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Further characterization of hydrophobin genes in genome of Flammulina velutipes
m y c o s c i e n c e 5 7 ( 2 0 1 6 ) 3 2 0 e3 2 5
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Further characterization of hydrophobin genes in genome of Flammulina velutipes Hong-Il Kim, Chang-Soo Lee, Young-Jin Park* Department of Biomedical Chemistry, Konkuk University, Chungju 27478, Republic of Korea
article info
abstract
Article history:
This study aimed to identify and further characterize hydrophobin genes in the whole-
Received 29 December 2015
genome sequence of Flammulina velutipes. Ten genes (Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5,
Received in revised form
Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10) sharing eight conserved cysteine residues were
25 April 2016
identified from 13 previously predicted putative hydrophobin genes in the F. velutipes
Accepted 29 April 2016
genome. Quantitative real-time PCR (qPCR) analysis showed that these genes were spe-
Available online 11 June 2016
cifically expressed in different developmental stages and tissues. Whereas all of the genes showed relatively higher levels of expression in the primordial stages of the fungus, most
Keywords: Fruit body development
of them were expressed at relatively low levels in the mycelial stage. © 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Pileus Stipe Winter mushroom
Flammulina velutipes, also known as winter mushroom or Enokitake, is a common white-rot basidiomycete distributed throughout temperate zones (Hughes et al. 1999). It is one of the six most actively cultivated mushroom species in the world (Psurtseva 2005). In the commercial production of mushrooms, the development of fruiting bodies is induced by controlling environmental factors such as temperature, light and humidity, as well as physical and chemical stimuli. Extensive reports on F. velutipes fruit body formation are available (Plunkett 1956; Kitamoto and Gruen 1976; Williams et al. 1985; Terashita et al. 1992) because this fungus readily forms fruit bodies under low-temperature and low-light conditions. Several studies have investigated the molecular basis of fruit body development in this fungus (Kim and Azuma 1999; Kim et al. 1999; Ando et al. 2001; Yamada et al. 2005). We recently determined the whole-genome sequence of F.
velutipes (Park et al. 2014). Therefore, genome-wide identification of the genes involved in the initiation of fruiting can now be studied to understand this process at the molecular level and develop efficient and reproducible methods of cultivating and inducing fruit body development in valuable basidiomycetes species. Several fruiting-specific or fruiting induction genes have been isolated in other basidiomycetes, such as Agaricus bisporus (De Groot et al. 1996), Schizophyllum commune (Mulder and re Wessels 1986) and Agrocybe aegerita (Salvado and Labare 1991). Some of these fungal genes belong to a family that encodes hydrophobic proteins called hydrophobins. In addition, two fruiting-specific or fruiting induction genes (fvh1 and Fvhyd1) encoding hydrophobins were isolated from a F. velutipes primordial complementary DNA (cDNA) library (Ando et al. 2001; Yamada et al. 2005).
* Corresponding author. Tel./fax: þ82 438403601. E-mail address: [email protected] (Y.-J. Park). http://dx.doi.org/10.1016/j.myc.2016.04.004 1340-3540/© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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Hydrophobins are small, secretory, hydrophobic fungusspecific proteins composed of four loops of disulfide bonds among eight highly conserved cysteine residues that selfassemble into amphipathic layers as hydrophilicehydrophobic interfaces (Ando et al. 2001; Yamada et al. 2005). Fungal strains usually contain several genes coding for hydrophobins, which play key roles in morphogenesis and fruit body development. Some of these hydrophobin genes are € sten and Wessels also highly expressed in fruiting bodies (Wo 1997). The aim of the present study was to further identify and characterize hydrophobin genes in F. velutipes by using our recently published whole-genome sequence information (Park et al. 2014). Herein, we describe 10 F. velutipes hydrophobin genes with deduced amino acid sequences and their regulation at the transcription level during fruiting body development. This comparative study of gene expression at various developmental stages and in different tissues provides valuable insights into the molecular basis of mushroom fruit body development.
Hydrophobin genes in the F. velutipes genome The F. velutipes KACC43778 dikaryotic strain was obtained from the Korean Agricultural Culture Collection (KACC; Rural Development Administration, Korea; http://www.genebank. go.kr/) and grown at 26 C on mushroom complete medium agar (0.2% peptone, 2% glucose, 0.2% yeast extract, 0.05% MgSO4, 0.046% KH2PO4, 0.1% K2HPO4, and 1.5% agar) for 2 wk. For genomic DNA and total RNA isolation from mycelia, a 300mL Erlenmeyer flask containing 50 mL mushroom complete medium was inoculated with fresh plugs from the plate (five mycelial plugs/flask) and incubated at 26 C for 2 wk without agitation. In our previous study, genome-wide identification of hydrophobin genes was carried out using several methods, including ab initio gene structure prediction (Fgenesh; http:// www.softberry.com), a homology-based approach (Fgeneshþ; http://www.softberry.com), and transcriptomebased gene identification (cufflinks; http://cufflinks.cbcb. umd.edu/manual.html) with the F. velutipes (KACC42780 monokaryotic strain) whole-genome sequence (AQHU00000000) (Park et al. 2014). In the present study, we performed additional gene prediction by using the AUGUSTUS tool (Stanke and Morgenstern 2005) with default parameters trained in Coprinopsis cinerea. The genes were compared by using BLAST (version 2.2.17) software with a series of protein databases, including the National Center for Biotechnology Information (NCBI) nucleotide (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) and non-redundant (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) sets, for functional annotation of the predicted genes. BLASTP searches within the NCBI-NR database of the amino acid sequences of F. velutipes genes, which were previously predicted by using combined approaches (Fgenesh, Fgeneshþ, and cufflinks), showed that 12 of the predicted proteins shared sequence similarity with fungal hydrophobins (Park et al. 2014). However, gene prediction performed with the AUGUSTUS tool indicated that the total number of hydrophobin genes in F. velutipes was smaller (10
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hydrophobin) than that predicted through combined approaches (Table 1). Nine putative hydrophobin genes were found in common among the genes predicted by both the combined approaches and the AUGUSTUS tool. Furthermore, one (Hyd-10) of these 10 putative genes was further identified and annotated as a hydrophobin gene in this study (Table 1). cDNA fragments encompassing all of the putative open reading frames (ORFs) were amplified with reverse transcriptase (RT)-PCR and their sequences were refined. For cDNA synthesis, samples were ground to a fine powder under liquid nitrogen by using a mortar and pestle and stored at 80 C. Total RNA was prepared from tissue samples (100 mg) with the TRIzol reagent (Invitrogen Life Technologies, USA) according to the manufacturer's instructions. Total RNA (10 mg) was treated for 30 min at 37 C with 1 U of RQ1 RNase-free DNase (Promega, Madison, WI, USA). Reverse transcription of RNA (1 mg) was performed in a 20-mL reaction volume with oligo-dT18 and ImProm-II reverse transcriptase (Promega, Madison, WI, USA). The reactions were incubated at 25 C for 5 min, 42 C for 60 min, and 70 C for 10 min to inactivate the reverse transcriptase. cDNA was sequenced with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Sequences were analyzed on an ABI Prism 3730 genetic analyzer (Applied Biosystems, Foster City, CA, USA), and sequence data were analyzed by using the Lasergene software (DNAStar Inc., Madison, WI, USA). The nucleotide and deduced amino acid sequences encoded by the hydrophobin genes were aligned by using the BioEdit program (http://www. mbio.ncsu.edu/bioedit/bioedit.html). The GenBank accession numbers of the sequences reported herein are KT868833 (Hyd1), KT868834 (Hyd-2), KT868835 (Hyd-3), KT868836 (Hyd-4), KT868837 (Hyd-5), KT868838 (Hyd-6), KT868839 (Hyd-7), KT868840 (Hyd-8), KT868841 (Hyd-9), and KT868842 (Hyd-10). Comparisons of the genomic and cDNA sequences of these genes revealed single ORFs of 363 bp, 315 bp, 315 bp, 366 bp, 336 bp, 345 bp, 336 bp, 384 bp, 393 bp, and 354 bp, respectively. In addition, comparisons of genomic DNA and cDNA showed that the genomic DNA of these genes had 3, 4, 4, 4, 4, 4, 4, 5, 5, and 4 introns, respectively, with an average intron size of 55 bp. All of the splicing sites followed the GTeAG rule (Supplementary Fig. S1). The signal peptide prediction of amino acid sequences encoded by hydrophobin genes in F. velutipes was carried out by using the SignalP 4.1 server (http://www.cbs.dtu.dk/ services/SignalP/). N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified with the NetNGlyc 1.0 server (http://www.cbs. dtu.dk/services/NetNGlyc/). Supplementary Fig. S2 shows the deduced amino acid sequences encoded by the 10 ORFs aligned with previously reported fungal hydrophobins (POH2 from Pleurotus ostreatus [GenBank AJ225061], HypB from A. bisporus [GenBank Y15941], Le-Hyd2 from Lentinula edodes [GenBank AF217808], SC3 from S. commune [GenBank XM_003031216], CoH1 from C. cinerea [GenBank XM_001834396], and fvh1 from F. velutipes [GenBank AB026721]). Hydrophobins are composed of four loops, formed by disulfide bonds, among eight highly conserved cysteine residues (Ando et al. 2001; Yamada et al. 2005). Among the 13 putative hydrophobin genes identified in F. velutipes, 10 genes (Hyd-1e10) were determined to encode the fungal
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Table 1 e Characteristics of predicted hydrophobin genes in Flammulina velutipes genome. Gene
Hyd-1 Hyd-2 Hyd-3 Hyd-4 Hyd-5 Hyd-6 Hyd-7 Hyd-8 Hyd-9 Hyd-10
Size Amino acids
gDNA
121 105 105 122 112 115 112 128 131 118
538 532 539 590 557 554 570 668 667 568
Distribution of exon
Potential glycosylation site
Predicted signal peptide residues (amino acid)
Accession no. (NCBI)
4 5 5 5 5 5 5 6 6 5
Asn-46 N.D. N.D. N.D. N.D. N.D. N.D. Asn-28, Asn-50 Asn-31, Asn-53 N.D.
21 20 20 20 17 17 23 20 23 20
KT868833 KT868834 KT868835 KT868836 KT868837 KT868838 KT868839 KT868840 KT868841 KT868842
N.D.: not detected; Asn: asparagine.
hydrophobin characteristics described above (Supplementary Fig. S2). Coding for eight cysteine residues for disulfide bridges was completely conserved in 10 hydrophobin genes from F. velutipes, as observed in other fungi (Supplementary Fig. S2). Coding for two potential N-glycosylation sites (Asn-Xaa-Ser/ Thr) was identified in Hyd-8 (Asn-28 and Asn-50) and Hyd-9 (Asn-31 and Asn-53), and one site was encoded in Hyd-1 (Asn46). Although Hyd-1 was predicted to encode a glycosylation site, the protein does not appear to be glycosylated. SC3 and POH2 are the only hydrophobins confirmed to be glycosylated (probably O-glycosylated) and to have a long threonine-rich ttir et al. region before their first cysteine residues (Asgeirsd o 1998; De Vocht et al. 1998). These stretches are encoded in Hyd-8 (Asn-28 and Asn-50) and Hyd-9 (Asn-31 and Asn-53; see Supplementary Fig. S2). Therefore, further studies are needed to evaluate the glycosylation of Hyd-8 and Hyd-9 proteins. The first 21, 20, 20, 20, 17, 17, 23, 20, 23, and 20 residues likely compose signal peptides in Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10, respectively. This characteristic is typical of extracellular enzymesdi.e., they consist of a positively charged amino terminus, a hydrophobic stretch, and small amino acid residues (Von Heijine 1985). These residues indicate that Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10 encode mature hydrophobins consisting of 100, 85, 85, 102, 95, 98, 89, 108, 108, and 98 amino acid residues, respectively (Supplementary Fig. S2). However, the amino acid sequences encoded by the other two putative hydrophobin genes did not fully contain either the eight highly conserved cysteine residues or the signal peptide cleavage site (data not shown). Moreover, Hyd-5 gene was predicted as two different hydrophobin genes in previous study (Park et al. 2014). The deduced amino acid sequence of Hyd-1 from F. velutipes showed absolute homology (100% identical) with that of Fv-hyd1 (GenBank AB126686) from Japanese F. velutipes strain MH092086 (Supplementary Fig. S2). Comparisons of deduced amino acid sequences revealed that Korean and Japanese F. velutipes strains have the same hydrophobin gene in their genomes. A hydropathy plot of the 10 hydrophobin genes highlighted a hydrophobic N-terminus, which could represent a signal sequence, and hydrophobic domains following the first cysteine residues. This feature could be observed in other hydrophobins such as SC3, CoH1, HypB, Le-Hyd2, and POH2 (Supplementary Fig. S3).
Developmentally regulated expression of hydrophobin genes in F. velutipes According to the results of a previous study, genes coding for hydrophobins are specifically regulated during the life cycle of basidiomycetes. Some of these genes are highly expressed at a specific developmental stagedi.e., the mature fruit body stage (Wessels 1997). To evaluate the specific expression pattern of the 10 hydrophobin genes identified in F. velutipes, we performed quantitative real-time (qPCR) analysis with total RNA harvested and isolated from cultures at three developmental stages (mycelia, primordia, and mature fruiting bodies) of F. velutipes. Mature fruiting bodies were subdivided into the two major tissues (stipe and pileus). For RNA isolation from primordia, budding (young fruiting bodies 2e5 mm in length) that appeared after 10 d of physical stimulation (induction) was collected. The sequence of each gene was obtained from the F. velutipes whole-genome sequence (AQHU00000000; NCBI) and used to design primers with Integrated DNA Technologies (https://eu.idtdna.com/Primerquest/Home/Index). RNA samples from three independent replicates were treated with DNase before cDNA synthesis. qPCR analysis was performed by using a RotorGene 6000 (Qiagen, Seoul, Korea) with 25-mL reaction mixtures containing 12.5 mL of a SensiFAST SYBR No-ROX kit (Bioline, Alexandria, Australia), 10 pM of each primer (Supplementary Table S1), and 25 ng cDNA template. The following reaction conditions were used: 3 min at 95 C followed by 40 cycles of 95 C for 5 s, 60 C for 10 s, and 72 C for 15 s. For normalization in the qPCR analysis, expression of the F. velutipes glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH; XM001834107) was used as an internal reference. The relative expression level of each gene was defined as DCt ¼ Cttarget CtGAPDH, which represented the difference between the transcript abundance of genes examined and the transcript abundance of GAPDH. Among the 10 hydrophobin genes, the deduced amino acid sequence of Hyd-1 showed absolute homology (100% identical) with that of a previously reported hydrophobin gene (Fv-hyd1) from a Japanese F. velutipes strain (Yamada et al. 2005). In addition, the fvh1 and Fv-hyd1 hydrophobin genes of F. velutipes were specifically expressed in the vegetative mycelium immediately after the induction of fruiting (1 d after
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induction) and after the pre-mature stage (primordium and mature fruiting bodies) (Ando et al. 2001; Yamada et al. 2005). As shown in Fig. 1, qPCR analysis showed that the hydrophobin genes were specifically expressed at different stages of F. velutipes development, and all of the genes were expressed at relatively higher levels during the primordial stages. By contrast, most of these genes were expressed at a relatively low levels at the mycelial stage of F. velutipes. Among the 10 hydrophobin genes, Hyd-1 showed the highest expression level in the fruit body during the primordial stage. This finding was consistent with the results of our previous study (Park et al. 2014). In accordance with the results of another previous report (Yamada et al. 2005), Fv-hyd1 was not expressed during the initial stage of fruiting but was expressed at the pre-mature stage and in mature fruiting bodies. Similarly, the transcription of Hyd-1 at the mycelial stage was lower than that in fruiting bodies, including both the pileus and the stipe. Moreover, qPCR results demonstrated tissue-restricted expression patterns for Hyd-1: the gene was expressed at higher levels in stipe than in pileus. Ando et al. (2001) also reported that fvh1 is highly expressed in vegetative mycelia 1 d after induction and during the initial stage of fruiting but is not expressed in mature fruiting bodies. The
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expression levels of fvh1 decrease during fruit body development. The expression of nine of the F. velutipes hydrophobin genes (Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10), which was reduced in fruiting bodies, showed patterns similar to that of fvh1. Thus, our results suggest that the transcription of the studied hydrophobin genes was regulated developmentally or tissue-specifically in F. velutipes during mushroom formation. The expression patterns of Hyd-2 and Hyd-3 were not fully evaluated by qPCR analysis because only one nucleotide substitution (A 4 G) was observed between these genes at position 118 in the cDNA sequence. Furthermore, in a previous study, we also identified two hydrophobin homologues (FvHyd1 and Fv-Hyd2) in an F. velutipes (ASI4019) cDNA library (Joh et al. 2009). These predicted hydrophobin genes were abundantly represented by redundancy analysis and specifically expressed at different development stages. The cDNA sequences of Fv-Hyd2 and Fv-Hyd1 corresponded with the F. velutipes cDNAs of fvh1 and Fv-hyd1, respectively (Ando et al. 2001; Yamada et al. 2005). However, the deduced amino acid sequences of nine of the hydrophobin genes (Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10) shared sequence similarity with fvh1, with identities in the range of
Fig. 1 e Expression patterns of 10 hydrophobin genes of Flammulina velutipes. Total RNAs were isolated from the mycelial, primordial stage and from the pileus and stipe of F. velutipes. The gene expression levels (arbitrary units) of these genes were normalized with GAPDH as an internal reference. Gene expression levels were quantified with quantitative real-time PCR.
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66.7e75.3%. In addition, the deduced amino acid sequence of Hyd-1 (Fv-hyd1) showed low similarity to that of fvh1 (only 48.8% identity), although the eight cysteine residues characteristic of hydrophobins were completely conserved in both sequences. This result indicates that the fvh1 homologue does not exist in the genome of the F. velutipes KACC42780 monokaryotic strain (AQHU00000000). Most basidiomycete species have multiple hydrophobin genes. For example, Van Wetter et al. (2000a,b) isolated four hydrophobin genes (SC1, SC3, SC4, SC6) from S. commune. The expression of multiple hydrophobin genes is developmentally regulated, as reported for S. commune (Dons et al. 1984), A. ttir bisporus (De Groot et al. 1999), and P. ostreatus (Asgeirsd o et al. 1998; Penas et al. 1998). The difference in expression patterns in various developmental stages and tissues in F. velutipes clearly implies a high level of specification of these genes during mushroom formation. In addition, the fruiting body-specific hydrophobin SC4 of S. commune localizes on the surface of passages (air channels) in fruiting body tissues, which indicates that the hydrophobicity of hydrophobins is key in keeping water away from the channel (Lugones et al. 1999). The promoter regions of these genes had putative TATA boxes (TATAA) located (at positions from 75 to 101) upstream of their start codons (Supplementary Fig. S4). The distance between the TATA box and the start codon is highly conserved in hydrophobin genes of basidiomycetesde.g., 103 bp in S. commune SC3 (Schuren and Wessels 1990) and 105 bp in A. bisporus abh1 (De Groot et al. 1996). CT-rich motifs, which are often found immediately upstream of the transcription start point of highly expressed filamentous fungal genes (Gurr et al. 1987), were also present at the expected positions in these genes. In addition, one or two additional CTrich regions were found upstream of the TATA boxes in the promoter regions of Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5, and Hyd-6 (Supplementary Fig. S4). Gurr et al. (1987) reported that CT-rich motifs are often found immediately upstream of the transcription start points of abundantly transcribed genes in Neurospora crassa and Aspergillus nidulans. Analysis of the genomic structure showed that Fv-hyd1 and fvh1 have CT-rich motifs both upstream and downstream of the TATA box (Ando et al. 2001; Yamada et al. 2005). SC3 and SC4 hydrophobin genes in S. commune also have CT-rich motifs immediately upstream of their transcription start points. In addition to having a CT-rich motif, SC4 accumulates in abundance in dikaryons (Mulder and Wessels 1986; Schuren and Wessels 1990). Similarly, five (Hyd-2, Hyd-3, Hyd-4, Hyd-5, and Hyd-6) of the 10 identified F. velutipes hydrophobin genes have CTrich motifs both upstream and downstream of the TATA box (Supplementary Fig. S4). However, four (Hyd-7, Hyd-8, Hyd-9, and Hyd-10) of the 10 hydrophobin genes have only one CTrich motif immediately upstream of their transcription start points. CT-rich motifs in the promoter region of these genes may be recognized by some other transcription-activating factors given the finding that the transcription of these genes was highly up-regulated in specific developmental stages or tissues in F. velutipes. In this genome-wide study of F. velutipes, we identified hydrophobin genes and further investigated their genomic structure and expression levels during fruit body formation. In
our previous study (Park et al. 2014), 12 predicted proteins sharing sequence similarity with fungal hydrophobin were identified in the F. velutipes genome by using the Fgenesh, Fgeneshþ, and cufflinks tools simultaneously. However, in the present study, 10 hydrophobin genes were predicted when we used the AUGUSTUS tool with the genome sequence of F. velutipes. The two separate approaches to hydrophobin gene identification in the F. velutipes genome identified 10 genes in common. Although additional analyses, including purification and localization, of these novel hydrophobins are required, the results of this study are valuable for understanding the molecular basis of mushroom formation and regulation of developmental stages in F. velutipes.
Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of Republic of Korea.
Acknowledgments We are sincerely grateful to the anonymous referees whose comments made our paper more accessible to the readers. The authors are grateful for comments and suggestions provided by Dr. Won-Sik Kong.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.myc.2016.04.004.
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Plunkett BE, 1956. The influence of factors of the aeration complex and light upon fruit-body form in pure cultures of an agaric and a polypore. Annals of Botany 20: 563e586. Psurtseva N, 2005. Modern taxonomy and medicinal value of the Flammulina mushrooms. The International Journal of Medicinal Mushrooms 7: 449e451; http://dx.doi.org/10.1615/ IntJMedMushrooms.v7.i3.870. re J, 1991. Isolation of transcripts preferentially Salvado JC, Labare expressed during fruit body primordial differentiation in the basidiomycete Agrocybe aegerita. Current Genetics 20: 205e210; http://dx.doi.org/10.1007/BF00326234. Schuren FHJ, Wessels JGH, 1990. Two genes specifically expressed in fruiting dikaryons of Schizophyllum commune: homologies with a gene not regulated by mating-type genes. Gene 90: 199e205; http://dx.doi.org/10.1016/0378-1119(90)90180-Y. Stanke M, Morgenstern B, 2005. AUGUSTUS: a web server for gene prediction in eukaryotes that allows user-defined constraints. Nucleic Acids Research 33: W465eW467; http://dx.doi.org/ 10.1093/nar/gki458. Terashita T, Ueda M, Yoshikawa K, Kono M, Shishiyama J, 1992. Changes in chitin and N-acetylglucosamine contents and chitolytic enzyme activities during the growth of Flammulina velutipes fruit bodies. Journal of The Japanese Society of Food Science and Technology 39: 827e835; http://dx.doi.org/10.3136/ nskkk1962.39.827. Van Wetter MA, Wosten HAB, Sietsma JH, Wessels JGH, 2000b. Hydrophobin gene expression affects hyphal wall composition in Schizophyllum commune. Fungal Genetics and Biology 31: 99e104; http://dx.doi.org/10.1006/fgbi.2000.1231. Van Wetter MA, Wosten HAB, Wessels JGH, 2000a. SC3 and SC4 hydrophobins have distinct roles in formation of aerial structures in dikaryons of Schizophyllum commune. Molecular Microbiology 36: 201e210; http://dx.doi.org/10.1046/j.1365-2958.2000.01848.x. Von Heijine G, 1985. Signal sequences. The limits of variation. Journal of Molecular Biology 184: 99e105; http://dx.doi.org/ 10.1016/0022-2836(85)90046-4. Wessels JGH, 1997. Hydrophobins: proteins that change the nature of the fungal surface. Advances in Microbial Physiology 38: 1e45; http://dx.doi.org/10.1016/S0065-2911(08)60154-X. Williams MAJ, Beckett A, Read ND, 1985. Ultrasturctural aspects of fruit body differentiation in Flammulina velutipes. In: Moore D, Casselton LA, Wood DA, Frankland JC (eds), Developmental biology of higher fungi. Cambridge Unversity Press, Cambridge, pp 429e450. € sten HAB, Wessels JGH, 1997. Hydrophobin, from molecular Wo structure to multiple functions in fungal development. Mycoscience 38: 363e374; http://dx.doi.org/10.1007/ BF2464099. Yamada M, Sakuraba S, Shibata K, Inatomi S, Okazaki M, Shimosaka M, 2005. Cloning and characterization of a gene coding for a hydrophobin, Fv-hyd1, specifically expressed during fruiting body development in the basidiomycete Flammulina velutipes. Applied Microbiology and Biotechnology 67: 240e246; http://dx.doi.org/10.1007/s00253-004-1776-2.
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Further characterization of hydrophobin genes in genome of Flammulina velutipes Hong-Il Kim, Chang-Soo Lee, Young-Jin Park* Department of Biomedical Chemistry, Konkuk University, Chungju 27478, Republic of Korea
article info
abstract
Article history:
This study aimed to identify and further characterize hydrophobin genes in the whole-
Received 29 December 2015
genome sequence of Flammulina velutipes. Ten genes (Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5,
Received in revised form
Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10) sharing eight conserved cysteine residues were
25 April 2016
identified from 13 previously predicted putative hydrophobin genes in the F. velutipes
Accepted 29 April 2016
genome. Quantitative real-time PCR (qPCR) analysis showed that these genes were spe-
Available online 11 June 2016
cifically expressed in different developmental stages and tissues. Whereas all of the genes showed relatively higher levels of expression in the primordial stages of the fungus, most
Keywords: Fruit body development
of them were expressed at relatively low levels in the mycelial stage. © 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
Pileus Stipe Winter mushroom
Flammulina velutipes, also known as winter mushroom or Enokitake, is a common white-rot basidiomycete distributed throughout temperate zones (Hughes et al. 1999). It is one of the six most actively cultivated mushroom species in the world (Psurtseva 2005). In the commercial production of mushrooms, the development of fruiting bodies is induced by controlling environmental factors such as temperature, light and humidity, as well as physical and chemical stimuli. Extensive reports on F. velutipes fruit body formation are available (Plunkett 1956; Kitamoto and Gruen 1976; Williams et al. 1985; Terashita et al. 1992) because this fungus readily forms fruit bodies under low-temperature and low-light conditions. Several studies have investigated the molecular basis of fruit body development in this fungus (Kim and Azuma 1999; Kim et al. 1999; Ando et al. 2001; Yamada et al. 2005). We recently determined the whole-genome sequence of F.
velutipes (Park et al. 2014). Therefore, genome-wide identification of the genes involved in the initiation of fruiting can now be studied to understand this process at the molecular level and develop efficient and reproducible methods of cultivating and inducing fruit body development in valuable basidiomycetes species. Several fruiting-specific or fruiting induction genes have been isolated in other basidiomycetes, such as Agaricus bisporus (De Groot et al. 1996), Schizophyllum commune (Mulder and re Wessels 1986) and Agrocybe aegerita (Salvado and Labare 1991). Some of these fungal genes belong to a family that encodes hydrophobic proteins called hydrophobins. In addition, two fruiting-specific or fruiting induction genes (fvh1 and Fvhyd1) encoding hydrophobins were isolated from a F. velutipes primordial complementary DNA (cDNA) library (Ando et al. 2001; Yamada et al. 2005).
* Corresponding author. Tel./fax: þ82 438403601. E-mail address: [email protected] (Y.-J. Park). http://dx.doi.org/10.1016/j.myc.2016.04.004 1340-3540/© 2016 The Mycological Society of Japan. Published by Elsevier B.V. All rights reserved.
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Hydrophobins are small, secretory, hydrophobic fungusspecific proteins composed of four loops of disulfide bonds among eight highly conserved cysteine residues that selfassemble into amphipathic layers as hydrophilicehydrophobic interfaces (Ando et al. 2001; Yamada et al. 2005). Fungal strains usually contain several genes coding for hydrophobins, which play key roles in morphogenesis and fruit body development. Some of these hydrophobin genes are € sten and Wessels also highly expressed in fruiting bodies (Wo 1997). The aim of the present study was to further identify and characterize hydrophobin genes in F. velutipes by using our recently published whole-genome sequence information (Park et al. 2014). Herein, we describe 10 F. velutipes hydrophobin genes with deduced amino acid sequences and their regulation at the transcription level during fruiting body development. This comparative study of gene expression at various developmental stages and in different tissues provides valuable insights into the molecular basis of mushroom fruit body development.
Hydrophobin genes in the F. velutipes genome The F. velutipes KACC43778 dikaryotic strain was obtained from the Korean Agricultural Culture Collection (KACC; Rural Development Administration, Korea; http://www.genebank. go.kr/) and grown at 26 C on mushroom complete medium agar (0.2% peptone, 2% glucose, 0.2% yeast extract, 0.05% MgSO4, 0.046% KH2PO4, 0.1% K2HPO4, and 1.5% agar) for 2 wk. For genomic DNA and total RNA isolation from mycelia, a 300mL Erlenmeyer flask containing 50 mL mushroom complete medium was inoculated with fresh plugs from the plate (five mycelial plugs/flask) and incubated at 26 C for 2 wk without agitation. In our previous study, genome-wide identification of hydrophobin genes was carried out using several methods, including ab initio gene structure prediction (Fgenesh; http:// www.softberry.com), a homology-based approach (Fgeneshþ; http://www.softberry.com), and transcriptomebased gene identification (cufflinks; http://cufflinks.cbcb. umd.edu/manual.html) with the F. velutipes (KACC42780 monokaryotic strain) whole-genome sequence (AQHU00000000) (Park et al. 2014). In the present study, we performed additional gene prediction by using the AUGUSTUS tool (Stanke and Morgenstern 2005) with default parameters trained in Coprinopsis cinerea. The genes were compared by using BLAST (version 2.2.17) software with a series of protein databases, including the National Center for Biotechnology Information (NCBI) nucleotide (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) and non-redundant (http://blast.ncbi.nlm.nih.gov/ Blast.cgi) sets, for functional annotation of the predicted genes. BLASTP searches within the NCBI-NR database of the amino acid sequences of F. velutipes genes, which were previously predicted by using combined approaches (Fgenesh, Fgeneshþ, and cufflinks), showed that 12 of the predicted proteins shared sequence similarity with fungal hydrophobins (Park et al. 2014). However, gene prediction performed with the AUGUSTUS tool indicated that the total number of hydrophobin genes in F. velutipes was smaller (10
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hydrophobin) than that predicted through combined approaches (Table 1). Nine putative hydrophobin genes were found in common among the genes predicted by both the combined approaches and the AUGUSTUS tool. Furthermore, one (Hyd-10) of these 10 putative genes was further identified and annotated as a hydrophobin gene in this study (Table 1). cDNA fragments encompassing all of the putative open reading frames (ORFs) were amplified with reverse transcriptase (RT)-PCR and their sequences were refined. For cDNA synthesis, samples were ground to a fine powder under liquid nitrogen by using a mortar and pestle and stored at 80 C. Total RNA was prepared from tissue samples (100 mg) with the TRIzol reagent (Invitrogen Life Technologies, USA) according to the manufacturer's instructions. Total RNA (10 mg) was treated for 30 min at 37 C with 1 U of RQ1 RNase-free DNase (Promega, Madison, WI, USA). Reverse transcription of RNA (1 mg) was performed in a 20-mL reaction volume with oligo-dT18 and ImProm-II reverse transcriptase (Promega, Madison, WI, USA). The reactions were incubated at 25 C for 5 min, 42 C for 60 min, and 70 C for 10 min to inactivate the reverse transcriptase. cDNA was sequenced with a BigDye Terminator Cycle Sequencing Kit (Applied Biosystems, Foster City, CA, USA) according to the manufacturer's instructions. Sequences were analyzed on an ABI Prism 3730 genetic analyzer (Applied Biosystems, Foster City, CA, USA), and sequence data were analyzed by using the Lasergene software (DNAStar Inc., Madison, WI, USA). The nucleotide and deduced amino acid sequences encoded by the hydrophobin genes were aligned by using the BioEdit program (http://www. mbio.ncsu.edu/bioedit/bioedit.html). The GenBank accession numbers of the sequences reported herein are KT868833 (Hyd1), KT868834 (Hyd-2), KT868835 (Hyd-3), KT868836 (Hyd-4), KT868837 (Hyd-5), KT868838 (Hyd-6), KT868839 (Hyd-7), KT868840 (Hyd-8), KT868841 (Hyd-9), and KT868842 (Hyd-10). Comparisons of the genomic and cDNA sequences of these genes revealed single ORFs of 363 bp, 315 bp, 315 bp, 366 bp, 336 bp, 345 bp, 336 bp, 384 bp, 393 bp, and 354 bp, respectively. In addition, comparisons of genomic DNA and cDNA showed that the genomic DNA of these genes had 3, 4, 4, 4, 4, 4, 4, 5, 5, and 4 introns, respectively, with an average intron size of 55 bp. All of the splicing sites followed the GTeAG rule (Supplementary Fig. S1). The signal peptide prediction of amino acid sequences encoded by hydrophobin genes in F. velutipes was carried out by using the SignalP 4.1 server (http://www.cbs.dtu.dk/ services/SignalP/). N-glycosylation sites (Asn-Xaa-Ser/Thr) were identified with the NetNGlyc 1.0 server (http://www.cbs. dtu.dk/services/NetNGlyc/). Supplementary Fig. S2 shows the deduced amino acid sequences encoded by the 10 ORFs aligned with previously reported fungal hydrophobins (POH2 from Pleurotus ostreatus [GenBank AJ225061], HypB from A. bisporus [GenBank Y15941], Le-Hyd2 from Lentinula edodes [GenBank AF217808], SC3 from S. commune [GenBank XM_003031216], CoH1 from C. cinerea [GenBank XM_001834396], and fvh1 from F. velutipes [GenBank AB026721]). Hydrophobins are composed of four loops, formed by disulfide bonds, among eight highly conserved cysteine residues (Ando et al. 2001; Yamada et al. 2005). Among the 13 putative hydrophobin genes identified in F. velutipes, 10 genes (Hyd-1e10) were determined to encode the fungal
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Table 1 e Characteristics of predicted hydrophobin genes in Flammulina velutipes genome. Gene
Hyd-1 Hyd-2 Hyd-3 Hyd-4 Hyd-5 Hyd-6 Hyd-7 Hyd-8 Hyd-9 Hyd-10
Size Amino acids
gDNA
121 105 105 122 112 115 112 128 131 118
538 532 539 590 557 554 570 668 667 568
Distribution of exon
Potential glycosylation site
Predicted signal peptide residues (amino acid)
Accession no. (NCBI)
4 5 5 5 5 5 5 6 6 5
Asn-46 N.D. N.D. N.D. N.D. N.D. N.D. Asn-28, Asn-50 Asn-31, Asn-53 N.D.
21 20 20 20 17 17 23 20 23 20
KT868833 KT868834 KT868835 KT868836 KT868837 KT868838 KT868839 KT868840 KT868841 KT868842
N.D.: not detected; Asn: asparagine.
hydrophobin characteristics described above (Supplementary Fig. S2). Coding for eight cysteine residues for disulfide bridges was completely conserved in 10 hydrophobin genes from F. velutipes, as observed in other fungi (Supplementary Fig. S2). Coding for two potential N-glycosylation sites (Asn-Xaa-Ser/ Thr) was identified in Hyd-8 (Asn-28 and Asn-50) and Hyd-9 (Asn-31 and Asn-53), and one site was encoded in Hyd-1 (Asn46). Although Hyd-1 was predicted to encode a glycosylation site, the protein does not appear to be glycosylated. SC3 and POH2 are the only hydrophobins confirmed to be glycosylated (probably O-glycosylated) and to have a long threonine-rich ttir et al. region before their first cysteine residues (Asgeirsd o 1998; De Vocht et al. 1998). These stretches are encoded in Hyd-8 (Asn-28 and Asn-50) and Hyd-9 (Asn-31 and Asn-53; see Supplementary Fig. S2). Therefore, further studies are needed to evaluate the glycosylation of Hyd-8 and Hyd-9 proteins. The first 21, 20, 20, 20, 17, 17, 23, 20, 23, and 20 residues likely compose signal peptides in Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10, respectively. This characteristic is typical of extracellular enzymesdi.e., they consist of a positively charged amino terminus, a hydrophobic stretch, and small amino acid residues (Von Heijine 1985). These residues indicate that Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10 encode mature hydrophobins consisting of 100, 85, 85, 102, 95, 98, 89, 108, 108, and 98 amino acid residues, respectively (Supplementary Fig. S2). However, the amino acid sequences encoded by the other two putative hydrophobin genes did not fully contain either the eight highly conserved cysteine residues or the signal peptide cleavage site (data not shown). Moreover, Hyd-5 gene was predicted as two different hydrophobin genes in previous study (Park et al. 2014). The deduced amino acid sequence of Hyd-1 from F. velutipes showed absolute homology (100% identical) with that of Fv-hyd1 (GenBank AB126686) from Japanese F. velutipes strain MH092086 (Supplementary Fig. S2). Comparisons of deduced amino acid sequences revealed that Korean and Japanese F. velutipes strains have the same hydrophobin gene in their genomes. A hydropathy plot of the 10 hydrophobin genes highlighted a hydrophobic N-terminus, which could represent a signal sequence, and hydrophobic domains following the first cysteine residues. This feature could be observed in other hydrophobins such as SC3, CoH1, HypB, Le-Hyd2, and POH2 (Supplementary Fig. S3).
Developmentally regulated expression of hydrophobin genes in F. velutipes According to the results of a previous study, genes coding for hydrophobins are specifically regulated during the life cycle of basidiomycetes. Some of these genes are highly expressed at a specific developmental stagedi.e., the mature fruit body stage (Wessels 1997). To evaluate the specific expression pattern of the 10 hydrophobin genes identified in F. velutipes, we performed quantitative real-time (qPCR) analysis with total RNA harvested and isolated from cultures at three developmental stages (mycelia, primordia, and mature fruiting bodies) of F. velutipes. Mature fruiting bodies were subdivided into the two major tissues (stipe and pileus). For RNA isolation from primordia, budding (young fruiting bodies 2e5 mm in length) that appeared after 10 d of physical stimulation (induction) was collected. The sequence of each gene was obtained from the F. velutipes whole-genome sequence (AQHU00000000; NCBI) and used to design primers with Integrated DNA Technologies (https://eu.idtdna.com/Primerquest/Home/Index). RNA samples from three independent replicates were treated with DNase before cDNA synthesis. qPCR analysis was performed by using a RotorGene 6000 (Qiagen, Seoul, Korea) with 25-mL reaction mixtures containing 12.5 mL of a SensiFAST SYBR No-ROX kit (Bioline, Alexandria, Australia), 10 pM of each primer (Supplementary Table S1), and 25 ng cDNA template. The following reaction conditions were used: 3 min at 95 C followed by 40 cycles of 95 C for 5 s, 60 C for 10 s, and 72 C for 15 s. For normalization in the qPCR analysis, expression of the F. velutipes glyceraldehyde-3-phosphate dehydrogenase gene (GAPDH; XM001834107) was used as an internal reference. The relative expression level of each gene was defined as DCt ¼ Cttarget CtGAPDH, which represented the difference between the transcript abundance of genes examined and the transcript abundance of GAPDH. Among the 10 hydrophobin genes, the deduced amino acid sequence of Hyd-1 showed absolute homology (100% identical) with that of a previously reported hydrophobin gene (Fv-hyd1) from a Japanese F. velutipes strain (Yamada et al. 2005). In addition, the fvh1 and Fv-hyd1 hydrophobin genes of F. velutipes were specifically expressed in the vegetative mycelium immediately after the induction of fruiting (1 d after
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induction) and after the pre-mature stage (primordium and mature fruiting bodies) (Ando et al. 2001; Yamada et al. 2005). As shown in Fig. 1, qPCR analysis showed that the hydrophobin genes were specifically expressed at different stages of F. velutipes development, and all of the genes were expressed at relatively higher levels during the primordial stages. By contrast, most of these genes were expressed at a relatively low levels at the mycelial stage of F. velutipes. Among the 10 hydrophobin genes, Hyd-1 showed the highest expression level in the fruit body during the primordial stage. This finding was consistent with the results of our previous study (Park et al. 2014). In accordance with the results of another previous report (Yamada et al. 2005), Fv-hyd1 was not expressed during the initial stage of fruiting but was expressed at the pre-mature stage and in mature fruiting bodies. Similarly, the transcription of Hyd-1 at the mycelial stage was lower than that in fruiting bodies, including both the pileus and the stipe. Moreover, qPCR results demonstrated tissue-restricted expression patterns for Hyd-1: the gene was expressed at higher levels in stipe than in pileus. Ando et al. (2001) also reported that fvh1 is highly expressed in vegetative mycelia 1 d after induction and during the initial stage of fruiting but is not expressed in mature fruiting bodies. The
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expression levels of fvh1 decrease during fruit body development. The expression of nine of the F. velutipes hydrophobin genes (Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10), which was reduced in fruiting bodies, showed patterns similar to that of fvh1. Thus, our results suggest that the transcription of the studied hydrophobin genes was regulated developmentally or tissue-specifically in F. velutipes during mushroom formation. The expression patterns of Hyd-2 and Hyd-3 were not fully evaluated by qPCR analysis because only one nucleotide substitution (A 4 G) was observed between these genes at position 118 in the cDNA sequence. Furthermore, in a previous study, we also identified two hydrophobin homologues (FvHyd1 and Fv-Hyd2) in an F. velutipes (ASI4019) cDNA library (Joh et al. 2009). These predicted hydrophobin genes were abundantly represented by redundancy analysis and specifically expressed at different development stages. The cDNA sequences of Fv-Hyd2 and Fv-Hyd1 corresponded with the F. velutipes cDNAs of fvh1 and Fv-hyd1, respectively (Ando et al. 2001; Yamada et al. 2005). However, the deduced amino acid sequences of nine of the hydrophobin genes (Hyd-2, Hyd-3, Hyd-4, Hyd-5, Hyd-6, Hyd-7, Hyd-8, Hyd-9, and Hyd-10) shared sequence similarity with fvh1, with identities in the range of
Fig. 1 e Expression patterns of 10 hydrophobin genes of Flammulina velutipes. Total RNAs were isolated from the mycelial, primordial stage and from the pileus and stipe of F. velutipes. The gene expression levels (arbitrary units) of these genes were normalized with GAPDH as an internal reference. Gene expression levels were quantified with quantitative real-time PCR.
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66.7e75.3%. In addition, the deduced amino acid sequence of Hyd-1 (Fv-hyd1) showed low similarity to that of fvh1 (only 48.8% identity), although the eight cysteine residues characteristic of hydrophobins were completely conserved in both sequences. This result indicates that the fvh1 homologue does not exist in the genome of the F. velutipes KACC42780 monokaryotic strain (AQHU00000000). Most basidiomycete species have multiple hydrophobin genes. For example, Van Wetter et al. (2000a,b) isolated four hydrophobin genes (SC1, SC3, SC4, SC6) from S. commune. The expression of multiple hydrophobin genes is developmentally regulated, as reported for S. commune (Dons et al. 1984), A. ttir bisporus (De Groot et al. 1999), and P. ostreatus (Asgeirsd o et al. 1998; Penas et al. 1998). The difference in expression patterns in various developmental stages and tissues in F. velutipes clearly implies a high level of specification of these genes during mushroom formation. In addition, the fruiting body-specific hydrophobin SC4 of S. commune localizes on the surface of passages (air channels) in fruiting body tissues, which indicates that the hydrophobicity of hydrophobins is key in keeping water away from the channel (Lugones et al. 1999). The promoter regions of these genes had putative TATA boxes (TATAA) located (at positions from 75 to 101) upstream of their start codons (Supplementary Fig. S4). The distance between the TATA box and the start codon is highly conserved in hydrophobin genes of basidiomycetesde.g., 103 bp in S. commune SC3 (Schuren and Wessels 1990) and 105 bp in A. bisporus abh1 (De Groot et al. 1996). CT-rich motifs, which are often found immediately upstream of the transcription start point of highly expressed filamentous fungal genes (Gurr et al. 1987), were also present at the expected positions in these genes. In addition, one or two additional CTrich regions were found upstream of the TATA boxes in the promoter regions of Hyd-1, Hyd-2, Hyd-3, Hyd-4, Hyd-5, and Hyd-6 (Supplementary Fig. S4). Gurr et al. (1987) reported that CT-rich motifs are often found immediately upstream of the transcription start points of abundantly transcribed genes in Neurospora crassa and Aspergillus nidulans. Analysis of the genomic structure showed that Fv-hyd1 and fvh1 have CT-rich motifs both upstream and downstream of the TATA box (Ando et al. 2001; Yamada et al. 2005). SC3 and SC4 hydrophobin genes in S. commune also have CT-rich motifs immediately upstream of their transcription start points. In addition to having a CT-rich motif, SC4 accumulates in abundance in dikaryons (Mulder and Wessels 1986; Schuren and Wessels 1990). Similarly, five (Hyd-2, Hyd-3, Hyd-4, Hyd-5, and Hyd-6) of the 10 identified F. velutipes hydrophobin genes have CTrich motifs both upstream and downstream of the TATA box (Supplementary Fig. S4). However, four (Hyd-7, Hyd-8, Hyd-9, and Hyd-10) of the 10 hydrophobin genes have only one CTrich motif immediately upstream of their transcription start points. CT-rich motifs in the promoter region of these genes may be recognized by some other transcription-activating factors given the finding that the transcription of these genes was highly up-regulated in specific developmental stages or tissues in F. velutipes. In this genome-wide study of F. velutipes, we identified hydrophobin genes and further investigated their genomic structure and expression levels during fruit body formation. In
our previous study (Park et al. 2014), 12 predicted proteins sharing sequence similarity with fungal hydrophobin were identified in the F. velutipes genome by using the Fgenesh, Fgeneshþ, and cufflinks tools simultaneously. However, in the present study, 10 hydrophobin genes were predicted when we used the AUGUSTUS tool with the genome sequence of F. velutipes. The two separate approaches to hydrophobin gene identification in the F. velutipes genome identified 10 genes in common. Although additional analyses, including purification and localization, of these novel hydrophobins are required, the results of this study are valuable for understanding the molecular basis of mushroom formation and regulation of developmental stages in F. velutipes.
Disclosure The authors declare no conflicts of interest. All the experiments undertaken in this study comply with the current laws of Republic of Korea.
Acknowledgments We are sincerely grateful to the anonymous referees whose comments made our paper more accessible to the readers. The authors are grateful for comments and suggestions provided by Dr. Won-Sik Kong.
Appendix A. Supplementary data Supplementary data related to this article can be found at http://dx.doi.org/10.1016/j.myc.2016.04.004.
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