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Contents lists available at SciVerse ScienceDirect

Fungal Genetics and Biology

journal homepage: www.elsevier .com/locate /yfgbi

Genes associated with lignin degradation in the polyphagous white-rot pathogenHeterobasidion irregulare show substrate-specific regulation

Igor A. Yakovlev a, Ari M. Hietala a, Pierre-Emmanuel Courty b, Taina Lundell c, Halvor Solheim a,Carl Gunnar Fossdal a,⇑a Norwegian Forest and Landscape Institute, P.O. Box 115, N-1431 Ås, Norwayb Botanical Institute, University of Basel, CH-4056 Basel, Switzerlandc Department of Food and Environmental Sciences, University of Helsinki, Viikki Biocenter 1, P.O. Box 56, FI-00014 Helsinki, Finland

a r t i c l e i n f o

272829303132333435363738

Article history:Received 29 March 2012Accepted 29 April 2013Available online xxxx

Keywords:Heterobasidion annosumLignin biodegradationNorway spruceLaccasesGMC oxidasesCellulose

3940414243444546

1087-1845/$ - see front matter � 2013 Published byhttp://dx.doi.org/10.1016/j.fgb.2013.04.011

⇑ Corresponding author. Fax: +47 64 94 29 80.E-mail address: foc@skogoglandskap.no (C.G. Foss

Please cite this article in press as: Yakovlev, I.Airregulare show substrate-specific regulation. Fu

a b s t r a c t

The pathogenic white-rot basidiomycete Heterobasidion irregulare is able to remove lignin and hemicel-lulose prior to cellulose during the colonization of root and stem xylem of conifer and broadleaf trees. Weidentified and followed the regulation of expression of genes belonging to families encoding ligninolyticenzymes. In comparison with typical white-rot fungi, the H. irregulare genome has exclusively the short-manganese peroxidase type encoding genes (6 short-MnPs) and thereby a slight contraction in the pool ofclass II heme-containing peroxidases, but an expansion of the MCO laccases with 17 gene models. Fur-thermore, the genome shows a versatile set of other oxidoreductase genes putatively involved in ligninoxidation and conversion, including 5 glyoxal oxidases, 19 quinone-oxidoreductases and 12 aryl-alcoholoxidases. Their genetic multiplicity and gene-specific regulation patterns on cultures based on definedlignin, cellulose or Norway spruce lignocellulose substrates suggest divergent specificities and physiolog-ical roles for these enzymes. While the short-MnP encoding genes showed similar transcript levels uponfungal growth on heartwood and reaction zone (RZ), a xylem defense tissue rich in phenolic compoundsunique to trees, a subset of laccases showed higher gene expression in the RZ cultures. In contrast, otheroxidoreductases depending on initial MnP activity showed generally lower transcript levels on RZ than onheartwood. These data suggest that the rate of fungal oxidative conversion of xylem lignin differsbetween spruce RZ and heartwood. It is conceivable that in RZ part of the oxidoreductase activities oflaccases are related to the detoxification of phenolic compounds involved in host-defense. Expressionof the several short-MnP enzymes indicated an important role for these enzymes in effective delignifica-tion of wood by H. irregulare.

� 2013 Published by Elsevier Inc.

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1. Introduction

Brown-, soft- and white-rot fungi are the most important wood-degrading organisms and play an important role in carbon andnitrogen cycling. While brown-rot is highly oxidative leading to ra-pid degradation of wood cellulose (Eastwood et al., 2011), thewood lignin is only partially modified (Yelle et al., 2008). Soft-rotfungi grow on wood in damp environments and secrete enzymesthat break down cellulose but have little or no effect on the woodlignin. On the contrary, white-rot basidiomycetes are the onlyorganisms able to efficiently decompose all components of wood,including the lignin heteropolymers (Hatakka, 1994; Kersten andCullen, 2007). Non-selective white-rot fungi simultaneouslydegrade all cell wall components in xylem whereas certainwhite-rot fungi, such as Ceriporiopsis subvermispora, Physisporinus

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., et al. Genes associated withngal Genet. Biol. (2013), http:/

rivulosus and Heterobasidion annosum s.l. tend to remove ligninand hemicellulose in advance of cellulose (Blanchette, 1984;Hakala et al., 2005; Hatakka and Hammel, 2010).

White-rot fungi are considered to decompose wood lignin via asynergistic action of oxidoreductive metalloenzymes encoded bymany fungal gene families (Lundell et al., 2010). In white-rot, thegeneration of organic radicals and oxidation of lignin are catalyzedby extracellular lignin-modifying enzymes (LMEs) including highredox-potential class II heme peroxidases (Hofrichter et al.,2010), peroxide-generating oxidases such as glyoxal oxidases andaryl-alcohol oxidases (Kersten and Cullen, 2007; Ferreira et al.,2009), and multicopper oxidases (MCOs) such as laccases (Courtyet al., 2009; Hildén et al., 2009; Hoegger et al., 2006). Biodegrada-tion of lignin is a co-metabolic process that requires access to addi-tional carbon sources to provide the needed energy. Thus thewhite-rot fungal conversion of lignin in wood coincides withhydrolysis of the storage and structural cell wall polysaccharides

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by carbohydrate-acting such as cellulases and hemicellulases (Hat-akka and Hammel, 2010; Lundell et al., 2010).

The white-rot basidiomycete H. annosum species complex is themost important cause of root and butt rot of conifers in the North-ern Hemisphere (Asiegbu et al., 2005). For the two Eurasian speciesH. annosum sensu stricto and Heterobasidion parviporum (Niemeläand Korhonen, 1998), both selective and non-selective wood lignindecay modes have been reported (Daniel et al., 1998). Besides theability to kill vascular cambium and the living cells of inner barkupon fungal penetration through roots, in conifer trees such asNorway spruce (Picea abies), Heterobasidion species can tolerateand presumably detoxify the polyphenols that accumulate as a treedefense response (reaction zone) opposing the expanding fungaldecay columns in the root and stem xylem (Hietala et al., 2009).

Heterobasidion irregulare (formerly H. annosum North AmericanP-type), (Otrosina and Garbelotto, 2010) is a North American spe-cies with a broad host range including conifers such as species inthe genera Pinus, Juniperus and Libocedrus but also angiosperms.Being a facultative parasite with a white-rot mode in the sapro-trophic phase and a necrotrophic pathogen in living host tissue,H. irregulare (Olson et al., 2012) supplements the list of genomesof wood-decaying basidiomycetes sequenced by the Joint GenomeInstitute (CA, USA), which also include the saprotrophic white-rotfungi Phanerochaete chrysosporium (Martinez et al., 2004) andPleurotus ostreatus (Ruiz-Dueñas et al., 2011), the litter-decompos-ing Agaricus bisporus (http://www.jgi.doe.gov), the primarilysaprotrophic Schizophyllum commune (Ohm et al., 2010), and thebrown-rots Postia placenta (Martinez et al., 2009) and Serpula lacry-mans (Eastwood et al., 2011).

Based on the CAZy Auxiliary Activities (www.cazy.org/Auxil-iary-Activities.html) database (Levasseur et al., 2008) and otherpublicly available information (Olson et al., 2012), we character-ized and performed qRT-PCR expression profiling of the gene fam-ilies coding for oxidative enzymes putatively involved in theconversion and degradation of lignin by H. irregulare on defined lig-nin and spruce cellulose media, and on natural lignocellulose sub-strates (heartwood and RZ defense wood).

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2. Materials and methods

2.1. Strain and culture conditions

The H. irregulare strain TC-32-1 subjected to genome sequenc-ing by JGI was cultured on four different media for gene transcriptlevel profiling. Hagem broth [0.5 g NH4NO3, 0.5 g KH2PO4, 0.5 gMgSO4 � 7H2O, 0.038 g MnSO4 � H2O, 0.8 mL Fe(II)Cl2 � 4H2O(1% aqueous solution) and 5 g malt extract per 1 L ddH2O] wasused as a basal liquid component for all the substrates. The pHwas adjusted to 4.5 with 1 M H2SO4, and after autoclaving, filter-sterilized thiamine HCl (0.1 mg per 1 L) was added. To preparethe inoculum, the fungus was grown on 2% malt extract agar for3 weeks at 21 �C in darkness. Conidia were resuspended from thecultures into liquid Hagem medium and collected. The concentra-tion of the suspension was adjusted to 300,000 conidia per mLusing a light microscope and Bürker cell calculation chamber. Inthe experimental set-up, either 2 g of crystalline cellulose (Sig-ma–Aldrich #22182), 0.5 g of Kraft alkaline lignin (Sigma–Aldrich#471003), 1 g of milled (IKA mill 10.2 impact grinding head, IKAWerke, Staufen, Germany) and gamma-sterilized Norway spruceheartwood or reaction zone xylem tissue was aseptically weighed,mixed with 9 mL of the inoculum suspension and poured into ster-ile Petri dish (diameter 9 cm). Due to the alkalinity of the Kraft lig-nin, the pH of the lignin/liquid Hagem medium was adjusted to pH4.5 with 1 M H2SO4 prior to incubation. Three replicates were pre-pared for each treatment. Free-living mycelium was grown in the

Please cite this article in press as: Yakovlev, I.A., et al. Genes associated withirregulare show substrate-specific regulation. Fungal Genet. Biol. (2013), http:/

closed Petri dishes for three weeks at 21 �C in the dark withoutshaking. The culture was snap-frozen in liquid nitrogen and storedat �80 �C until further analysis.

2.2. Gene annotation, manual curation and peptide structure analyses

The sequencing, assembly and annotation of the H. irregularegenome (Olson et al., 2012) were performed by DOE Joint GenomeInstitute (JGI) using the whole-genome shotgun sequencing meth-od. All H. irregulare (referred as H. annosum at the JGI FungalGenomics Program internet resource) gene sequences are available(http://genome.jgi-psf.org/Hetan2/Hetan2.home.html). Selectionof class II heme-containing peroxidases, laccases and hydrogenperoxide-generating gene models was based on expressed se-quence tag (EST) support, completeness, and homology to a cu-rated set of proteins. Additionally, a range of gene sequencesavailable from fungi at the National Center for Biotechnology Infor-mation (NCBI) GenBank (http://www.ncbi.nlm.nih.gov/) and UNI-PROT (http://expasy.org/) were used to probe the H. irregularegenome database using BLASTN, TBLASTN, and BLASTP algorithmsat the JGI Fungal Genomics Program pipeline and servers. Theputative homologs were characterized based on conserved proteindomains, identities, and E-values in comparison with known pro-teins from other fungal species.

Signal peptides were predicted with SignalP 3.0 (http://www.cbs.dtu.dk/services/SignalP) (Dyrløv Bendtsen et al., 2004).Amino-acid multiple sequence alignments of specific conservedprotein motif regions were designed using WebLogo 3 application(http://weblogo.threeplusone.com/create.cgi) (Crooks et al., 2004).Conserved protein domains were analyzed using the Swissprotprosite (http://us.expasy.org/prosite) (Gasteiger et al., 2003) andInterProScan (http://www.ebi.ac.uk/InterProScan). Similarities/identities between selected pairs of protein sequences were calcu-lated using MatGAT (Matrix Global Alignment Tool) (http://bitin-cka.com/ledion/matgat/).

2.3. RNA isolation, cDNA synthesis and real-time quantitative RT-PCR

Prior to RNA extraction, culture samples were ground in liquidnitrogen. Total RNA was extracted from 100 mg of the pulverizedcultures using the RiboPure™ Kit (Ambion, Austin, TX, USA,#1924) following manufacturer’s recommendations. DNA was re-moved from the total RNA samples using the DNA-Free™ Kit(Ambion, #1906) according to the manufacturer’s protocol andstored at �80 �C until further use. Transcript level profiling ofH. irregulare genes was performed using a two-step quantitativeRT-PCR (qRT-PCR) procedure. Total RNA was quantified with themicro-volume spectrophotometer NanoDrop 2000 (Thermo Scien-tific, Wilmington, DE, USA) and then reverse-transcribed (300 ngper reaction) using the TaqMan Reverse Transcription kit (AppliedBiosystems, Carlsbad, CA, USA, #8080234) in 50-ll-reaction vol-ume. cDNA was used as template in the qRT-PCR reactions withgene-transcript specific primers designed using Primer 3 (http://frodo.wi.mit.edu/primer3/). The following criteria were used:product size inferior to 120 bp, GC percentage between 40% to60%, and melting temperature about 70 �C (Tables S1 and S2). Tar-get gene expression was normalized by using the H. irregulare actin(HiAct, ID 147003/408649), a-tubulin (HiaTub, ID 62388) and ubiq-uitin-conjugating enzyme 2 (HiUbc2, ID 145590) as constitutivelyexpressed housekeeping genes. Reactions of qRT-PCR were runusing the 7500 Fast Real-time PCR System (Applied Biosystems,Carlsbad CA, USA).qRT-PCR amplification was performed in a25-ll-reaction volume, using 2 ll of a threefold diluted cDNA solu-tion as template, 12.5 ll of 1� SYBR Green master mix and 200 nMof each primer. The following cycling parameters were applied:95 �C for 3 min and then 40 cycles of 95 �C for 30 s, 60 �C for

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1 min and 72 �C for 30 s. A control with no cDNA was run for eachprimer pair. For data analysis, the geometric mean of the biologicalreplicates (n = 3) was calculated. The primer efficiency ranged be-tween 90% and 110%.

Absolute quantification method was performed by using 7500-system SDS software. Data were further processed in MS Excel andadditionally analyzed using RT2 Profiler PCR Array Data Analysisweb portal from SABiosiences/Qiagen (Frederick, MD, USA)(http://pcrdataanalysis.sabiosciences.com/pcr/arrayanalysis.php)using portal defaults for custom arrays.

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3. Results

3.1. Lignin-modifying class II and other peroxidases

In the H. irregulare genome (Olson et al., 2012), eight putativeclass II fungal heme-containing, secreted peroxidase encodinggenes were identified (Table 1). Six of the eight genes (MnP1–3and MnP5–7) code for short-type of manganese peroxidases(short-MnPs, EC 1.11.1.13; Hildén et al., 2005; Hofrichter et al.,2010; Sundaramoorthy et al., 1994), while MnP4 (renamed toGPx1) encodes a low-redox potential peroxidase, while the last po-tential member (MnP8) is not transcribed and likely a pseudogene(Table S4, Fig. S4). Further information about the short-MnPs andall other sequences studied in this work, such as the JGI ProteinID, number of predicted exons, reading frame length and numberof aa in their predicted proteins, is provided in Table S1 andSupplement 4. In addition to the class II peroxidases, additionalheme-containing, extracellular peroxidases were detected in theH. irregulare genome: one DyP-type dye-decolorizing peroxidase(Protein ID 40020; DyP1), and four heme-thiolate peroxidases(Protein ID 170062, 146733, 126765, 156524; HTP1-4) similar toaromatic peroxygenases (APO) and chloroperoxidases (CPO) (Hof-richter et al., 2010). No genes encoding for lignin peroxidases (LiPs,EC 1.11.1.14) or versatile peroxidases (VPs, EC 1.11.1.16) werefound.

3.2. Multicopper oxidases (MCOs)

The H. irregulare draft genome sequence identified 18 genemodels coding for proteins with conserved multicopper oxidases(MCOs) domains (Table 1, Tables S1 and S3.1–S3.3). According tocriteria used by Hoegger et al. (2006), 16 gene models belongto laccases sensu stricto (Lcc1–Lcc8, Lcc10–Lcc16, Lcc18), and oneto the Fet3 ferroxidases (FeOx1, formerly annotated as Lcc9 andFet3), while the remaining Lcc17 with only one copper-binding sig-nature sequence is likely not a MCO encoding gene. Additionaldescriptions of the MCOs are given in Supplement 3.

3.3. Extracellular H2O2 generation and redox enzyme genes

We identified five gene models coding for putative glyoxal oxi-dase (Table 1 and Supplement 5). The predicted protein sequencescontain conserved residues coordinating the copper atom andforming the radical redox site of Glox enzyme similarly to P. chry-sosporium glyoxal oxidase 1) (Cys70, Tyr135, Tyr377, His378, andHis471) (Kersten and Cullen, 2007; Whittaker et al., 1999). Theputative Glox5 with the longest aa sequence contains four tandemcopies of a WSC domain (for cell wall integrity and stress compo-nent) in the N-terminus (Fig. S5 and Table 5S), notably Glox5 lacksa signal peptide suggesting that it is not secreted.

Thirty-five putative genes coding for glucose-methanol-choline(GMC) oxidoreductases (GOr), these including aryl-alcohol oxi-dases (AAO) and choline dehydrogenases (ChD) (Ferreira et al.,2005, 2009), were found in the H. irregulare genome (Table 1). All

Please cite this article in press as: Yakovlev, I.A., et al. Genes associated withirregulare show substrate-specific regulation. Fungal Genet. Biol. (2013), http:/

the predicted mature proteins contained the GMC oxidoreductasesignature 1 or 2 or both, except ChD2 (Table S6). Most of themwere predicted to be secretory proteins, except AAO7, ChD1,ChD3, ChD6, GOr 1 to GOr3, GOr6, GOr7, GOr10 to GOr12, GOr14and GOr15.

The 19 genes homologous to quinone oxidoreductases (QOr)were found in the H. irregulare genome (Table 1 and S7). The Hi-QOr1-19 encoded no secretory signal peptide as expected for intra-cellular function. .

3.4. Other putative lignin-converting genes

In the H. irregulare genome, we identified three (Supplement 8)hypothetical (Hyp) lignin converting genes highly induced in H.parviporum (Hp) during incipient decay of Norway spruce trees(Yakovlev et al., 2008). Post genome sequencing, HpHyp1corre-sponds to HiCup1, that belongs to 2-oxoglutarate (2OG) andFe(II)-dependent oxygenase superfamily (Cupin clan), HpHyp2 toHiMtc1 defined as a mitochondrial carrier protein 1, while HpHyp3corresponding to a H. irregulare gene encoding a hypothetical pro-tein with similarity to Choline dehydrogenase and relatedflavoproteins.

Two secreted cellobiose dehydrogenase encoding genes, con-taining the common cellobiose dehydrogenase/carbohydrate-bind-ing 9 domain (IPR015922), were predicted in the H. irregularegenome (Table 1). CDH1 contains one glucose-methanol-choline(GMC) oxidoreductase domain, whereas CDH2 in turn harnessesa carbohydrate-binding module 1 (CBM1).

3.5. Regulation of gene expression

Regarding manganese peroxidases, except for the down-regu-lated MnP3, the short-MnPs and the unusual GPx1 showed thehighest transcript levels upon fungal growth on the two sprucewood substrates (Fig. 1 and S1D). In comparison to the celluloseculture, MnP2-6 and MnP7 showed over twofold increase in genetranscript level on the woody substrates (Fig. S1D and Table 2).Heartwood induced additional upregulation of MnP1 while hostdefence xylem, reaction zone (RZ), induced additional up-regula-tion of GPx1 and MnP5 in comparison to cellulose. Among the genesup-regulated on the woody substrates, MnP2 showed the highestinduction level, the gene being clearly up-regulated also duringgrowth on lignin in comparison to the cellulose culture (Fig. S1Dand Table 2).

The woody substrates induced the highest transcript levels ofthe multicopper oxidases as well (Fig. 1 and S1A). In comparisonto the cellulose culture, fungal growth on heartwood and host de-fense RZ showed over twofold increase in transcript levels for thelaccases Lcc3, -4, -8, -10, -12, -13, -14, -15, -16 and for the ferroxi-dase FeOx1, the RZ induced additional up-regulation for Lcc1, 5and 6 as well. The transcript levels of Lcc5, -10, -15 and FeOx1showed over twofold increase upon fungal growth on the definedlignin medium in comparison to cellulose (Fig. S1A).

The glyoxal oxidase encoding genes Glox1 and Glox4 showedover twofold increase upon fungal growth on the woody substratescompared to the defined cellulose culture (Fig. S1E). RZ inducedup-regulation of Glox3 as well, whereas only Glox4 showed overtwofold increase in transcript level in the lignin culture in compar-ison to cellulose.

The aryl alcohol oxidase encoding genes AAO5, -9, -10, -11,and -12 showed over twofold increase upon fungal growth onthe woody substrates compared to the cellulose culture, heart-wood showing similar up-regulation of AAO1, -3, -4 and -6 as well(Fig. S1C). All the potential AAO encoding genes showed highertranscript levels on heartwood culture than on RZ culture. Thetranscript levels of AAO1, -5, -6 and -9 showed over twofold

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Table 1Number of genes putatively involved in lignin degradation in the genomes of several wood degrading basidiomycetes (based on cluster analyses in JGI genome browserb and theCoprinopsis cinerea databasec, and according to Floudas et al. (2012)F).

Gene familiesa Heterobasidionirregulare

Phanerochaetechrysosporium

Pleurotusostreatus

Schizophyllumcommune

Postia placenta Coprinopsis cinerea Laccaria bicolor

White-rotnecrotroph

White-rotsaprotroph

White-rotsaprotroph

White-rotsaprotroph

Brown-rotsaprotroph

Soil and leaf-littersaprotroph

Ectomycorrhi-zal,symbiotic

Genome sizee(Mbp) 33,6 35,1 34,3 38,5 90.9f 37,5 64.9f

Number of genes 13,405 10,048 11,603 13,210 9113 13,544 19,036Total Multicopper

oxidases (MCOs)18(17 g) 5 12 6 7 (5g) 17 15

Laccases 16 – 12 6 7 (5g) 17 9Ferroxidases 1 1 – – – – 2Ascorbate oxidases – – – – – – 4Others (1i) 4 – – – – –Total Class II

peroxidases7 16h 9 0 1 1 1

Lignin (LiP) – 10 – – – – –Manganese (MnP) – 5 – – – – –Versatile (VP) – – 3 – – – –Short–MnP 6 – 6 – – – –Low–redox – 1 – – 1 1 –Others 1 – – – – – 1Glyoxal oxidases 5 7 16 2 2 6 14Quinone reductases 19 24 17 19 13 4 23GMC oxidoreductasesd 35 34 40 21 21(45h) 19 13AAO 12 – – – – 11 –GOr 16 – – – – 1 –ChD 7 – – – – 1 –Pdh – – – – – 6 –Cellobiose

dehydrogenases2(1g) 2(1g) 1 3(1g) 0 2(1g) 0

a AAO – Aryl-alcohol oxidase; GOr – Glucose-methanol-choline oxidoreductase; ChD – Choline dehydrogenase; pdh – pyranose dehydrogenase.b http://gp-edge1.jgi-psf.org:1080/clustering/pages/cluster/clusters.jsf?organism=Hetan2.c http:/www.broadinstitute.org/annotation/genome/coprinus_cinereus/MultiHome.html.d Classification differing among different sources.e Haploid, if not otherwise depicted.f Dikaryon.g Floudas et al. (2012).h Martínez et al. (2009).i Lcc17 with only one copper-binding signature sequence.

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increase on lignin culture in comparison to cellulose culture(Fig. S1C).

The maximum transcript levels of the two genes potentiallyencoding cellobiose dehydrogenases were observed on heartwood,both genes showing over twofold increase in transcript level on theheartwood culture in comparison to cellulose alone (Fig. S1F andTable 2). A drastic down-regulation of the H. irregulare CDH1 andto a lesser extent for CDH2 was observed on the lignin substrate.

In general, maximum transcript levels of quinone oxidoreductaseencoding genes were observed in the heartwood culture, most ofthese genes showing comparably low transcript levels on the RZand the defined substrates (Fig. S1B and Table 2). Compared to thecellulose culture, QOr1, -2, -3, -7, -11 and 13 showed over twofold in-crease in transcript levels upon fungal growth on the natural sub-strates. Heartwood showed up-regulation of QOr5, -10 and 14 aswell, whereas only QOr10 showed over twofold increase in transcriptlevel on the lignin culture in comparison to the cellulose culture.

The three additional genes, encoding 2-oxoglutarate synthase,mitochondrial carrier protein 1, and the unknown Hyp3 with sim-ilarity to Choline dehydrogenase, all showed over twofold increasein transcript levels on the woody substrates in comparison to cel-lulose, maximum induction being observed in the heartwood cul-ture. None of the three genes was upregulated over twofold onlignin in comparison to the cellulose culture (Fig. S1F and Table 2).

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4. Discussion

Pathogenicity of the white-rot basidiomycetes in the H. anno-sum s.l. complex is manifested by an ability to challenge active

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bark defenses and the unique secondary xylem defense tissue[reaction zone wood (RZ)] characterized by polyphenols that havebeen actively deposited in tracheid and ray cell lumens by the liv-ing sapwood cells (e.g. Hietala et al., 2009). Furthermore, the tra-cheid cell wall lignin is a feeding obstacle that H. annosum s.l.must overcome during the dominant saprotrophic growth phasein heartwood. The ligninolytic repertoire of H. irregulare and tran-script level profiling of these genes upon colonization of coniferheartwood and defense reaction zone xylem described here sup-plement previous research that was primarily focused on lignocel-lulose degradation by wood decay fungi associated solely withdead trees and lumber.

4.1. Lignin-modifying class II and other peroxidases

In H. irregulare, six of the seven class II peroxidase genes encodefor the short hybrid-type of MnP enzymes (short-MnPs) (Hildénet al., 2005; Hofrichter et al., 2010), which is strikingly differentfrom the situation in the model white-rot species P. chrysosporiumwhich has five genes coding for the typical long MnP enzymes(long-MnPs) with the C-terminal extension (Martinez et al., 2004;Sundaramoorthy et al., 1994). Similar to long-MnP enzymes, theshort-MnPs oxidize Mn2+ ions to chelated Mn3+ complexes, whichas diffusible agents may mediate enzyme-generated oxidation atsome distance from the fungal hyphae (Hatakka et al., 2003; Hof-richter, 2002; Maijala et al., 2003). Existence and expression of theshort-MnPs seems to be common in the white-rot basidiomycetesPhlebia radiata (Hildén et al., 2005), P. brevispora, P. rivulosus (Hakalaet al., 2006), P. ostreatus (Ruiz-Dueñas et al., 2011) (Table 1) and T.versicolor (Floudas et al., 2012), and in the litter-decomposing agaric

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Fig. 1. The heat map with dendrograms showing transcript regulation of selected lignocellulose active H. irregulare genes during fungal growth on different substrates: Cel –cellulose; Lgn – lignin; RZ – powdered reaction zone wood of Norway spruce; HW – powdered heartwood wood of Norway spruce.

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mushroom A. bisporus (Lankinen et al., 2005). Based on protein se-quence similarity, the H. irregulare short-MnPs are structurally morerelated to VP and LiP than to the classic long-MnPs. The short-MnPsinclude the complete Mn-ion binding site but lack the exposed tryp-tophan codon present in LiPs and VPs.

Please cite this article in press as: Yakovlev, I.A., et al. Genes associated withirregulare show substrate-specific regulation. Fungal Genet. Biol. (2013), http:/

Class II secreted fungal peroxidases form a sister lineage toother heme-containing peroxidases, such as the DyP-type andheme-thiolate peroxidase (HTP; chloroperoxidase-like) superfami-lies, all recently re-classified according to protein sequence andcatalytic function (Hofrichter et al., 2010; Lundell et al., 2010).

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Table 2Genes showing over twofold increase in transcript levels upon fungal growth onlignin (Lgn), heartwood (HW) and reaction zone (RZ) media in comparison to cellulosemedium.

Gene families Number of genes up-regulated

Number of genesup-regulated onboth HW and RZ

Lgn HW RZ

MCOsLaccases (16)a 3 9 12 9Ferroxidases (1) 1 1 1 1Class II peroxidasesShort–MnP (6) 1 4 5 3Other (1) – – – –Fungal oxidasesGlyoxal oxidases (5) 1 2 3 2Quinone reductases (19) 1 9 6 6AAO (12) 4 9 5 5Cellobiose dehydrogenases (2) – 2 – –

a The number of genes in each gene family is given in brackets.

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One DyP-type and four heme-thiolate peroxidase encoding geneswere also found in the H. irregulare genome. Catalytically, theDyP and heme-thiolate peroxidases are partially overlapping withthe fungal class II peroxidases in their ability to oxidize lignin-likedimeric model compounds and veratryl alcohol (Hofrichter et al.,2010), and thereby these novel oxidoreductases may well be in-cluded in the pool of lignin-modifying enzymes.

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4.2. Multicopper oxidases (MCOs)

Employing the classification suggested by Hoegger et al. (2006),16 genes encoding proteins similar to laccases sensu stricto wereidentified in the draft genome of H. irregulare. In addition, we iden-tified one short laccase related gene (Lcc17) and one gene (FeOx1,former Lcc9) coding for a potential ferroxidase. This is the highestnumber of genes encoding for laccases among the white-rot basid-iomycete genomes sequenced to date (Table 1), and it is similar tothe laccase multiplicity reported for the coprophilic mushroom C.cinerea (17 Lcc; (Kilaru et al., 2006). That 12 of 19 of the laccasesare up-regulated on the phenol rich RZ (Table 1), hint at a rolefor these laccases in metabolizing or detoxifying phenolics suchas lignans in this host defense tissue.

Iron has an important role in lignin degradation. The H. irregulareferroxidase gene (FeOx1/Lcc9) is located within less than 400 bp ofan iron permease gene, which is homologous to S. cerevisiae ftr1.The fet3/ftr1-based transport system, required for high-affinity ironuptake, has been described in the white-rot fungus P. chrysosporium(Larrondo et al., 2007) and has also predicted in the ectomycorrhi-zal fungus Laccaria bicolor (Courty et al., 2009).

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4.3. Extracellular H2O2 production and redox enzyme genes

An important component of the ligninolytic systems of white-rot fungi is hydrogen peroxide which is required as an oxidant inperoxidative reactions. We found more than 60 fungal oxidases(i.e. the copper radical oxidases and GMC oxidoreductases) whichcan play a role in generation of extracellular H2O2 (Table S1). Gly-oxal oxidase and aryl-alcohol oxidase (AAO) generate extracellularH2O2 by oxidizing aldehydes released during lignin breakdown orsynthesized de novo by fungi (Ferreira et al., 2009; Kersten and Cul-len, 2007; Ruiz-Dueñas and Martínez, 2009). Five glyoxal oxidaseand 12 AAO gene models were identified in the H. irregulare gen-ome. This number is similar to that found in other white-rot fungi,providing H. irregulare with a large repertoire of enzymes that en-able the use of an array of reducing substrates.

Please cite this article in press as: Yakovlev, I.A., et al. Genes associated withirregulare show substrate-specific regulation. Fungal Genet. Biol. (2013), http:/

Fungal quinone oxidoreductase (QOr) and cellobiose dehydro-genase (CDH, EC 1.1.99.18) are also linked to lignin degradation(Gomez-Toribio et al., 2009; Guillén et al., 1997; Varela et al.,2000). In the brown-rot basidiomycete Gloeophyllum trabeum, aquinone reductase may drive extracellular Fenton systems via re-dox cycling of secreted fungal phenols and quinones (Cohenet al., 2002; Hammel and Cullen, 2008; Jensen et al., 2001). Thehigh number of genes encoding quinone oxidoreductases in thegenome of H. irregulare (19) is similar to that found in saprophyticwhite rot and brown rot fungi (Table 1). CDH, generating highlyreactive hydroxyl radicals that participate in lignocellulose depoly-merisation (Henriksson et al., 2000a; Kersten and Cullen, 2007), isable to use cellobiose as reducing substrate while donating elec-trons to quinones and may oxidize phenolic intermediates formedduring lignin decomposition (Henriksson et al., 2000a,b). In linewith such a role, the H. irregulare CDH1 transcript level increaseon woody substrates containing both lignin and cellulose but dropdramatically when cellulose is not present, such as on the ligninsubstrate devoid of cellulose. The expression of the other potentialH. irregulare cellobiose dehydrogenase CDH2 show a similar trendbut the drop on substrate lacking cellulose is strikingly less dra-matic than for CDH1, suggesting that CDH2 has less importancein providing cellobiose derived radicals toward lignocellulosedegradation.

4.4. Transcript regulation of ligninolytic genes during colonization ofheartwood and defense xylem

No obvious differences were observed in the regulation of geneexpression of the short-MnP genes between the Norway spruce RZand HW cultures, the latter having low extractive content. How-ever, the genes encoding enzymes generating extracellular H2O2

(i.e. AAO, GlOx) from lignin degradation products as well as theother profiled gene products participating in lignocellulose degra-dation (two CDH and majority of the 19 QOR) were highly ex-pressed in HW in comparison to RZ. This finding agrees with thedata of Nagy et al. (2012) who showed that the rate of selectivedelignification of RZ by H. parviporum is slow compared to thatof heartwood. In vitro, the short-MnP3 from P. radiata was ableto oxidize and convert phenolic and aromatic compounds dis-solved from milled pine wood, and the process was enhanced inthe presence of lipids and Mn2+ ions (Hofrichter et al., 2001). Theenzyme also demonstrates some Mn-independent peroxidationactivity against phenolic compounds and amines, thus catalyticallyresembling versatile peroxidases (VP) (Hatakka et al., 2003; Hein-fling et al., 1998). The protein 3D structural similarity of fungalshort-MnPs and VPs (Hildén et al., 2005; Ruiz-Dueñas et al.,2011) furthermore suggests a coinciding action on wood polyphe-nols, lignans and lignins. Taken together the substrate-specifictranscript level profiles of the short-MnPs of H. irregulare and theother profiled genes downstream MnP activity on lignin, it isconceivable that the fungus employs short-MnP in the degradationof defense polyphenols as well as lignified tissues per se.

Most lignin-degrading white-rot fungi produce laccases in addi-tion to heme-containing peroxidases (Hatakka, 1994; Hoeggeret al., 2006; Lundell et al., 2010; Niku-Paavola et al., 1990). The lig-nin degrading ability of a laccase-deficient mutant of the white-rotfungus P. cinnabarinus was impeded (Bermek et al., 1998). In thework reported here, most of the laccase encoding genes were sim-ilarly over-expressed on both HW and RZ, and their substrate-spe-cific transcript level patterns generally corresponded well withthose of the short-MnPs and generic peroxidase, implying a role,together with the class II peroxidases, in degradation of wood lig-nin. In contrast, the subset of laccases (Lcc1, Lcc3, Lcc5, Lcc6 andLcc13) over-expressed on RZ in comparison to HW may participatein detoxification of host defense-associated polyphenols that

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impregnate the secondary cells walls and cell lumens. It has beenproposed previously that Heterobasidion laccases participate indetoxification of the xylem defense associated lignans (e.g. Johans-son et al., 1976), and that the ability of laccases to alter the toxicityof phenols is related to the formation of phenoxy radicals (e.g. Bol-lag et al., 1988). Recently, Areskogh et al. (2010) and Maijala et al.(2012) have shown in laboratory conditions that fungal laccasesoxidized some phenolic compounds (i.e. phenolic dimers and olig-omers) commonly present in the RZ of Norway spruce, a processresulting in the formation of polymeric compounds. The pH of Nor-

way spruce RZ is approximately 7, heartwood typically showing apH close to 5 (Shain 1971), and most of the characterized fungallaccases have clearly acidic pH optima (Baldrian, 2006; Hoeggeret al., 2006). It is thus noteworthy that the RZ induced Lcc6 andLcc13 show affinity with the neutral pH active Lcc4 of the corticoidbasidiomycete Thanathephorus cucumeris (anamorph Rhizoctoniasolani) (Wahleithner et al., 1996), suggesting a role for these twoenzymes in the modification the RZ.

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5. Conclusions

The first overview of the H. irregulare genome revealed no obvi-ous features in gene number or organization in comparison to otherwhite-rot fungi in order to suggest any higher efficiency or selectiv-ity for lignin or lignocellulose degradation (Olson et al., 2012). How-ever, the H. irregulare genome showed a high number of fungaloxidases and an expansion of the MCOs. Moreover, in H. irregularas with the white-rot species P. ostreatus, the exclusively short-MnPs and numerous laccases likely play a fundamental role indecomposition of wood lignin. The observed gene-specific expres-sion patterns on RZ compared to H. irregulare growth on heartwood,and cellulose alone, suggest divergent roles for individual membersof these oxidoreductive enzyme families in wood degradation andelimination of host tree defense phenolics. Response of H. irregulareto host RZ included up-regulation of individual laccases, which iscompatible with the need to detoxify the phenols such as lignansthat impregnate this defense tissue. In contrast, the other oxidore-ductases involved in lignin modification, such as the hydrogen per-oxide producing glyoxal oxidase and aryl-alcohol oxidases showedgenerally lower transcript levels on spruce RZ than on heartwood.This is in agreement with the slow delignification rate of RZ by Het-erobasidion species observed in previous studies.

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6. Uncited references

Giardina et al. (2010), Hoegger et al. (2004), Kuan and Tien(1993), Kumar et al. (2003), Larkin et al. (2007), Messerschmidtand Huber (1990), Mäkelä et al. (2010), Notredame et al. (2000)and Tamura et al. (2011).

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Acknowledgments

We thank Åke Olson (Swedish University of AgriculturalSciences) for providing the H. irregulare strain TC-32-1 for thisstudy, which was supported by the Norwegian Research Council(Grants #315052; 135901). Heterobasidion genome sequence datawere produced by the US Department of Energy Joint GenomeInstitute http://www.jgi.doe.gov/ in collaboration with the usercommunity. Pierre-Emmanuel Courty gratefully acknowledgesthe Swiss National Science Foundation for current support(PZ00P3 136651). Taina Lundell is grateful for the research ProjectOx-Red grant #113833 from the Academy of Finland.

Please cite this article in press as: Yakovlev, I.A., et al. Genes associated withirregulare show substrate-specific regulation. Fungal Genet. Biol. (2013), http:/

Appendix A. Supplementary material

Supplementary data associated with this article can be found, inthe online version, at http://dx.doi.org/10.1016/j.fgb.2013.04.011.

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lignin degradation in the polyphagous white-rot pathogen Heterobasidion/dx.doi.org/10.1016/j.fgb.2013.04.011