Environment Health Techniques Phytochromes in basidiomycetes
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Short Communication Genomewide analysis of phytochrome proteins in the phylum Basidiomycota L. Lavın1, Lucıa Ramırez2, Antonio G. Pisabarro2 and Jose A. Oguiza2 Jose 1
2
Genome Analysis Platform, Functional Genomics Unit, CIC bioGUNE & CIBERehd, Bizkaia Technology Park, Derio, Spain Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, Pamplona, Spain
Phytochromes are photoreceptor proteins involved in the detection of the red and far-red regions of the visible light spectrum. Fungal phytochromes are hybrid histidine kinases with a conserved domain architecture composed of an N-terminal photosensory module and a C-terminal regulatory output module that includes the histidine kinase and response regulator receiver domains. In this study, we have analyzed the distribution, domain architecture, and phylogenetic analysis of phytochrome proteins in 47 published genome sequences among the phylum Basidiomycota. Genome analysis revealed that almost every genome of basidiomycetes contained at least one gene encoding a phytochrome protein. Domain architecture of fungal phytochromes was completely conserved in the identified phytochromes of basidiomycetes, and phylogenetic analysis clustered these proteins into clades related with the phylogenetic classification of this fungal phylum. Keywords: Basidiomycetes / Phytochrome / Genomic analysis / Comparative genomics / Histidine kinase Received: February 2, 2015; accepted: March 20, 2015 DOI 10.1002/jobm.201500078
Introduction Light is an important environmental signal for almost all cellular organisms. Photoreceptor proteins absorb light of specific wavelengths through specialized low-molecular-weight organic molecules known as chromophores [1, 2]. With the exception of the Saccharomycotina yeasts, the majority of fungal species can sense and respond to light over a broad spectrum range, from ultraviolet to far-red light [3, 4]. Phytochromes are photoswitchable photosensors that utilize a bilin chromophore for the detection of the red and far-red regions of the visible light spectrum. Phytochromes can carry out a reversible photoconversion between the biological inactive red light-absorbing (Pr) form and the active far-red light absorbing (Pfr) form [5]. All currently characterized phytochromes act A. Oguiza, Genetics and Microbiology Correspondence: Dr. Jose Research Group, Department of Agrarian Production, Public University of Navarre, Pamplona 31006, Spain E-mail: [email protected] Phone: þ34 948 169757 Fax: þ34 948 169732 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
as dimers, and each subunit contains a covalently attached chromophore [6]. The domain architecture of phytochromes is composed of two functionally structural regions: an N-terminal photosensory module that harbors the chromophore attachment site, and a C-terminal regulatory output module that is involved in dimerization of phytochrome polypeptide chains and in transmitting the light signal [5–7]. The photosensory module comprises three conserved domains: a Per/ Arndt/Sim (PAS) domain, a cGMP phosphodiesterase/ adenylyl cyclase/FhlA (GAF) domain, and a phytochrome (PHY) domain [5, 6]. Phytochromes are found in a broad range of organisms and have been organized into five major functional groups: Phy (plant and green algae phytochrome), BphP (bacteriophytochrome), Cph (cyanobacterial phytochrome), Fph (fungal phytochrome), and Dph (diatom phytochrome) [5–8]. Fph proteins contain a variable N-terminal extension (NTE) preceding the PAS-GAF-PHY domains of the photosensory module. In addition, Fphs are hybrid HKs with a C-terminal regulatory output module comprised of a HK domain and a response regulator receiver (RR REC) domain [5, 6, 8–12].
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Fphs show a sporadic distribution across the fungal kingdom [4, 12]. These photoreceptors have been identified in the genomes of several ascomycetes and basidiomycetes, but not in ascomycete yeasts and some few species of basidiomycete yeasts. Most of the fungi contain a single Fph, although in some species two or three Fph copies have been found [4, 6, 9, 11–15]. Not much is known about the functions of these proteins in fungi. In the ascomycete Aspergillus nidulans, the FphA protein acts as red light photoreceptor and is required to trigger asexual development and repress sexual development under red-light conditions [9, 10]. In basidiomycetes, molecular analysis of Fphs has been only performed in Cryptococcus neoformans and Lentinula edodes. The function of C. neoformans Fph (Tco3/Phy1) remains uncharacterized and the phenotype for the mutant strain is unknown [16]. Transcriptome analysis of L. edodes has shown that the Fph may also serve as photoreceptors in the light-induced brown-film formation on the surface of mature mycelium [17]. Here, we have performed an exhaustive screening to search for Fph proteins in 47 complete genome sequences of basidiomycetes analyzing their distribution, domain architecture, and phylogenetic relationships. This study constitutes a basis for future analysis to understand the role of Fph proteins in the phylum Basidiomycota.
Materials and methods Identification and analysis of Fph proteins To identify Fph proteins, we utilized the published genome sequence information of 47 basidiomicetes available via the MycoCosm portal of Joint Genome Institute (JGI) website (http://genome.jgi.doe.gov/programs/fungi/index.jsf) [18]. These included species belonging to the subphyla Agaricomycotina (34), Pucciniomycotina (4) and Ustilaginomycotina (7), and to the Basidiomycota incertae sedis (2) (Table 1). Genes coding for Fphs were identified using the pipeline web server BASID2CS [13]. A Hidden Markov Model (HMM) targeting Fph proteins (Tco3) was used to search against the proteomes of basidiomycetes, and hits with an E-value below a defined cutoff (1018) were extracted. In addition, genome sequences of basidiomycetes lacking Fphs were also investigated using TBLASTN [19]. Functional domains of Fph proteins were identified by search the Conserved Domain Databases (CDD) with Reversed Position Specific BLAST [20] and InterPro Scan 5 [21]. Amino acid sequences were aligned with Clustal Omega using the default parameter settings [22]. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Phylogenetic analysis The part comprising the GAF and PHY domains (equivalent to amino acids 914–1264 of C. neoformans Tco3) in the alignment of the selected Fph protein sequences of basidiomycetes was chosen for construction of a phylogenetic tree using the Phylogeny.fr platform [23]. Ambiguous regions of the alignment were removed with Gblocks v0.91b [24] using the default settings. A phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program v3.0 aLRT [25, 26] using the default settings. Reliability for internal branch was assessed using the bootstrapping method with 100 bootstrap replicates. Graphical representation and edition of the phylogenetic tree were performed with TreeDyn v198.3 [27].
Results and discussion Fph proteins in basidiomycetes Genome analysis revealed that almost every genome of basidiomycetes examined in this study contained at least one gene encoding a Fph protein, with the exception of Agaricus bisporus, Laccaria bicolor, Moniliophthora perniciosa, Malassezia globosa, M. sympodialis, and Tilletiaria anomala whose genomes lack Fphs (Table 1). Previously, it has been shown that Fphs are also absent in several ascomycete yeasts [4, 12]; but the only basidiomycete yeasts without Fhps were the Malassezia species associated with a number of skin disorders (M. globosa, M. sympodialis). The absence of Fphs in A. bisporus, L. bicolor, M. perniciosa, and T. anomala might be related to their complex lifecycles (T. anomala is a dimorphic fungus with hyphal and yeast phases) or the adaptation to specific environments (such as the symbiotic mycorrhizal association of L. bicolor or the humic-rich leaf-litter environment of A. bisporus). Furthermore, Auricularia subglabra and the halophilic fungi Wallemia icthyophaga and W. sebi contained two genes encoding Fph proteins, and Jaapia argillacea three Fph genes (Table 1). Despite the reduced genome sizes and number of predicted genes in Wallemia species, expansion of several protein families has been observed that may represent an adaptation to the osmotolerant lifestyle [28, 29]. Fph genes of both Wallemia species were located near each other on the same chromosome at a distance of 470 bp in W. icthyophaga and 304,487 bp in W. sebi. However, the identity among these Fph proteins was very low (33% in W. icthyophaga and 35% in W. sebi). Similarly, the J. argillacea Fph genes Jaar1_30166 and Jaar1_149003
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Table 1. Fph proteins in basidiomycetes. Subphylum
Organism
Fph
Agaricomycotina
Agaricus bisporus var. bisporus (H97) v2.0 Armillaria mellea DSM 3731 Auricularia subglabra v2.0
— Armme1_12663 Aurde3_1_1200027 Aurde3_1_1213812 Botbo1_35351 Cersu1_111992 Conpu1_104552 Copci1_2565 Cryne_JEC21_1_1449 (Tco3/Phy1) Dacsp1_65545 Dicsq1_106677 Fibra1_7724 Fomme1_97970 Fompi3_1163343 Galma1_238000 Glotr1_1_116494 Hetan2_9148 Jaaar1_30166 Jaaar1_149003 Jaaar1_176751 — — Ompol1_328 Phaca1_106758 Phchr2_2989301 Pirin1_75622 PleosPC15_2_25802 PosplRSB12_1_1160067 Punst1_118301 Rhiso1_9520 Schco3_76719 SerlaS7_3_2_109488 Stehi1_66042 Trave1_69400 Treme1_33037 Volvo1_118802 Wolco1_98767 Mellp1_39867 Mixos1_92045 Pucgr1_31737 Pucst1_498118 — — Psean1_1_84677 Psehu1_1564 Spore1_2232 — Ustma1_5732 Walic1_2774 Walic1_2775 Walse1_59424 Walse1_59425
Botryobasidium botryosum v1.0 Ceriporiopsis (Gelatoporia) subvermispora B Coniophora puteana v1.0 Coprinopsis cinerea Cryptococcus neoformans var. neoformans JEC21 Dacryopinax sp. DJM 731 SSP1 v1.0 Dichomitus squalens v1.0 Fibroporia radiculosa TFFH 294 Fomitiporia mediterranea v1.0 Fomitopsis pinicola FP-58527 SS1 v3.0 Galerina marginata v1.0 Gloeophyllum trabeum v1.0 Heterobasidion annosum v2.0 Jaapia argillacea v1.0
Pucciniomycotina
Ustilaginomycotina
Basidiomycota incertae sedis
Laccaria bicolor v2.0 Moniliophthora perniciosa FA553 Omphalotus olearius Phanerochaete carnosa HHB-10118-Sp v1.0 Phanerochaete chrysosporium RP-78 v2.2 Piriformospora indica DSM 11827 Pleurotus ostreatus PC15 v2.0 Postia placenta MAD-698-R-SB12 v1.0 Punctularia strigosozonata v1.0 Rhizoctonia solani AG-1 IB Schizophyllum commune H4-8 v3.0 Serpula lacrymans S7.3 v2.0 Stereum hirsutum FP-91666 SS1 v1.0 Trametes versicolor v1.0 Tremella mesenterica Fries v1.0 Volvariella volvacea V23 Wolfiporia cocos MD-104 SS10 v1.0 Melampsora laricis-populina v1.0 Mixia osmundae IAM 14324 v1.0 Puccinia graminis Puccinia striiformis f. sp. tritici PST-130 Malassezia globosa Malassezia sympodialis ATCC 42132 Pseudozyma antarctica T-34 Pseudozyma hubeiensis SY62 Sporisorium reilianum SRZ2 Tilletiaria anomala UBC 951 v1.0 Ustilago maydis Wallemia ichthyophaga EXF-994 Wallemia sebi v1.0
also mapped relatively close in the same chromosome at a distance of 32,733 kb. None of the Fphs have been functionally characterized for any species of basidiomycetes and consequently their roles remain unknown. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Domain architecture of Fph proteins in basidiomycetes The domain arrangement of Fph proteins was analyzed by searching CDD [20]. All the identified Fph proteins of basidiomycetes completely conserved the Fph domain
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architecture composed of an N-terminal photosensory module with a concatenation of a variable NTE and PASGAF-PHY domains attached to a C-terminal regulatory output module with HK and RR REC domains (Fig. 1). Despite of having the typical domain architecture of Fphs, some of the specific domains were absent in Armme1_12663, Dacsp1_65545, and Pucgr1_31737 proteins, probably due to an incorrect prediction of these gene models during the genome annotation process. Multiple sequence alignment of the photosensory module showed that key amino acid residues of the PAS, GAF, and PHY domains were highly conserved over the Fph proteins of basidiomycetes (Fig. 1). It has been proposed that large NTEs of Fphs might be unstructured or proteolytically removed to facilitate protein folding [5]. The PAS–GAF–PHY domains have been predicted to function as the light sensory
input module by forming the pocket necessary for chromophore binding and generating the appropriate contacts for stabilizing the Pr and Pfr forms. PAS domains participate in protein–protein interactions, signal transfer, or directly sensing perceived stimuli such as visible light, oxygen, redox potential, or some other stimuli [30]. GAF domains bind small ligands, such as cyclic nucleotides (cAMP and cGMP) and chromophores [5, 14]. Fph proteins use biliverdin as the chromophore that is attached covalently to a positionally conserved cysteine (Cys) residue within the PAS domain [5, 8, 9, 12]. As previously observed in other Fphs, the chromophore attachment site within the PAS domain was preserved in all the Fphs of basidiomycetes (Fig. 1). PHY domain is distantly related to PAS and specific to phytochrome proteins [6]. The output activity of phytochromes is probably controlled by a
Figure 1. (A) Scheme of the domain architecture of Fphs. The indicated domains are as follows: NTE, N-terminal extension; PAS, Per-Arnt-Sim domain; GAF, cGMP phosphodiesterase/adenylyl cyclase/FhlA domain; PHY, phytochrome domain; HisKA, dimerization/phosphoaceptor domain; HATPase_c, HK-type ATPase catalytic domain; REC, receiver domain. (B) Sequence alignment of part of the PAS, GAF, and PHY domains located in the N-terminal photosensory module of basidiomycetes Fphs. The conserved chromophore attachment site (C) upstream of the PAS domain, D-I-P motif in the GAF domain and P-R-X-S-F motif in the tongue region of the PHY domain are marked by asterisks. Black and grey boxes denote identical and similar residues respectively. Proteins corresponding to incorrectly predicted Fph gene models of basidiomycetes were not included in the multiple sequence alignment. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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light-induced conformational change in the photosensory module. Recently, analysis of the BphP crystal structure from Deinococcus radiodurans has shown an evolutionarily conserved “tongue” of the PHY domain which is in contact with the chromophore, and refolding of this tongue is essential to the structural change during the transition between Pr and Pfr forms [31]. The key amino acid motifs D-I-P in the GAF domain and P-R-X-S-F (where X denotes any amino acid) in the tongue region of the PHY domain were also conserved among all the Fph proteins of basidiomycetes (Fig. 1).
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The C-terminal regulatory output module of all Fphs in basidiomycetes included the HK and RR REC domains, indicating that these proteins were hybridHKs [5, 8–12] (Fig. 1). HKs are key components of two-component system (TCS) signal transduction pathways that are based on phosphotransfer reactions between histidine (His) and aspartate (Asp) residues in signaling domains of TCS proteins [32]. The HK domain is formed by the highly conserved HisKA (dimerization and phosphoaceptor His residues) and HATPase_c (HK-like ATPase catalytic) domains (Fig. 1).
Figure 2. Maximum likelihood phylogenetic tree based on the GAF and PHY domains of Fphs from basidiomycetes. Numbers at nodes are bootstrap values. Clusters corresponding to the different subphyla of basidiomycetes are indicated by dashes besides the tree. The horizontal bar represents the scale for the average number of substitutions per site. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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The HATPase_c domain is responsible for binding ATP and catalyzing autophosphorilation of a conserved His residue found within the HisKA domain in response to a perceived signal. The phosphate is subsequently transferred to the Asp residue in the RR REC domain. In the ascomycete A. nidulans, the FphA phytochrome is a functional hybrid-HK that autophosphorylates in response to red-light stimulation [10]. The conserved phosphoaceptor His and Asp residues necessary to function as hybrid-HKs were found in all the Fphs of basidiomycetes (data not shown). Phylogenetic analysis of Fphs in basidiomycetes Phylogenetic analysis of the GAF and PHY domains separated the Fphs into clades related with the phylogenetic classification of the phylum Basidiomycota [33]: the first clade included the Basidiomycota incertae sedis species, and the second, third and fourth clades included species belonging to the subphyla Ustilagomycotina, Pucciniomycotina and Agaricomycotina, respectively (Fig. 2). Concluding remarks The identification and analysis of Fph proteins is a major interest in the phylum Basidiomycota to understand how these fungi sense and respond to light. Genome analysis showed that at least one gene encoding a Fph protein is highly conserved in the majority of basidiomycetes species; although, there are some species that lack Fphs (A. bisporus, L. bicolor, M. perniciosa, M. globosa, M. sympodialis, and T. anomala). All the identified Fph proteins of basidiomycetes shared a common domain architecture including an N-terminal photosensory module and a C-terminal regulatory output module. This study broadens the knowledge on the Fph proteins of basidiomycetes and provides a starting point for future experiments to understand the role they play in the biology of these fungi.
Acknowledgments
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This work was supported by research projects AGL201130495 of the Spanish National Research Plan, BioethanolEuroinnova from the Autonomous Government of Navarre, and by additional institutional support from the Public University of Navarre.
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Conflict of interest
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The authors declare that there is no conflict of interest. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
[15] Defosse, T.A., Sharma, A., Mondal, A.K., Duge de Bernonville, T. et al., 2015. Hybrid histidine kinases in pathogenic fungi, Mol. Microbiol., 95, 914–924.
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[17] Tang, L.H., Jian, H.H., Song, C.Y., Bao, D.P. et al., 2013. Transcriptome analysis of candidate genes and signaling pathways associated with light-induced brown film formation in Lentinula edodes. Appl. Microbiol. Biotechnol., 97, 4977–4989. [18] Grigoriev, I.V., Nikitin, R., Haridas, S., Kuo, A. et al., 2014. MycoCosm portal: Gearing up for 1000 fungal genomes. Nucleic Acids Res., 42, D699–D704. [19] Altschul, S.F., Madden, T.L., Sch€affer, A.A., Zhang, J. et al., 1997. Gapped BLAST and PSI-BLAST: A new generation of protein database search programs. Nucleic Acids Res., 25, 3389–3402. [20] Marchler-Bauer, A., Anderson, J.B., DeWeese-Scott, C., Fedorova, N.D. et al., 2003. CDD: A curated Entrez database of conserved domain alignments. Nucleic Acids Res., 31, 383–387. [21] Jones, P., Binns, D., Chang, H.Y., Fraser, M. et al., 2014. InterProScan 5: Genome-scale protein function classification. Bioinformatics, 30, 1236–1240.
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ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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Short Communication Genomewide analysis of phytochrome proteins in the phylum Basidiomycota L. Lavın1, Lucıa Ramırez2, Antonio G. Pisabarro2 and Jose A. Oguiza2 Jose 1
2
Genome Analysis Platform, Functional Genomics Unit, CIC bioGUNE & CIBERehd, Bizkaia Technology Park, Derio, Spain Genetics and Microbiology Research Group, Department of Agrarian Production, Public University of Navarre, Pamplona, Spain
Phytochromes are photoreceptor proteins involved in the detection of the red and far-red regions of the visible light spectrum. Fungal phytochromes are hybrid histidine kinases with a conserved domain architecture composed of an N-terminal photosensory module and a C-terminal regulatory output module that includes the histidine kinase and response regulator receiver domains. In this study, we have analyzed the distribution, domain architecture, and phylogenetic analysis of phytochrome proteins in 47 published genome sequences among the phylum Basidiomycota. Genome analysis revealed that almost every genome of basidiomycetes contained at least one gene encoding a phytochrome protein. Domain architecture of fungal phytochromes was completely conserved in the identified phytochromes of basidiomycetes, and phylogenetic analysis clustered these proteins into clades related with the phylogenetic classification of this fungal phylum. Keywords: Basidiomycetes / Phytochrome / Genomic analysis / Comparative genomics / Histidine kinase Received: February 2, 2015; accepted: March 20, 2015 DOI 10.1002/jobm.201500078
Introduction Light is an important environmental signal for almost all cellular organisms. Photoreceptor proteins absorb light of specific wavelengths through specialized low-molecular-weight organic molecules known as chromophores [1, 2]. With the exception of the Saccharomycotina yeasts, the majority of fungal species can sense and respond to light over a broad spectrum range, from ultraviolet to far-red light [3, 4]. Phytochromes are photoswitchable photosensors that utilize a bilin chromophore for the detection of the red and far-red regions of the visible light spectrum. Phytochromes can carry out a reversible photoconversion between the biological inactive red light-absorbing (Pr) form and the active far-red light absorbing (Pfr) form [5]. All currently characterized phytochromes act A. Oguiza, Genetics and Microbiology Correspondence: Dr. Jose Research Group, Department of Agrarian Production, Public University of Navarre, Pamplona 31006, Spain E-mail: [email protected] Phone: þ34 948 169757 Fax: þ34 948 169732 ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
as dimers, and each subunit contains a covalently attached chromophore [6]. The domain architecture of phytochromes is composed of two functionally structural regions: an N-terminal photosensory module that harbors the chromophore attachment site, and a C-terminal regulatory output module that is involved in dimerization of phytochrome polypeptide chains and in transmitting the light signal [5–7]. The photosensory module comprises three conserved domains: a Per/ Arndt/Sim (PAS) domain, a cGMP phosphodiesterase/ adenylyl cyclase/FhlA (GAF) domain, and a phytochrome (PHY) domain [5, 6]. Phytochromes are found in a broad range of organisms and have been organized into five major functional groups: Phy (plant and green algae phytochrome), BphP (bacteriophytochrome), Cph (cyanobacterial phytochrome), Fph (fungal phytochrome), and Dph (diatom phytochrome) [5–8]. Fph proteins contain a variable N-terminal extension (NTE) preceding the PAS-GAF-PHY domains of the photosensory module. In addition, Fphs are hybrid HKs with a C-terminal regulatory output module comprised of a HK domain and a response regulator receiver (RR REC) domain [5, 6, 8–12].
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Fphs show a sporadic distribution across the fungal kingdom [4, 12]. These photoreceptors have been identified in the genomes of several ascomycetes and basidiomycetes, but not in ascomycete yeasts and some few species of basidiomycete yeasts. Most of the fungi contain a single Fph, although in some species two or three Fph copies have been found [4, 6, 9, 11–15]. Not much is known about the functions of these proteins in fungi. In the ascomycete Aspergillus nidulans, the FphA protein acts as red light photoreceptor and is required to trigger asexual development and repress sexual development under red-light conditions [9, 10]. In basidiomycetes, molecular analysis of Fphs has been only performed in Cryptococcus neoformans and Lentinula edodes. The function of C. neoformans Fph (Tco3/Phy1) remains uncharacterized and the phenotype for the mutant strain is unknown [16]. Transcriptome analysis of L. edodes has shown that the Fph may also serve as photoreceptors in the light-induced brown-film formation on the surface of mature mycelium [17]. Here, we have performed an exhaustive screening to search for Fph proteins in 47 complete genome sequences of basidiomycetes analyzing their distribution, domain architecture, and phylogenetic relationships. This study constitutes a basis for future analysis to understand the role of Fph proteins in the phylum Basidiomycota.
Materials and methods Identification and analysis of Fph proteins To identify Fph proteins, we utilized the published genome sequence information of 47 basidiomicetes available via the MycoCosm portal of Joint Genome Institute (JGI) website (http://genome.jgi.doe.gov/programs/fungi/index.jsf) [18]. These included species belonging to the subphyla Agaricomycotina (34), Pucciniomycotina (4) and Ustilaginomycotina (7), and to the Basidiomycota incertae sedis (2) (Table 1). Genes coding for Fphs were identified using the pipeline web server BASID2CS [13]. A Hidden Markov Model (HMM) targeting Fph proteins (Tco3) was used to search against the proteomes of basidiomycetes, and hits with an E-value below a defined cutoff (1018) were extracted. In addition, genome sequences of basidiomycetes lacking Fphs were also investigated using TBLASTN [19]. Functional domains of Fph proteins were identified by search the Conserved Domain Databases (CDD) with Reversed Position Specific BLAST [20] and InterPro Scan 5 [21]. Amino acid sequences were aligned with Clustal Omega using the default parameter settings [22]. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Phylogenetic analysis The part comprising the GAF and PHY domains (equivalent to amino acids 914–1264 of C. neoformans Tco3) in the alignment of the selected Fph protein sequences of basidiomycetes was chosen for construction of a phylogenetic tree using the Phylogeny.fr platform [23]. Ambiguous regions of the alignment were removed with Gblocks v0.91b [24] using the default settings. A phylogenetic tree was reconstructed using the maximum likelihood method implemented in the PhyML program v3.0 aLRT [25, 26] using the default settings. Reliability for internal branch was assessed using the bootstrapping method with 100 bootstrap replicates. Graphical representation and edition of the phylogenetic tree were performed with TreeDyn v198.3 [27].
Results and discussion Fph proteins in basidiomycetes Genome analysis revealed that almost every genome of basidiomycetes examined in this study contained at least one gene encoding a Fph protein, with the exception of Agaricus bisporus, Laccaria bicolor, Moniliophthora perniciosa, Malassezia globosa, M. sympodialis, and Tilletiaria anomala whose genomes lack Fphs (Table 1). Previously, it has been shown that Fphs are also absent in several ascomycete yeasts [4, 12]; but the only basidiomycete yeasts without Fhps were the Malassezia species associated with a number of skin disorders (M. globosa, M. sympodialis). The absence of Fphs in A. bisporus, L. bicolor, M. perniciosa, and T. anomala might be related to their complex lifecycles (T. anomala is a dimorphic fungus with hyphal and yeast phases) or the adaptation to specific environments (such as the symbiotic mycorrhizal association of L. bicolor or the humic-rich leaf-litter environment of A. bisporus). Furthermore, Auricularia subglabra and the halophilic fungi Wallemia icthyophaga and W. sebi contained two genes encoding Fph proteins, and Jaapia argillacea three Fph genes (Table 1). Despite the reduced genome sizes and number of predicted genes in Wallemia species, expansion of several protein families has been observed that may represent an adaptation to the osmotolerant lifestyle [28, 29]. Fph genes of both Wallemia species were located near each other on the same chromosome at a distance of 470 bp in W. icthyophaga and 304,487 bp in W. sebi. However, the identity among these Fph proteins was very low (33% in W. icthyophaga and 35% in W. sebi). Similarly, the J. argillacea Fph genes Jaar1_30166 and Jaar1_149003
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Table 1. Fph proteins in basidiomycetes. Subphylum
Organism
Fph
Agaricomycotina
Agaricus bisporus var. bisporus (H97) v2.0 Armillaria mellea DSM 3731 Auricularia subglabra v2.0
— Armme1_12663 Aurde3_1_1200027 Aurde3_1_1213812 Botbo1_35351 Cersu1_111992 Conpu1_104552 Copci1_2565 Cryne_JEC21_1_1449 (Tco3/Phy1) Dacsp1_65545 Dicsq1_106677 Fibra1_7724 Fomme1_97970 Fompi3_1163343 Galma1_238000 Glotr1_1_116494 Hetan2_9148 Jaaar1_30166 Jaaar1_149003 Jaaar1_176751 — — Ompol1_328 Phaca1_106758 Phchr2_2989301 Pirin1_75622 PleosPC15_2_25802 PosplRSB12_1_1160067 Punst1_118301 Rhiso1_9520 Schco3_76719 SerlaS7_3_2_109488 Stehi1_66042 Trave1_69400 Treme1_33037 Volvo1_118802 Wolco1_98767 Mellp1_39867 Mixos1_92045 Pucgr1_31737 Pucst1_498118 — — Psean1_1_84677 Psehu1_1564 Spore1_2232 — Ustma1_5732 Walic1_2774 Walic1_2775 Walse1_59424 Walse1_59425
Botryobasidium botryosum v1.0 Ceriporiopsis (Gelatoporia) subvermispora B Coniophora puteana v1.0 Coprinopsis cinerea Cryptococcus neoformans var. neoformans JEC21 Dacryopinax sp. DJM 731 SSP1 v1.0 Dichomitus squalens v1.0 Fibroporia radiculosa TFFH 294 Fomitiporia mediterranea v1.0 Fomitopsis pinicola FP-58527 SS1 v3.0 Galerina marginata v1.0 Gloeophyllum trabeum v1.0 Heterobasidion annosum v2.0 Jaapia argillacea v1.0
Pucciniomycotina
Ustilaginomycotina
Basidiomycota incertae sedis
Laccaria bicolor v2.0 Moniliophthora perniciosa FA553 Omphalotus olearius Phanerochaete carnosa HHB-10118-Sp v1.0 Phanerochaete chrysosporium RP-78 v2.2 Piriformospora indica DSM 11827 Pleurotus ostreatus PC15 v2.0 Postia placenta MAD-698-R-SB12 v1.0 Punctularia strigosozonata v1.0 Rhizoctonia solani AG-1 IB Schizophyllum commune H4-8 v3.0 Serpula lacrymans S7.3 v2.0 Stereum hirsutum FP-91666 SS1 v1.0 Trametes versicolor v1.0 Tremella mesenterica Fries v1.0 Volvariella volvacea V23 Wolfiporia cocos MD-104 SS10 v1.0 Melampsora laricis-populina v1.0 Mixia osmundae IAM 14324 v1.0 Puccinia graminis Puccinia striiformis f. sp. tritici PST-130 Malassezia globosa Malassezia sympodialis ATCC 42132 Pseudozyma antarctica T-34 Pseudozyma hubeiensis SY62 Sporisorium reilianum SRZ2 Tilletiaria anomala UBC 951 v1.0 Ustilago maydis Wallemia ichthyophaga EXF-994 Wallemia sebi v1.0
also mapped relatively close in the same chromosome at a distance of 32,733 kb. None of the Fphs have been functionally characterized for any species of basidiomycetes and consequently their roles remain unknown. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
Domain architecture of Fph proteins in basidiomycetes The domain arrangement of Fph proteins was analyzed by searching CDD [20]. All the identified Fph proteins of basidiomycetes completely conserved the Fph domain
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architecture composed of an N-terminal photosensory module with a concatenation of a variable NTE and PASGAF-PHY domains attached to a C-terminal regulatory output module with HK and RR REC domains (Fig. 1). Despite of having the typical domain architecture of Fphs, some of the specific domains were absent in Armme1_12663, Dacsp1_65545, and Pucgr1_31737 proteins, probably due to an incorrect prediction of these gene models during the genome annotation process. Multiple sequence alignment of the photosensory module showed that key amino acid residues of the PAS, GAF, and PHY domains were highly conserved over the Fph proteins of basidiomycetes (Fig. 1). It has been proposed that large NTEs of Fphs might be unstructured or proteolytically removed to facilitate protein folding [5]. The PAS–GAF–PHY domains have been predicted to function as the light sensory
input module by forming the pocket necessary for chromophore binding and generating the appropriate contacts for stabilizing the Pr and Pfr forms. PAS domains participate in protein–protein interactions, signal transfer, or directly sensing perceived stimuli such as visible light, oxygen, redox potential, or some other stimuli [30]. GAF domains bind small ligands, such as cyclic nucleotides (cAMP and cGMP) and chromophores [5, 14]. Fph proteins use biliverdin as the chromophore that is attached covalently to a positionally conserved cysteine (Cys) residue within the PAS domain [5, 8, 9, 12]. As previously observed in other Fphs, the chromophore attachment site within the PAS domain was preserved in all the Fphs of basidiomycetes (Fig. 1). PHY domain is distantly related to PAS and specific to phytochrome proteins [6]. The output activity of phytochromes is probably controlled by a
Figure 1. (A) Scheme of the domain architecture of Fphs. The indicated domains are as follows: NTE, N-terminal extension; PAS, Per-Arnt-Sim domain; GAF, cGMP phosphodiesterase/adenylyl cyclase/FhlA domain; PHY, phytochrome domain; HisKA, dimerization/phosphoaceptor domain; HATPase_c, HK-type ATPase catalytic domain; REC, receiver domain. (B) Sequence alignment of part of the PAS, GAF, and PHY domains located in the N-terminal photosensory module of basidiomycetes Fphs. The conserved chromophore attachment site (C) upstream of the PAS domain, D-I-P motif in the GAF domain and P-R-X-S-F motif in the tongue region of the PHY domain are marked by asterisks. Black and grey boxes denote identical and similar residues respectively. Proteins corresponding to incorrectly predicted Fph gene models of basidiomycetes were not included in the multiple sequence alignment. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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light-induced conformational change in the photosensory module. Recently, analysis of the BphP crystal structure from Deinococcus radiodurans has shown an evolutionarily conserved “tongue” of the PHY domain which is in contact with the chromophore, and refolding of this tongue is essential to the structural change during the transition between Pr and Pfr forms [31]. The key amino acid motifs D-I-P in the GAF domain and P-R-X-S-F (where X denotes any amino acid) in the tongue region of the PHY domain were also conserved among all the Fph proteins of basidiomycetes (Fig. 1).
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The C-terminal regulatory output module of all Fphs in basidiomycetes included the HK and RR REC domains, indicating that these proteins were hybridHKs [5, 8–12] (Fig. 1). HKs are key components of two-component system (TCS) signal transduction pathways that are based on phosphotransfer reactions between histidine (His) and aspartate (Asp) residues in signaling domains of TCS proteins [32]. The HK domain is formed by the highly conserved HisKA (dimerization and phosphoaceptor His residues) and HATPase_c (HK-like ATPase catalytic) domains (Fig. 1).
Figure 2. Maximum likelihood phylogenetic tree based on the GAF and PHY domains of Fphs from basidiomycetes. Numbers at nodes are bootstrap values. Clusters corresponding to the different subphyla of basidiomycetes are indicated by dashes besides the tree. The horizontal bar represents the scale for the average number of substitutions per site. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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The HATPase_c domain is responsible for binding ATP and catalyzing autophosphorilation of a conserved His residue found within the HisKA domain in response to a perceived signal. The phosphate is subsequently transferred to the Asp residue in the RR REC domain. In the ascomycete A. nidulans, the FphA phytochrome is a functional hybrid-HK that autophosphorylates in response to red-light stimulation [10]. The conserved phosphoaceptor His and Asp residues necessary to function as hybrid-HKs were found in all the Fphs of basidiomycetes (data not shown). Phylogenetic analysis of Fphs in basidiomycetes Phylogenetic analysis of the GAF and PHY domains separated the Fphs into clades related with the phylogenetic classification of the phylum Basidiomycota [33]: the first clade included the Basidiomycota incertae sedis species, and the second, third and fourth clades included species belonging to the subphyla Ustilagomycotina, Pucciniomycotina and Agaricomycotina, respectively (Fig. 2). Concluding remarks The identification and analysis of Fph proteins is a major interest in the phylum Basidiomycota to understand how these fungi sense and respond to light. Genome analysis showed that at least one gene encoding a Fph protein is highly conserved in the majority of basidiomycetes species; although, there are some species that lack Fphs (A. bisporus, L. bicolor, M. perniciosa, M. globosa, M. sympodialis, and T. anomala). All the identified Fph proteins of basidiomycetes shared a common domain architecture including an N-terminal photosensory module and a C-terminal regulatory output module. This study broadens the knowledge on the Fph proteins of basidiomycetes and provides a starting point for future experiments to understand the role they play in the biology of these fungi.
Acknowledgments
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This work was supported by research projects AGL201130495 of the Spanish National Research Plan, BioethanolEuroinnova from the Autonomous Government of Navarre, and by additional institutional support from the Public University of Navarre.
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The authors declare that there is no conflict of interest. ß 2015 WILEY-VCH Verlag GmbH & Co. KGaA,Weinheim
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