J. Microbiol. Biotechnol. (2011), 21(4), 347–358 doi: 10.4014/jmb.1010.10048 First published online 15 January 2011

Analysis of Expressed Sequence Tags from the Wood-Decaying palustris and Identification of Potential Genes Involved in the Decay Process

Karim, Nurul1,2, Hajime Shibuya1, and Taisei Kikuchi1*

1Forestry and Forest Products Research Institute, Tsukuba, Ibaraki 305-8687, Japan 2Department of Biochemistry and Molecular Biology, Jahangirnagar University, Savar, Dhaka-1342, Bangladesh Received: October 22, 2010 / Revised: December 17, 2010 / Accepted: January 1, 2011

Fomitopsis palustris, a brown-rot basidiomycete, causes destructive decay of wooden structures. Whereas white-rot the most destructive type of decay in wooden structures. fungi degrade all components of plant cell walls including In spite of its great economic importance, very little , , and , the brown-rot fungi information is available at the molecular level regarding employ a different approach. Although they modify lignin its complex decay process. To address this, we generated extensively, the products remain in situ as a polymeric over 3,000 expressed sequence tags (ESTs) from a cDNA residue [31, 50]. Rapid depolymerization of cellulose is a library constructed from F. pa l u str is . Clustering of 3,095 distinguishing feature of brown-rot [24], but the biochemical high-quality ESTs resulted in a set of 1,403 putative unigenes mechanisms and underlying genetics of this process are comprising 485 contigs and 918 singlets. Homology searches poorly understood. based on BlastX analysis revealed that 78% of the F. Like other brown-rot basidiomycetes, F. palustris secretes palustris unigenes had a significant match to proteins an array of enzymes to hydrolyze cellulose and deposited in the nonredundant databases. A subset of F. of the wood [15, 17-20]. Several lines of evidence palustris unigenes showed similarity to the carbohydrate- have suggested that F. palustris produces a mixture of active enzymes (CAZymes), including a range of glycosyl capable of yielding soluble sugars from crystalline hydrolase (GH) family proteins. Some of these CAZyme- cellulose [52, 53, 55]. However, to date, molecular biological encoded genes were previously undescribed for F. palustris information regarding its cellulolytic system is scarce as but predicted to have potential roles in biodegradation of most of previous investigations have focused on the wood. Among them, we identified and characterized a induction, purification, and activity of extracellular crude gene (FpCel45A) encoding the GH family 45 endoglucanase. enzymes. The sequences of only a few of these enzymes Moreover, we also provided functional classification of have been cloned and characterized as the glycosyl 473 (34%) of F. palustris unigenes using the Gene Ontology hydrolase (GH) family of proteins [40, 42, 54] and found hierarchy. The annotated EST data sets and related analysis in the Carbohydrate-Active Enzymes (CAZymes) Web may be useful in providing an initial insight into the server (http://www.cazy.org) [14]. Systematic molecular- genetic background of F. palustris. based studies on F. palustris and its cellulolytic enzymes Keywords: ESTs, Fomitopsis palustris, brown-rot fungus, are greatly needed to achieve a better understanding of its GH family 45 endoglucanase, Gene Ontology complex biodegradative mechanism. Since the sequencing of the genome of Saccharomyces cerevisiae [11], the number of available fungal genome sequences has increased by an order of magnitude. Over 40 Brown-rot fungi, such as Fomitopsis palustris (formerly Tyromyces palustris complete fungal genomes have been publicly released, with called ) are common inhabitants of an equal number currently being sequenced, representing forest ecosystems and are also largely responsible for the the widest sampling of genomes from any eukaryotic kingdom [10]. The availability of genome data for the white- *Corresponding author Phanerochaete chrysosporium Phone: +81 29 873 3211; Fax: +81 29 873 1543; rot fungus has greatly E-mail: [email protected] facilitated the identification of the extracellular proteins 348 Karim et al. produced during cellulose degradation by the wood-rotting 1.0% peptone, 0.1% yeast extract, 0.75% (w/v) Buna Kraft pulp, fungi [28]. Furthermore, recently reported draft genome 0.01% Na2HPO4, 0.01% MgSO4, 0.03% CaCl2, 0.001% FeSO4·7H2O, information for the brown-rot fungus, Postia placenta, has 0.0001% MnSO4·4H2O, 0.0001% ZnSO4·7H2O, 0.0002% CuSO4·5 H2O, and 0.0002% CoCl2, in a 500-ml Erlenmeyer flask. The culture was provided unparalleled opportunities for investigating unique o mechanisms of lignocellulose conversion [29]. An unusual then incubated at 26 C for 7 days on a reciprocating shaker at 150 rpm. Mycelia were collected by filtration, washed with sterile repertoire of GHs, with a few conventional cellulases, has o water, and kept at -80 C until RNA extraction. been identified in this fungus, and such resources should aid the discovery of similar genes in other brown-rot fungi. Isolation of Total RNA, cDNA Synthesis, and cDNA Library Consistent with the fact that two different types of wood- Construction rotting fungi (i.e., white-rot and brown-rot) contain a large For RNA isolation, the mycelia were ground into powder under number of CAZyme-encoding genes [28, 29], the classical liquid nitrogen with a pestle and mortar. Total RNA was isolated cellulases and hemicellulases described so far in F. palustris using Sepazol I super and the CTAB treatment according to the [15, 17-20, 40, 42, 55] may represent a small fraction of manufacturer’s instructions (Nacalai Tesque). Analysis of the total RNA the potential genes involved in the degradation of plant on a denaturing agarose gel showed a smear from 50 to 3,000 bp with cell wall in this organism. Investigations two distinct bands of ribosomal RNA. The RNA quality was also at the molecular level could be helpful in identifying more confirmed by evaluating the RNA quality indicator (RQI) using an CAZyme-encoding genes in F. palustris, potentially contributing Experion Biorad system. The RQI was 8.7, indicating that high- quality total RNA was used for construction of the cDNA library. towards our understanding of its decay process. Poly(A)+ RNA was extracted from total RNA using FastTrack Although whole genome sequencing provides the most MAG micro mRNA Isolation kits (Invitrogen). cDNA was synthesized abundant information on an organism’s genetic makeup, using the SMART PCR cDNA amplification method (CLONTECH, the process is both expensive and time-consuming [36]. As Palo Alto, CA, USA) with a NotI oligo-dT primer (5'-AACTGGAA an alternative, expressed sequence tags (ESTs), which are GAATTCGCGGCCGCAGGAATTTTTTTTTTTTTTTTTT-3'). The generated by one-shot random sequencing of a cDNA SalI/SmaI adaptors (TAKARA) were added to double-stranded library, are used to obtain essential functional information cDNA, which was digested with NotI, and size fractionated using a about the genome. ESTs, because of their derivation from cDNA Size Fractionation Column (Invitrogen). The cDNA-containing mRNAs, can provide an efficient and powerful molecular fractions were pooled and ethanol precipitated. Inserts were Not Sal tool for discovering novel genes and generating information directionally cloned into I and I sites of the pSPORT1 vector, Escherichia coli α about their expression in organisms with no history of and transformed into DH5 cells. genetic research [41]. The application of EST analysis to EST Generation study a wide range of fungal species has led to the large- Individual transformants (n=3,424) from the plasmid library were scale identification of active genes [9, 23, 30, 32, 51]. picked and transferred into 96-well plates containing 0.5 ml of LB To give initial insight into the poorly understood genetic medium, containing 100 µg/ml ampicillin. Plates were incubated background of F. palustris, we generated over 3,000 ESTs overnight at 37oC. A small aliquot of each culture was stored at -80oC and identified 1,403 unigenes from this fungus. We performed after being mixed with the same volume of 25% glycerol in LB. similarity searches on these unigenes to putatively identify Plasmid DNA was isolated and purified using FB glass fiber plates genes with functions that are important in the biodegradation (Millipore) according to the glass bead method described previously of wood. Moreover, functional annotation based on the Gene [8]. cDNA inserts were sequenced from the 5' end using the M13- Ontology (GO) classification system was also provided for T7 primer (5'-TAATACGACTCACTATAGGG-3') and the BigDye a subset of the F. palustris unigenes. terminator ver. 3.1 kit (Applied Biosystems) on an ABI 3100 DNA sequencer (Applied Biosystems). Raw sequence trace data from the 3100 sequencer were processed in an automated pipeline, the MATERIALS AND METHODS trace2dbEST package [33]. Before submitting them to the public database (dbEST), sequences were processed to assess quality, remove Fungal Strains and Culture Conditions vector and adaptor sequences, identify contaminants and cloning The wood-destroying basidiomycete F. palustris, strain FFPRI 0507 artifacts, and determine any similarities by Blast searches. (NBRC 30339), was used in this study. This fungus was cultured and maintained on potato glucose agar (PDA) at 26oC. The fungal Clustering and Sequence Analysis inocula were prepared from a single, well-grown colony, removed Clustering was performed using PartiGene, a software pipeline designed with a cork borer (6 mm diameter) from the agar. A plug of mycelia to analyze and organize EST data sets [33]. Sequences were clustered was inoculated into 20 ml of potato dextrose broth liquid medium in into groups (putative genes) on the basis of sequence similarity using a 100-ml Erlenmeyer flask and cultured at 26oC for 7 days on a CLOBB [34]. Clusters were assembled to yield consensus sequences reciprocating shaker at 150 rpm. After incubation, the fresh mycelia using Phrap (P. Green, unpublished data). All contigs and singlets were harvested, homogenized using a homogenizer, and resuspended resulting from this automated clustering were considered to be the in 5 ml of sterile water. Then, 1 ml of mycelial suspension was best estimation of a minimal gene set for our EST library, and we inoculated into 100 ml of liquid media containing 2.0% (w/v) xylose, called this set “unigenes.” Each unigene sequence was subjected to ANALYSIS OF ESTS FROM THE WOOD-DECAYING FUNGUS F. PALUSTRIS 349

BlastX analysis against the GenBank nonredundant (nr) protein Bank of Japan (DDBJ) under the accession numbers AB508815 and database [1]. AB508816, respectively.

Identification of a GH Family 45 Endoglucanase Gene: Sequence and Phylogenetic Analyses RESULTS A unigene comprising three EST clones showed high similarity with the GH family 45 endoglucanases (EGs) during BlastX analysis. One Generation of ESTs from the F. palustris cDNA Library of the EST clones, containing full-length cDNA, was identified and ESTs were generated from the cDNA library constructed resequenced in both directions using universal primers (T7 and SP6) and from F. palustris (Table 1). To estimate the average length a designed primer, Fpcel45-1F (5'-GAGGTGTGGTGCGGCCAGAC-3'), of the cDNA inserts, 32 clones were randomly chosen and in order to obtain a full-length cDNA sequence. The genomic coding region of the cDNA clone was obtained by PCR amplification from their inserts were amplified using the SP6 and T7 universal F. palustris genomic DNA, using a pair of gene-specific primers primers designed against the vector. The amplified products flanking the open reading frame (ORF). These primers were designated were then separated by agarose gel electrophoresis. The Cel45A-0F (5'-GGAGCCTCCCTGAGGAACC-3') and Cel45A-0R estimated insert sizes were between 400 bp and 1.8 kb, (5'-TTGTCGAATATACACATTCACG-3'). Genomic DNA was prepared with an average size of about 1.3 kb. A total of 3,424 using a Fungal DNA Isolation Kit, according to the manufacturer’s cDNA clones were randomly isolated and single-pass instructions (Omega). PCR products were cloned using the pGEM-T sequenced from the 5' end using the T7 primer in order to Easy vector (Promega) and sequenced using standard protocols. generate ESTs. The sequences were trimmed to remove Signal peptide prediction was made using the SignalP program [5]. vector sequences, adaptor sequences, poly(A) tails, and O A search for the presence of -glycosylation sites was performed low-quality sequences, and filtered for a minimum length with NetOGlyc version 3.1 (http://www.cbs.dtu.dk/services/NetOGlyc/). (150 bp). This resulted in a total of 3,095 high-quality The theoretical pI and molecular weight of proteins were predicted ESTs, which gave an overall success rate of 90%. The using the Compute pI/MW tool available in the ExPASy server (http:// br.expasy.org/tools/pi_tool.html). Sequence alignment was performed average length of submitted ESTs was 504 nt, with the using a multiple alignment program, MAFFT version 6 (http:// longest and shortest lengths being 668 and 153 nt, align.bmr.kyushu-u.ac.jp/mafft/software/about.html), and output was respectively. The total submitted nucleotides were calculated produced using BOXSHADE 3.21 (http://www.ch.embnet.org/software/ to be 1.56 million. BOX_form.html). Phylogenetic analysis was performed using the software MEGA Cluster Formation and Analysis version 4.0 [43] and included amino acid sequences of the F. To identify overlapping EST sequences, improve base palustris GH family 45 EG, the NCBI sequences with significant accuracy and transcript length, and produce nonredundant similarity in BlastP searches, and the fungal enzymes in the GH EST data for further functional annotation, the 3,095 ESTs family 45 on the CAZy server (http://www.cazy.org). Sequences were from the F. palustris library were grouped by sequence aligned by MAFFT (v. 6) configured for highest accuracy (MAFFT with E-INS-i option). Prior to the phylogenetic analysis, signal peptide identity into clusters. Based upon regions of nucleotide sequences and other N and C terminal extensions peculiar to identity, EST sequences were merged into contiguous individual taxa were excluded. In total, 165 characters were used for consensus sequences (contigs). “Contig” member ESTs are the phylogenetic analysis. The neighbor-joining phylogenetic tree derived from identical transcripts, whereas “cluster” member was constructed using the Poisson correction substitution model. ESTs may derive from the same gene but represent

Gene Ontology The F. palustris unigene sequences were classified into GO functional Table 1. F. palustris cDNA/ ESTs library statistics. categories [2] based on BlastX similarities to known genes in the 5 GenBank nr protein databases and using the Blast2GO annotation Titer of cDNA library (pfu/ml) 1.1×10 tool (http://www.blast2go.de/) [7] with an E-value cut-off of 1e-05 Average cDNA insert size 1.3 kb Sequence reactions 3424 and summarized according to their biological processes, molecular a functions, and cellular components. A node sequence filter in the Number of quality sequences (QS) 3,095 (90%) GO graph was used: 20 for biological process, 15 for molecular Average length of QS 504 bp function, and 5 for cellular component. Pie charts were generated to Number of contigs 485 represent the three-level categorizations of F. palustris unigenes into Number of singlets 918 b different GO classes. Total number of unique sequences (unigenes) 1403 Total unigenes with significant similarity 1,097 (78%)c Sequence Data Deposition Unigenes with no significant similarity 306 (22%) All EST sequences described in this article have been deposited in a Calculated from total sequence reactions. the dbEST division of GenBank under the accession numbers bNumber of putative unigenes equals the number of contigs plus the GR367900-GR370994. The mRNA and gDNA sequences of the F. number of singlets. palustris GH family 45 EG have been deposited in the DNA Data cCalculated as percentage of total unigenes with similarity at E <10-5. 350 Karim et al. different transcript splice isoforms (i.e., ESTs form contigs, unigene set) with an E value lower than 1e-05 (significant contigs form clusters). Cluster analysis suggested that the match) (Table 1). Out of the remaining 306 unigenes (22%) 3,095 ESTs were assembled into 485 contigs, grouped into with no significant similarity, 45 sequences (3% of total 403 clusters (enclosing sequence with minor sequence unigenes) matched with nr proteins with higher E values variation), and 918 remained as singlets (non-overlapping (between 1e-05 and 1e-01). Out of all unigenes, 261 (19%) EST sequences), representing a final set of 1,403 putative gave no BlastX hits when the E-value cut-off was set as unigenes (Table 1). The contigs varied from 2 to 234 ESTs. high as 1e-01. Of these, some could be unique to the genus The majority of contigs were composed of 10 or fewer Fomitopsis or even to F. palustris. ESTs. The redundancy of the F. palustris EST library was calculated to be 70% (number of EST assembled into Transcript Abundance and Highly Represented Genes contigs/total number of ESTs), which seemed to be reasonable A high level of representation in a cDNA library usually compared with the redundancy obtained in other fungal correlates with high transcript abundance in the original EST libraries (49.5-85%) [32, 37, 47, 51]. By eliminating biological sample [3], although artifacts of library construction redundancy during contig building, the total number of can result in a selection for or against representation of nucleotides used for further analysis was reduced from some transcripts. The F. palustris unigenes were ranked 1.56 million to 0.78 million. In addition, this process according to the number of contributing ESTs, and the top significantly increased the length of assembled transcript 20 contigs are summarized in Table 2. Each of these sequences from 506±91 nucleotides for submitted ESTs contigs contained 12 or more EST copies and represented alone to 595±321 nucleotides for contigs. The longest 21% of the total number of ESTs obtained. Eleven of the sequence generated also increased from 668 to 2,672 contigs had significant matches to assign functionality nucleotides. based on BlastX (E<10-5) against the nr database, and all of them had homologs in fungal species. Of these, 5 BlastX Analysis of F. palustris Unigenes transcripts showed homology to proteins with unknown To identify homologs of the 1,403 unigenes derived from functions, including two hypothetical proteins from the F. palustris, each unigene sequence was queried against human pathogen Cryptococcus neoformans [26]. The largest the NCBI nr protein database using the BlastX algorithm contig (unigene ID FPC00005_3), containing 234 ESTs, was [1]. This resulted in 1,097 matches (78% of the total similar to a small heat shock protein from the basidiomycete

Table 2. The most abundantly represented transcripts in the F. palustris cDNA library. Top BlastX hit in nonredundant protein databasea No. Unigene ID ESTs Best identity descriptor Accession No. E value 1 FPC00005_3 234 Small heat shock protein [Laccaria bicolor] AAM78595 3e-43 2 FPC00032_1 53 No hit -- -- 3 FPC00036_1 52 ZIP-like iron-zinc transporter [Laccaria bicolor] XP_001885404 1e-108 4 FPC00034_3 47 No hit -- -- 5 FPC00018_2 27 No hit -- -- 6 FPC00061_1 27 Predicted protein [Coprinopsis cinerea] XP_001830117 6e-12 7 FPC00047_1 26 NAD-dependent formate dehydrogenase [Ceriporiopsis subvermispora] BAF98207 1e-129 8 FPC00137_1 21 Synaptobrevin [Schizosaccharomyces japonicus] XP_002173453 5e-32 9 FPC00149_1 20 Hypothetical protein [Coprinopsis cinerea] XP_001839717 1e-65 10 FPC00018_1 19 No hit -- -- 11 FPC00021_2 17 No hit -- -- 12 FPC00085_1 16 No hit 13 FPC00009_1 14 CipC1 protein, concanamycin-induced protein C [Laccaria bicolor] XP_001879340 3e-74 14 FPC00870_1 14 No hit -- -- 15 FPC00064_1 13 No hit -- -- 16 FPC00120_1 13 Predicted protein [Laccaria bicolor] XP_001880848 1e-36 17 FPC00238_2 13 No hit -- -- Ectomycorrhiza-regulated CFEM domain-containing protein 18 FPC00245_1 13 XP_001886750 4e-98 [Laccaria bicolor] 19 FPC00038_1 12 Hypothetical protein [Cryptococcus neoformans] XP_569977 7e-25 20 FPC00087_1 12 Hypothetical protein [Cryptococcus neoformans] XP_778235 3e-21 aTop BlastX hits with E value lower than 10-5 are shown. ANALYSIS OF ESTS FROM THE WOOD-DECAYING FUNGUS F. PALUSTRIS 351 fungus, Laccaria bicolor [27]. Three other documented involved in the degradation of plant cell wall polysaccharides, proteins of this fungus were also present as the top-BlastX as well as those involved in the synthesis of fungal cell hits of three abundantly represented transcripts of F. palustris. walls (Table 3). A total of 44 ESTs distributed in 22 These were a ZIP-like iron-zinc transporter, concanamycin- unigenes (2% of total) were identified with significant induced protein C, and ectomycorrhiza-regulated CFEM similarity to the CAZyme-encoded genes, which included domain-containing protein. Other abundant ESTs included ranges of GHs, two glycosyl transferases (GT), and one synaptobrevin, an intrinsic membrane protein of small carbohydrate esterase (CE) family protein. The synaptic vesicles [4], and the metabolic enzyme NAD- homologs that were identified included the previously dependent formate dehydrogenase. The latter protein-encoded published GH family 12 EG from F. palustris [40] and a gene was identified as the best match with the gene from GH family 45 EG from white-rot fungus P. chrysosporium the white-rot fungus Ceriporiopsis subvermispora [48]. [16]. Both cellulase genes were identified as contigs. The Nine of the 20 most abundantly expressed transcripts features of the sequence of the GH family 45 EG from F. from F. palustris had no significant similarity to any palustris are discussed in more detail below. sequence in the nr protein database based on the E-value The transcript encoding two major hemicellulose-degrading cut-off of less than 1e-5. Of these, we found homologous enzymes, β-xylosidase and β-1,4-xylanase, was identified sequences for six unigenes using a low BlastX stringency as the best match with the genes from P. placenta, the first (E=0.01), but failed to get any hits for the remaining brown-rot fungus to have its genome sequenced [29]. three highly expressed ESTs (Unigene IDs FPC00032_1, Although the transcript encoding β-xylosidase contains a FPC00021_2, and FPC00064_1). Finally, we compared single EST, the β-1,4-xylanase unigene was identified as a these three contigs against the NCBI EST-others database contig of five individual ESTs. Other hemicellulase homologs but received no BlastN hits with the E-value cut-off set as that were identified included α-mannosidase, α-1,6 mannase, high as 1e-01, indicating that these are more likely to be and α-1,2-mannosyl transferase. These enzymes are involved Fomitopsis-specific genes and may encode proteins with in the degradation of polysaccharides of mannose. Moreover, functions unique to this fungal group. enzyme homologs that could degrade the polysaccharides other than cellulase and hemicellulase were β-1,3-glucanase, Identification of F. palustris Transcripts Similar to α-1,3-glucanase, α-amylase, and polygalacturonase (PG). CAZymes Homologs of two different types of genes, chitin The well-known efficiency of F. palustris to degrade wood synthase and β-1,3-glucanosyltransferase, that could encode led us to search the unigenes that showed significant enzymes important in biosynthesis of the fungal cell wall, similarity to genes encoding potential CAZymes. BlastX were identified. In addition to the classical CAZymes, analysis allowed us to identify the enzyme homologs which potentially act on plant cell wall polysaccharides,

Fig. 1. The nucleotide and deduced amino acid sequences of cDNA encoding F. palustris Cel45A. The predicted secretion signal sequence is underlined. Predicted positions of the five intron sequences identified are indicated by darkened triangles. Primers used for obtaining the full-length cDNA sequence and genomic amplification are indicated by arrows. Three possible O-glycosylation sites are marked with an asterisk. 352 Karim et al. value E EED82889 2e-23 a ] XP_002477260 7e-45 ] BAG68300 1e-58 ] XP_568714 6e-47 ] XP_001886822 2e-09 ] XP_001873806 2e-12 Postia placenta ] CAB96110 1e-35 ] XP_002391565] 4e-30 ] XP_001884146 1e-26 ] ABO42285 1e-55 ] BAF49602 1e-89 ] XP_001888816 2e-29 ] XP_001829066 2e-31 ] XP_001829066 ] EED82341 7e-76 ] EED84312 1e-117 Laccaria bicolor Laccaria bicolor ] XP_002471666 1e-46 ] EED82234 2e-39 ] EED84213] 2e-06 ] EED85542 2e-68 ] XP_002469716 4e-66 ] XP_002473119 6e-129 ] EED85010 3e-41 ]] XP_002472422 XP_002470910 9e-22 1e-35 ] EED80923 1e-56 Agaricus bisporus Cryptococcus neoformans Phanerochaete chrysosporium Phanerochaete Laccaria bicolor ] Postia placenta Postia Fomitopsis palustris Laccaria bicolor Postia placenta Top BlastX hit in nonredundant protein database Top Postia placenta Postia placenta Moniliophthora perniciosa Postia placenta Postia placenta Coprinopsis cinerea Postia placenta Postia placenta Postia placenta Postia placentaPostia placentaPostia Postia placenta Phanerochaete chrysosporiumPhanerochaete Postia placenta -Acetylhexosaminidase [ Best descriptor identity Accession No. deacetylase from esterase carbohydrate family CE10 [ N CAZy CAZy family FPC00816_1 3 GH45 protein [ Endoglucanase V-like FPC00336_1FPC00651_1 1 1 GH16 GH17 Glycoside hydrolase family 16 protein [ protein [ Hypothetical FPC00830_1FPC01071_1 1 1 GH16 GH16 protein [ Hypothetical protein [ Hypothetical FPC01225_1 1 GH16 protein [ Hypothetical FPC01241_1 1 GH16 protein [ Hypothetical FPC00234_1FPC00655_1 4 1 -- -- Predicted protein [ Predicted protein [ transcripts similar to carbohydrate-active enzymes (CAZymes). F. palustris F.

1,4-Endoglucanase FPC00104_1 2 GH12 Endo-1,4-beta-glucanase [ - -Xylosidase-1,4-Xylanase-Mannosidase FPC00747_1 FPC00792_1 1 5 FPC00103_1 GH3 3 GH10 Beta-xylosidase [ GH47 Endo-1,4-beta-xylanase [ protein [ Hypothetical -1,3-Endoglucanase FPC00144_1 1 GH16 protein [ Hypothetical -1,2-Mannosyl transferase FPC01095_1 1 GT15 transferase family Glycosyl 15 protein [ -1,6-Mannanase FPC00805_1 2 GH76 Endo-1,6-alpha-mannosidase [ -Amylase FPC01067_1 1 GH13 Alpha-amylase [ -1,3-Glucanase FPC00670_1 2 GH71 protein [ Hypothetical -Hexosaminadase FPC00882_1 1 GH20 -1,3-Glucanosyltransferase FPC00096_1 3 GH72 1,3-Beta-glucanosyltransferase [ Top BlastX hits with E value lower than 10-5 are shown. are 10-5 than lower E value with hits BlastX Top Table 3. Enzyme/proten name Unigne ID ESTs a β β β β β β β β β β Chitin synthase FPC01444_1 1 GT2 Putativechitinsynthase enzyme [ PolygalacturonaseCarbohydrate esterase FPC00643_1 FPC00636_1 1 1 GH28 CE10 protein [ Hypothetical β Glucoronyl hydrolase FPC00503_1 3 GH88hydrolase-like protein [ d-4,5-Unsaturated-glucuronyl Expansin-like protein FPC00154_1 1 -- Expansin family protein [ ANALYSIS OF ESTS FROM THE WOOD-DECAYING FUNGUS F. PALUSTRIS 353 we identified genes encoding expansin-like proteins in the The gene encoding GH family 45 EG from F. palustris F. palustris data set. Expansins and expansin-like proteins was identified as a contig of three individual ESTs (Table 3). have been found in wood decay fungi [28, 29] and it is The full-length cDNA sequence of F. palustris EG, thought that these proteins disrupt noncovalent bonds in designated FpCel45A, was obtained from the plasmid the plant cell wall, enhancing the activity of other plant clone corresponding to the EST sequence. The complete cell-wall-degrading enzymes such as cellulases. cDNA was 807 bp in length [including poly(A)] and contained an ORF of 206 amino acids with an ATG start Isolation and Sequence Analysis of a Gene Encoding codon at position 25 and a TGA stop codon at position 643 GH Family 45 Endoglucanase (Fig. 1). A signal peptide of 22 amino acids was predicted The extracellular production of several EGs from F. using the SignalP program [5] at the N terminus of the palustris has been reported [15, 19, 52], and two small deduced FpCel45A polypeptide. Three possible O- EGs belonging to the GH family 12 have been cloned and glycosylation sites were predicted using the NetOGlyc characterized previously [40, 42]. However, there is no server. The molecular mass of the putative mature protein information about GH family 45 proteins for this fungus or was 18.6 kDa and the theoretical pI value was 3.85. No any other brown-rot fungi. Recently, an EG gene belonging putative cellulose-binding domain was identified in this to GH family 45 was cloned from the white-rot basidiomycete sequence. P. chrysosporium [16], and this enabled us to identify the A genomic clone of FpCel45A was obtained by PCR similar gene in F. palustris (Table 3). amplification using gene-specific primers (Cel45A-0F and

Fig. 2. Unrooted phylogenetic tree of selected fungal GH family 45 proteins generated using maximum likelihood analysis. GenBank accession numbers of sequences showing significant alignments with FpCel45A in the BlastP analysis (Subfamilies B and C) and fungal enzymes in the GH family 45 from the CAZy server (http://www.cazy.org) (subfamily A) are shown in brackets.The bootstrap values are calculated from 1,000 replicates. The scale bar represents 10 substitutions per 100 amino acid positions. 354 Karim et al.

Cel45A-0R) and genomic DNA as a template. The principles of GO: biological process, molecular function, FpCel45A genomic DNA product was 945 bp in length and cellular component. The three-level categorizations of from the ATG to the stop codon. The position of exon/ unigenes into different GO classes are shown in Fig. 3A-3C, intron boundaries were determined by aligning the genomic and a complete listing of GO mappings assigned for the F. sequence with the corresponding cDNA sequence. Five palustris unigenes is provided in Table S1 (supplementary introns were identified in FpCel45A (Fig. 2) with lengths materials). ranging from 53 to 67 bp, a feature commonly found in When analyzed by biological process, the majority of fungi [25]. All introns were bordered by canonical cis- annotations (56%) were involved in metabolism, followed splicing sequences [25]. by biosynthesis (12%) (Fig. 3A). The remainder (32%) was A protein homology search, BlastP, using the deduced distributed among several processes, including transport, amino acid sequence of FpCel45A, indicated that FpCel45A housekeeping functions, growth, and the regulation of was most similar to the EG V-like protein (PcCel45A) from white-rot fungus P. chrysosporium [16]. FpCel45A shared 64.5% identity with PcCel45A. Although no putative conserved domain was found for FpCel45A by BlastP search, multiple sequence alignment of FpCel45A with the best matches obtained from BlastP analysis revealed that this protein has low similarity to other fungal GH family 45 EGs (20-22% identity) and some uncharacterized fungal proteins (hypothetical or predicted proteins; 18- 22% identity) (Fig. S1, in supplementary material). Based on the comparison with Hypocrea jecorina (Trichoderma reesei) EGV [38], and conservation in other GH family 45 sequences, one of the catalytic residues of FpCel45A may be Asp 139 (Asp 140 for FpCel45A). However, another possible catalytic residue (Asp 27 in H. jecorina EGV) is missing for both EGs (FpCel45A and PcCel45A) but conserved in other GH family 45 EGs, indicating that both FpCel45A and PcCel45A are different from known GH family 45 EGs. A phylogenetic tree was generated for the sequences showing significant alignment with FpCel45A on the BlastP search, including PcCel45A (subfamily C), fungal GH family 45 EGs (subfamily B), and fungal enzymes of the GH family 45 on the CAZy server (subfamily A) (Fig. 2). FpCel45A clustered into subfamily C with the PcCel45A from P. chrysosporium. This clade also includes hypothetical proteins from Coprinopsis cinerea (EAU91056), Ustilago maydis (XP_761686), and a predicted protein from P. placenta (EED77380). In terms of amino acid sequence, FpCel45A was clearly distinct from subfamily A, but showed some similarity to subfamily B, as expected from the results of the Blast search.

Functional Classification of Unigenes Based on GO Assignments F. palustris unigenes were annotated according to GO guidelines [2] with Blast2GO, a universal Web-based annotation application [7]. Of the 1,097 sequences with BlastX hits in the nr protein database with an E value lower than 1e-05, Fig. 3. Functional annotation of F. palustris unigenes based on mapping of GO terms or annotation was not possible for Gene Ontology (GO) categorization. 387 and 217 sequences, respectively. The remaining 473 Sequences were evaluated for their predicted involvement in biological processes (A), molecular functions (B), and cellular components (C). Data (34% of total unigenes) sequences were successfully annotated are presented at level three GO categorization. Note that a single gene can and mapped to one or more of the three organizing be assigned to more than one category in the GO classification system. ANALYSIS OF ESTS FROM THE WOOD-DECAYING FUNGUS F. PALUSTRIS 355 development. Hydrolases, oxidoreductases, and transferases library included a gene encoding a ZIP-like iron-zinc comprised 32% of the total number of molecular functions transporter (Table 2). The functional roles of various genes identified by GO analysis (Fig. 3B). Hydrolases, which encoding potential ZIP family proteins have been studied utilize water molecules to break chemical bonds, perform a extensively in S. cerevisiae [56-58]. The finding of such a broad range of functions in fungi. As a well-known wood- gene in the highly represented transcripts of F. palustris decaying fungus, F. palustris is thought to secrete a large supports the possibility that, like many other fungi, F. number of extracellular hydrolases to degrade cell wall palustris is very active in ion transportation and ZIP polysaccharides. EGs and some other GH family proteins, transporters may have a predominant role in such processes. listed in Table 3, were included as hydrolases in the major Considering the fact that wood-rotting fungi are equipped GO class, “molecular function.” Oxidoreductases catalyze with a large number of CAZyme-encoded genes to degrade the transfer of electrons between molecules. A number of plant cell wall polysaccharides, we searched and identified the annotated unigenes of F. palustris could encode enzymes a subset of F. palustris unigenes with significant similarities involved in primary and secondary metabolism (e.g., several to genes encoding enzymes potentially involved in the dehydrogenases, cytochrome c oxidase), as well as the biodegradation of wood (Table 3). In addition to typical detoxification of compounds such as reactive oxygen cellulases and hemicellulases, a substantial number of β- species, superoxide and hydrogen peroxide. The F. palustris 1,3-glucanase genes that could encode proteins of GH unigenes were predicted to encode enzymes with transferase family 16 (six cases) or GH family 17 (one case) were activities, including many proteins kinases (Fig. 3B). The identified. Similar genes have been cloned and characterized vast majority of annotated unigenes were predicted to from white-rot fungus P. chrysosporium [22]. Several β- encode intracellular proteins within the “cellular component” 1,3-glucanase genes have also been predicted in the genome class (Fig. 3C). sequence of the brown-rot basidiomycete P. placenta [29]. Since β-1,3-D-glucan is the main structural component of fungal cell walls, β-1,3-glucanases appear to be key DISCUSSION enzymes in cell wall lysis [46]. However, β-1,3-D-glucan can also be present as a wood cell wall polysaccharide in Brown-rot basidiomycetes cause the most destructive type varying amounts (2-4%), and it is likely that this enzyme of wood decay and are important contributors to biomass has a functional role in the decay process. recycling [12, 24]. F. palustris causes typical brown-rot, is An unigene of F. palustris had significant homology to an important copper-tolerant fungus, and has been used as α-amylase from the white-rot basidiomycete P. chrysosporium a Japanese Industrial Standard (JIS) fungus for testing of [39]. By acting at random locations along the starch chain, wood preservatives efficacy [45]. In this study, an EST α-amylase breaks down the α-1,4-glycosidic bonds of approach was successfully applied to gain an initial insight long-chain carbohydrates. The F. palustris cDNA library into the gene content of this organism and to provide a also contained a homologous sequence to the gene encoding preliminary assessment of the transcripts involved in the a pectin-degrading enzyme, PG. A large number of PGs biodegradation of wood. The generation of 3,095 high- have been isolated and characterized from several fungal quality ESTs and their subsequent clustering allowed us to species including white- and brown-rot fungi [6, 13, 49]. identify 1,403 unigenes (Table 1), which corresponded to a PGs are thought to participate early in the wood decay new gene discovery rate of 45% (1,403/3,095). Taking into process because of their hydrolytic effects on pit membranes account the number of protein-coding genes in other resulting in increased wood permeability [44]. The production basidiomycete fungi, such as U. maydis (6,522) [21], C. of PG during incipient brown-rot may be crucial to rapid neoformans (7,302) [26], P. chrysosporium (10,048) [28], and efficient colonization of the wood. C. cinerea (13,544) (http://www.broad.mit.edu/annotation/ Analysis of F. palustris ESTs led to the identification of genome/coprinus_cinereus/MultiHome.html), and L. bicolor β-1,4-EG genes belonging to GH families 12 and 45, (20,614) [27], the full data set of unigenes identified in this respectively (Table 3). The former enzyme has recently study may represent approximately 7-22% of F. palustris been cloned and characterized from F. palustris [40]. To genes. date, however, no EG belonging to GH family 45 has been Special attention was paid to the largest unigenes, as reported for this fungus. Moreover, no GH family 45 gene they most likely correspond to highly expressed genes in F. has yet been predicted from the sequenced genome of the palustris. One notable observation in the BlastX results of brown-rot fungus P. placenta [29]. We therefore cloned the highly represented transcripts was the absence of homologous full-length cDNA and genomic sequences of the putative sequences of structural genes (Table 2). The only metabolic GH family EG from F. palustris. To the best of our enzyme identified in highly represented F. palustris knowledge, the FpCel45A identified in this study was the transcripts was NAD-dependent formate dehydrogenase. first GH family 45 EG gene from any brown-rot fungi. The Transcripts abundantly represented in the F. palustris presence of introns in the gDNA of FpCel45A (Fig. 1) 356 Karim et al. indicated that this gene originated from a eukaryote and palustris is a valuable addition to our knowledge regarding not from contamination of prokaryotes. the cellulolytic enzyme systems of this fungus and may FpCel45A has a molecular mass of 18 kDa and is one of facilitate the search for similar genes in other brown-rot the smallest EGs, being devoid of a cellulose-binding fungi. This annotated EST data set will provide a good domain. Although the enzymatic activity has not been resource to explore the molecular basis of this fungus. proven for the EG we describe here, we tentatively named this EG FpCel45A on the basis of high sequence similarity with the functionally characterized GH family 45 EG Acknowledgments (PcCel45A) from white-rot fungus P. chrysosporium. Because of its low similarities with the EGs belonging to The authors thank Asuka Shichiri for technical assistance. the two major subfamilies (A and B) of GH family 45, N.K. is supported by the Japan Society for the Promotion PcCel45A has been placed in a new subfamily, C [16]. The of Science (JSPS) Postdoctoral Fellowship for foreign sequence alignments and subsequent phylogenetic analysis researchers. T.K. has been funded by the Japanese Ministry clearly suggested that FpCel45A from F. palustris could of Education, Science, Sports and Culture, Grant-in-Aid also be a member of subfamily C, as it was more distinct for Encouragement of Young Scientists (B) 18780032. from known GH family 45 EGs and monophyletic with PcCel45A (Fig. 2). The sequence similarity of the predicted protein (EED77380) from brown-rot fungus P. placenta REFERENCES with FpCel45A and PcCel45A (Fig. S1 in supplementary materials) and its phylogenetic position in subfamily C 1. Altschul, S. F., T. L. Madden, A. A. 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