCB19) and PHYTOCHROME KIANSE SUBSTRATE 4(PKS4) in order to inactivate both proteins (de Wit et al., 2016; Fankhauser and Christie, 2015; Liscum et al., 2014). In Arabidopsis, primary blue light signaling involves two protein families, NPH3/RPT2-Like (NRL) and PKS. They are responsible for the early steps of phot-mediated signaling pathways for phototropism. NONPHOTOTROPIC HYPOCOTYL 3 (NPH3), ROOT PHOTOTROPISM 2 (RPT2), PKS1, PKS2 and PKS4 are plasma membrane-associated proteins and directly interact with phototropins (Goyal et al., 2013). For example, NPH3 is essential for normal phototropic response including phototropism under a broad range of blue-light intensity. Structurally, NPH3 encodes a protein containing N-terminal Broad Complex [Tramtrack, and Bric à Brac (BTB)] domain that

is important for the interaction with CULLIN3 (CUL3), a component of CULLIN3-based E3 ubiquitin ligase complexes (CLR3), and C-terminal coiled –coil domain directly interacting with N-terminal LOV domain of PHOT1. This interaction leads to clathrin-mediated PHOT1-relocalization into the cytoplasm and then regulates the subcellular localization of phototropism signaling components including polar auxin transporters (Goyal et al., 2013; Liscum et al., 2014). Another example, ROOT PHOTOTROPISM2 (RPT2) is responsible for hypocotyl phototropism under high light intensity condition (Liscum et al., 2014). Moreover, phototropin autophosphorylation and cascade of phosphorylation gradient process may contribute to drive lateral auxin redistribution during phototropism mechanism. The lateral auxin gradient across hypocotyl is controlled by polar auxin transporters (PAT). There are three polar auxin transporters (PAT) families including AUXIN RESISTANT 1 (AUX1) family, ABC transporters (primarily ABCB19), and auxin efflux carriers of the PIN-FORMED (PIN) family, all are involved in phototropism (Goyal et al., 2013). The PIN-FORMED (PIN) proteins are defined as auxin efflux carriers and localized at plasma membrane and PIN-FORMED (PIN) activity is controlled by phosphorylation of two kinase proteins, PINOD (PID) and D6 PROTEIN KINASE (D6PKs) (de Wit et al., 2016; Fankhauser and Christie, 2015; Liscum et al., 2014; Weller et al., 2017). The PID-dependent phosphorylation mediated PIN3 polarization is involved in asymmetric auxin distribution during hypocotyl positive phototropism and root negative phototropic response (Ding et al., 2011; Zhang et al., 2013). However, pin3 null mutant displays a partial in phototropic responses in both the hypocotyl and root. These results indicate that additional auxin transporters might be participated in lateral auxin gradient in light and controlled by the PHOT-mediated mechanism. In addition to PIN proteins, ABC

transporter family (three type: ABCB1, ABCB4 and ABCB19) function as auxin transporter but they exhibit less polar intracellular localization than PIN proteins.

(Liscum et al., 2014). In ABC transporter family, the function of ABCB19 interlinked between auxin movement and light –activated phosphorylation by PHOT1 have been reported (de Wit et al., 2016; Liscum et al., 2014). The reports support the functional incorporation between ABCB19 and PIN1 in order to mediate longitude auxin transporter through ABCB19-dependent stabilization of PIN membrane localization mechanism. In contrast to pin mutants, abcb19 mutant has been shown to have the increased auxin concentration in the hypocotyl and appeared to promote the phototropism. In response to red light, etiolated seedlings may enhance the magnitude and acceleration of blue light-induced phototropism, indicating that phytochrome is partially involved in phototropism. By genetic studies, phytochrome not only enhances the transcriptional level of positive regulators involved in phototropism, such as INDOLE-3-ACETIC ACID 19 (IAA19), PKS1, and RPT2, but also downregulates the levels of ABCB19. This indicates that phytochromes promote phototropism through multiple mechanisms in both nucleus and cytosol. Besides phototropins, crytotochrome is a blue light receptor responsible for enhancing phototropism by induction of RPT2 expression. In fact, not only phototropin but also phytochrome and cryptochrome interplay the formation of auxin distribution in plants through various transporter proteins required for phototropism (de Wit et al., 2016; Goyal et al., 2013; Liscum et al., 2014).

1.5 Photobiology and the basic light transduction pathways in fungi

Light is an important environmental signal that influences physiological and developmental processes in fungi. Light can induce the developmental transitions in fungal life cycle, for example resetting of circadian clock, pigmentation, reproductive process, phototropism, and direction of ascospore dispersal (Idnurm et al., 2010). Based on genome-wide microarray and RNA-sequencing studies, ranging from 3% up to 31%

of the expressed genes are potentially regulated by light and categorized into early and late light responsive genes. These results indicate that regulation of various cellular and physiological processes are enriched in response to light in fungi (Chen et al., 2009;

Wu et al., 2014).

Among different wavelengths in the visible spectrum, blue light is considered as the most spectrum that influences fungal photomorphogenesis; however, red light affects the activation of several light signaling pathways in Aspergillus nidulans. In contrast, model fungi including Saccharomyces cerevisiae, Schizosaccharomyces pombe, and pathogen Microsporum lack photoreceptor genes for light sensing. Therefore, the analyses of physiology, biochemical response as well as characterization of the genes and proteins are crucial for studying and dissecting the light-sensing pathway. In fungi, light affects several output pathways and processes such as changes of nutrient supply, temperature, hormones, and mechanical damages. Perception of light by their photoreceptors has allowed them to respond and acclimatize to environmental light conditions (Belozerskaya et al., 2012; Idnurm and Heitman, 2005; Idnurm et al., 2010).

Effect of common light on fungi is characterized by their physiological changes and respective phenotypes including alteration of sexual/asexual reproduction, and pigmentation such as carotenoid or melanin (Idnurm et al., 2010). Conidiation

controlled by several environment factors and is regulated by the light-mediated circadian clock mechanism. Fungal conidia serve as specialized cells that can function as resting or dispersal propagules. The mechanisms leading to conidial formation have been intensively studied in many fungi including Aspergillus nidulans and Neurospora crassa in response to light. For example, Neurospora crassa contains a group of conidiation (con) genes that are preferentially expressed during asexual development and mediated by blue light (Lauter and Russo, 1991; Leeder et al., 2011). Moreover, blue light induced sporulations have been reported in serval fungal species such as Trichroderma atroviride, A. nidulans, and Paecilomyces fumosoroseus; however, photo-inhibition of sporulation have been found in Alternaria solani (Corrochano, 2007;

Fischer et al., 2016; Lukens 1963). Moreover, previous studies have showed that sexual development of zygomycete Phycomyces blakesleeanus is inhibited by light (Corrochano, 2007). Therefore, the balance and interplay between asexual and sexual reproduction in fungi and light rhythm are essential for regulating reproductive style across fungal kingdom (Fischer et al., 2016). In basidiomycetes Cyathus stercoreus, light is requires to initiate, at least development until hyphal knots formation, the fruiting body development (Lu, 1965). Similarity, blue /UV light plays an important role for controlling the growth, sexual filamentation as well as virulence in the pathogenic yeast Cryptococcus neoformans (Idnurm and Heitman, 2005; Lu et al., 2005). In ascomycetes, light stimuli play a crucial in the sexual cycle of Hypocrea jecorina, whereas, stroma formation is inhibited by incubating under constant light due to blocking of light-induced asexual conidiation (Chen et al., 2012; Seidl et al., 2009).

The number of Neurospora fruiting body is abundantly produced in darkness; however, in early fruiting body stage, protoperithecia formation is greatly induced by blue light,

indicating that light is necessary for initial stimulation of pre-fruiting bodies (female structure) (Fischer et al., 2016; Innocenti et al., 1983; Sokolovsky et al. ,1992). In addition to light, nitrogen metabolite and G proteins play an important role in regulating the formation of female reproductive structures (Leeder et al., 2011; Li et al., 2007).

The sexual development of A. nidulan is inhibited by red and blue light, whereas far-red light enhances sexual development (Dyer and O'Gorman, 2012). Recently, light induced fruiting body formation has been reported in well-known edible and medicinal fungus, Ganoderma lucidum (basidiomycete) and Cordyceps militaris (ascomycete) (Wang et al., 2011; Xu et al., 2017; Yang et al., 2016). Beside fruiting body development, the direction of peritheical beak toward light, called phototropism, has been previously studied in N. crassa and Sordaria fimicola (Harding and Melles, 1983; Ingold and Hadland, 1959). Similar to positive phototropism of perithecial beak, the asexual reproductive structures, sporangiophores, of P. blakesleeanus, Mucor circinelloides as well as Pilobolus crystallinus display and bend toward unilateral blue light (Fischer et al., 2016). Otherwise, fungi obviously protect themselves against harsh environment (i.e light) by generating some barriers such carotenoid and melanin pigment. For example, light-induced carotenoid production is likely to protect themselves against excessive ultraviolet (UV) exposure. Since the carotenoid defective mutants are more sensitive than wide type (Idnurm et al., 2010; Luque et al., 2012; Moliné et al., 2009). Such phenomenon has been reported in serval fungi such as P. blakesleeanus (Cerda-Olmedo, 2001), M. circinelloides (Velayos et al., 2000), Fusarium fujikuri (Avalos and Schrott, 1990), N. crassa (Harding et al., 1969), and S. fimicola (Ingold and Hadland, 1959).

1.6 Photoreceptor proteins and signal transductions in fungi Fungal photoreceptors

Through the Mycota genome, there are three major types of photoreceptors opsins, phytochrome and cryptochrome in fungi. In addition, fungi also contain small proteins comprised of light-sensing LOV domain that widely regulate many physiological and developmental processes. Based on function and sequence similarity, they are identified and defined as blue light photoreceptor proteins. Recently, fungal genome sequencing databases allow researchers to identify photoreceptor proteins across fungal kingdom.

Opsin

Opsins or rhodopsins are membrane-bound photoreceptors containing seven transmembrane α-helices that bind to retinal chromophore through a conserved lysine residue. Opsin is essential for animal vision. The first fungal opsin was described in Neurospora. Neurospora NOP1 protein that exhibits slow photocycle and long-lived intermediate function was defined as a photosensory protein. Neurospora nop-1 mutant has been showed to display minor defects in vegetative growth and the pattern of conidiation in the presence of a mitochondrial ATPase inhibitor (or when mitochondrial ATP synthesis is impaired). NOP-1 proteins also participate in the modulation of carotenogenesis and repression of conidiation-specific gene expression (Bieszke et al., 2007; Idnurm et al., 2010). Recently, the biological function of NOP-1 does not exclude the modulator of light response; however, nop-1 mutant exhibits the de-repressor transcriptional response of several genes under light condition (Chen et al., 2009; Olmedo et al., 2010). In Leptosphaeria maculans, a rhodopsin protein acts as proton ion transporters; however, the role of light-dependent proton pump of this

organism has been yet uncovered till recently (Fischer et al., 2016). Fusarium fujikuroi genome contains two opsin genes, carO and opsA, that have been characterized. Both opsin genes are induced by light. The nop-1 mutant in N. crassa as well as carO and opsA mutants in F. fujikuroi do not display apparent changes in phenotypes including unaltered morphology, pigmentation, and conidiation; however opsA mutant exhibits a significant decrease in mRNA levels of genes required for carotenoid pathway (Estrada and Avalos, 2009). Fungal opsin-like genes have been further identified in several species including A. nidulan and T. atroviride (Fischer et al., 2016); however, the distribution of opsins in fungi is occurred sporadically.

Phytochromes

Phytochromes are composed of multi-domian proteins combined with tetrapyrrole chromophore in response to red and far-red wavelengths. The proteins moiety contains an N-terminal photosensory region with three domains: PAS, GAF, and PHY similar to those of plant and bacteria phytochromes. Upon absorption of red light, the chromophore, linear tetrapyrrole, autocatalytically binds to cysteine in the PAS domain and undergoes a structure change. As a result, the conformation and activity changes lead to alternation of the spectroscopic property. The absorption of chromophore is able to shift to far-red light. The interconversion of the chromophore exhibits the absorption between red and far red light. In fact, phytochrome has two photo- interconversion forms, which are the Pr and Pfr. The Pr form is responsible for absorption of red light and can be immediately converted to Pfr, whereas Pfr absorbs far-red light and is quickly reversible back to Pr. Both PHY-2 of Neurospora and FphA of A. nidulans can bind to biliverdin as a chromophore to display Pr form (Fuller et al., 2015). The c–terminal phytochrome comprises of a histidine-kinase domain containing

ATP binding and a substrate domain, functioning as a regulator domain. A. nidulan undergoes the asexual development under red light while displays sexual reproduction in the dark. These indicate that light governs the reproduction processes. A. nidulan phytochrome is autophosrylated by red light and the autophosrylated FphA protein consequently regulates in asexual-sexual transition and secondary metabolite biosynthesis in response to light (Blumenstein et al., 2005; Brandt et al., 2008; Idnurm et al., 2010). Moreover, FphA physically interacts with LreB (a WC-2 homolog), which is already bound to LreA (a WC-1 homolog in Neurospora), through the C-terminal regulatory domains. Comparably, the histidine kinase domain of FphA interacts with velvet (VeA) which plays a role in the activation of sexual development and inhibition of asexual development. These large complex interactions widely regulate and mediate reproductive development as well as secondary metabolism in Aspergillus fungus (Calvo, 2008; Purschwitz et al., 2009). The red light receptor has been identified and characterized N. crassa and C. neoformans (Fischer et al., 2016). Recently, Neurospora phy2 was found to bind either biliverdin or phycocyanobilin with a photocycle in vitro (Froehlich et al., 2005) and presumably plays a role in the adaptation of fast asexual growth and initiation of sexual reproduction in exposed postfire environment (Wang et al., 2016b). Nevertheless, the most current knowledge of fungal phytochrome function is mainly obtained using A. nidulans as a model to understand the relationships between red light sensing and its global regulation (Fischer et al., 2016; Idnurm et al., 2010).

Cryptochromes and photolyases

Cryptochromes (CRY) are plant blue light photoreceptors that are similar to photolyases, a DNA damage repairing enzymes. Fungal cryptochrome consists of an N-terminal photolyase-related region (PHR) that binds noncovalently to two

chromophores, flavin adenine dinucleotide (FAD) and 5,10-methenyltetrahydrofolate (MTHF)/pterin, and variable C-terminal domain. Functionally, cryptochrome bound to FAD is essential for catalysis and light-harvesting chromophore, whereas cryptochrome interacting with MTHF acts as an antenna to harvest light in order to initiate the photoreaction process. Cryptochromes are required for photosensory regulation of growth, development, cell signaling and circadian rhythm, and magnetoreceptors (Liedvogel and Mouritsen, 2010). Based on evolutionary comparisons and functional analysis, cryptochromes are likely derived from a CDP photolyase ancestor. CRY-DASH type (Cryptochrome-Drosophila, Arabidopsis, Synechocystis, Human) is widely found in eukaryotes and prokaryotes. In fact, CDP photolyase functioning as a light-activated DNA repair protein is essential for all living cell. The CRY-DASH displays subtle to no photolyase activity for requirement of double stranded DNA whereas it shows a photolyase activity toward cyclobutane pyrimidine dimers repair in single-stranded DNA. In fungi, photosensory functions of photolyase-related region (PHR) are initially found in T. atroviride and further identified in N. crassa, F. fujikuroi, and A.

nidulans. The Trichoderma photolyase phr1 gene has been shown to regulate its own expression in the light condition probably functioning as a negative modulator of the BLR orthologues of WC proteins (Berrocal-Tito et al., 2007). Other species such as Trichoderma reesei, Trichoderma photolyase CRY-1 protein also shows DNA repair capability (Guzman-Moreno et al., 2014). Neurospora cry transcripts are induced by light in a WC-1 dependent manner (Fischer et al., 2016; Idnurm et al., 2010).

Neurospora CRY is involved in the regulation of circadian clock and function as a secondary oscillation, so called the CRY-dependent oscillator (CDO), in constant illumination (Nsa et al., 2015). A. nidulans has a cryptochrome/ photolyase type that

has dual function for DNA-repairing enzyme and photoreceptor. A. nidulans CPD photolyase CryA exhibits DNA repair activity and acts as a negative regulator during sexual development (Bayram et al., 2008).

1.7 Other photoreceptor: the flavin based white collar photoreceptor 1.7.1 History of White collar-1 orthologues

Among three classes of photoreceptors, numerous fungi contain a blue light sensing gene, White collar 1 (wc-1), in their genome. wc-1 photoreceptor was firstly cloned from the bread mold Neuropora crassa (Ballario et al., 1996). N. crassa wild type exhibits bright orange color in both conidia and mycelia under light condition, and the pigment is induced by light through the carotenoid biosynthesis. Phenotypically, white-collar is named according to the appearance of mutant phenotype. Carotenoid pigments are normally produced in conidia of wc-1 mutants; however, pigments in mycelia fail to accumulate the carotenoid in response to light illumination due to mutation. Therefore, wc-1 mutants display white collar at the interface of aerial fungal mass and underneath mycelia on the top of agar slant (Fuller et al., 2015). WC-1 plays roles in many physiological and developmental processes in N. crassa including circadian clock (Baker et al., 2012a; Heintzen and Liu, 2007) and light induced pigment production (Harding and Turner, 1981), sporulation (Belozerskaya et al., 2012), phototropism (Harding and Melles, 1983) and light-induced protoperithecia formation (Degli-Innocenti and Russo, 1984).

1.7.2 The structure of White collar proteins

The Neurospora WC-1 protein contains three PAS (Per-Arnt-Sim) domains and Zn-finger DNA binding domain defined as a GATA-like transcription factor (Froehlich

et al., 2002; He et al., 2002). However, many WC-1 proteins in the basidiomycetes, for example, Cryptococcus neoformans, Ustilago maydis, Phanerochaete chrysosporium, Coprinus cinereus, Schizophyllum commune, and Ganoderma lucidum, lack the zinc-finger DNA-binding motif domain (Fuller et al., 2015; Xu et al., 2017). At N-terminus, the first PAS domain is a special class of PAS domain called a LOV (light, oxygen and voltages) domain, by which WC-1 non-covalently binds to flavin adenine dinucleotide (FAD) as a chromophore. The LOV-domain containing proteins function as light sensors in Neurospora. Neurospora WC-1 protein interacts with its partner proteins, WC-2 protein, through their PAS domain and form a heterodimer called White Collar Complex (WCC), to regulate light-mediated gene expression. Similar to wc-1 mutant, wc-2 mutants also display blind phenotype. WC-2 protein contains PAS and GATA Zn-finger domains but it has lost the LOV domain. Therefore, WC-2 is essential for light response but not for light sensing. The Zn-finger domain of WC-2 is required for light induced binding of WCC to DNA. Moreover, the DNA binding domain for circadian functions is requires both WC-2 and WC-1 (Fischer et al., 2016; Froehlich et al., 2002;

Fuller et al., 2015; Wang et al., 2016a).

1.7.3 Light activated WCC complex in Neurospora

Upon light activation, LOV domain undergoes the transiently covalent cysteinyl adduct formation between flavin-thiol and conserved cysteine of LOV domain (Fischer et al., 2016). Light-activated WCC contributes chromosome remodeling at the light-responsive element (LRE) close to the transcriptional start of numerous genes such as Frequency (frq), vivid (vvd) and others (Belden et al., 2007). Through chromatin remodeling events, the light activated-histone H3 acetylation is mediated by histone acetyltransferase, NGF-1. This process may be recruited by the WCC to form the

light-WCC (L-light-WCC). L-light-WCC is associated with light-responsive region (LRR) in the promoter of light responsive genes including al-3 and vvd for histone acetyltransferase (Grimaldi et al., 2006). In addition to histone acetyltransferase, histone H3 lysine 9 trimethylation (H3K9me3) by DIM-5 appears to occur in the heterochromatin at frq promoter, which results in the mute of light response and repression of circadian output (Ruesch et al., 2015)

Both WCs are phosphorylated in dark, whereas hyperphosphorylation appears to occur upon light activation. In fact, WC-1 in the dark is not sufficient to turn on light responsive gene activity due to their phosphorylation, which inhibits the LRE-binding activation of dark WC complex (D-WCC). Light–WCC (L-WCC) is rapidly

Both WCs are phosphorylated in dark, whereas hyperphosphorylation appears to occur upon light activation. In fact, WC-1 in the dark is not sufficient to turn on light responsive gene activity due to their phosphorylation, which inhibits the LRE-binding activation of dark WC complex (D-WCC). Light–WCC (L-WCC) is rapidly