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Environmental Microbiology (2014) 16(6), 1654–1667

doi:10.1111/1462-2920.12321

Transcriptomic profiles of Heterobasidion annosum under abiotic stresses and during saprotrophic growth in bark, sapwood and heartwood

Tommaso Raffaello,1,2† Hongxin Chen,1,3† Annegret Kohler4 and Fred O. Asiegbu1,2,3* 1 University of Helsinki, Department of Forest Sciences, Latokartanonkaari 7, 00014, Helsinki, Finland. 2 Viikki Doctoral Programme in Molecular Biosciences (VGSB), Viikinkaari 9, 00014, Helsinki, Finland. 3 Helsinki Graduate Program in Biotechnology and Molecular Biology (GPBM), Viikinkaari 9, 00014, Helsinki, Finland. 4 UMR 1136 INRA/Université de Lorraine, Interactions Arbres/Micro-organismes, INRA, Institut National de la Recherche Agronomique, Centre INRA de Nancy, 54280, Champenoux, France. Summary The success of many wood decaying fungi lies in their ability to overcome unfavourable environmental conditions within and outside of litter and wood debris. Although so much has been learned about the ecology, taxonomy and physiology of several wood decaying basidiomycete fungi, the molecular basis for their survival in a diverse range of substrates and ecological habitats has been very little studied. Using the wood decay fungus (Heterobasidion annosum s.s.) as a model, we investigated its transcriptomic response when exposed to several environmental stressors (high and low temperature, osmotic stress, oxidative stress and nutrient starvation) and during growth on specific pine wood compartments (bark, sapwood and heartwood). Among other genes and pathways, we documented the specific induction of the major facilitator superfamily 1 and cytochrome P450 families at low temperature, and protein kinases together with transcription factors during starvation. On the other hand, during saprotrophic growth, we observed the induction of many glycosyl hydrolases, three multi-copper oxidases (MCO), five manganese peroxidases (MnP) and one oxidoreductase which are specific for wood degradation. This is the first study Received 9 July, 2013; accepted 26 October, 2013. *For correspondence. E-mail [email protected]; Tel. (+358) 9191 58109; Fax (+358) 9191 58100. †Contributed equally as first authors.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd

providing insights on the potential mechanisms for adaptation to abiotic stresses and pine heartwood degradation in H. annosum s.s. Introduction Wood degradation by microorganisms is of vital importance as this process is central to nutrient and carbon cycling. Ecologically, the availability of different utilizable organic substances is pivotal in maintaining biodiversity and dynamics of community development, functioning and microbial succession in the ecosystem. Several functional groups of fungi (necrotrophs, mycorrhizal, saprotrophic wood and litter decomposers) play central roles in this ecological process (Boddy, 1993). Although so much has been done to further our understanding of ecophysiological factors determining wood decay patterns and the consequences to interspecific fungal interaction (Boddy, 1993), very little is known on a genomic level on the response of fungi to environmental heterogeneity (e.g. growth on complex substrates). Heterobasidion annosum sensu stricto (s.s.) used primarily as a representative model in this study is one of the main cause of root and butt rot disease of conifers and the most economically important diseases of forest trees in the Northern Hemisphere (Asiegbu et al., 2005). In addition to parasitic life style, this fungus can also live as a saprotroph on dead wood tissues, which contributes substantially to nutrient recycling by returning vital nutrients locked up within wood tissues back to the soil (Filip and Morrison, 1998). Like all wood decayers, Heterobasidion root rot influences species composition, ecosystem diversity, stand structure, stand density and direction and rate of forest succession (Garbelotto et al., 1998), and provides habitat for a wide variety of animals (Filip and Morrison, 1998). In nature, H. annosum s.s. has been reported to persist and survive for decades in dead roots left underground after stump removal (Lygis et al., 2004). In these dead tree tissues, the fungus continues to degrade all the major components of wood including lignin (Blanchette, 1991; Daniel et al., 1998). During in vivo growth within woody tissues, it is known that the fungus secretes a wide range of enzymes which are used to exploit a variety of carbon

Heterobasidion annosum sources such as starch, cellulose, pectin and lignin (Asiegbu et al., 2005). The ability to grow as a saprotroph on lignocellulosic biomass provides the fungus with sufficient nutrients vital for its survival. Recently, the genome of a closely related North American species, Heterobasidion irregulare was sequenced (Olson et al., 2012).The analysis revealed that the H. irregulare genome encoded a repertoire of lignocellulose degrading active enzymes including 179 glycoside hydrolases (GHs), eight manganese peroxidases (MnPs) and 17 multicopper oxidases (MCOs) (Davies and Henrissat, 1995; Floudas et al., 2012; Olson et al., 2012). In this earlier publication (Olson et al., 2012), we focused on understanding fundamental differences in the two fungal strategies (parasitism and saprotrophic) important for nutrient acquisition. However, the aspects on the ability of Heterobasidion species to respond to several abiotic stresses was not addressed as well as the behaviour of the fungus during growth on specific wood substrates (heartwood, sapwood and bark). In this study, we took advantage of the availability of genome of H. irregulare to set up a transcriptomic microarray analysis for H. annosum s.s. where different conditions mostly abiotic stressors and growth in heartwood not strictly addressed in Olson and colleagues (2012) were tested: particularly growth on separated wood materials like pine bark, sapwood and heartwood. However, despite the importance of GHs, MnPs and MCOs in wood degradation as outlined in the earlier publication (Olson et al., 2012), a detailed investigation on which of these genes are actively induced during saprotrophic growth on sapwood, bark and heartwood is still missing in the literature, and very little is known about genes and pathways involved in the specific responses to unfavourable environmental stresses. Therefore, by using a microarray gene expression profiling, we evaluated how primary metabolic transcription was affected due to responses to environmental cues and growth on diverse wood tissues. The availability of global transcriptomic information could provide the much needed insight on possible adaptive mechanisms of this wood rotting fungus. Materials and methods Fungal strain and growth conditions H. annosum s.s. (isolate 03012) was kindly provided by Kari Korhonen, METLA Finland. For the control, the fungus was first grown on solid malt extract glucose (MEG) media (0.5% malt extract, 0.5% glucose and 2% agar) at 20°C for 2 weeks. A 0.5 cm × 0.5 cm agar block was then cut from the plate and transferred to 100 ml liquid MEG media. The fungus was grown at 20°C for another 2 weeks, harvested by filtration and immediately frozen in liquid nitrogen. Three biological replicates were prepared for

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each condition, and total RNA was extracted according to previous studies (Raffaello et al., 2012). Growth at various temperatures, osmotic conditions, oxidative stress and nutrient starvation To reduce the complexity of environmental treatments and conditions, the experiments on abiotic stresses were performed in simple laboratory media. The effect of temperature was studied partly because the fungi is known to survive low to moderate temperature regimes prevalent in Nordic countries and it is one of the abiotic factors often exploited in the management of Heterobasidion infection. In the present study, for the growth of H. annosum s.s. at 8°C or 27°C, the fungus was grown in liquid MEG medium for 2 weeks for a reasonable fungal biomass to accumulate and incubated for 3 weeks at the respective temperature. The effect of osmotic conditions on the growth and survival of the fungus was necessitated because of high accumulation of the ion Ca2+ often observed in decayed wood (Oliva et al., 2011). For the osmotic conditions, the fungus was grown as in the control followed by exposure to 0.5 M of either CaCl2 or NaCl and incubated for 60 min. For H. annosum s.s. in oxidative conditions, the fungus was grown in liquid MEG medium for 2 weeks followed by exposure to 5 mM of H2O2 and incubated for 60 min. Finally, for H. annosum s.s. in nutrient starvation condition, the fungus was grown as in the control: the mycelia was then washed three times with autoclaved Milli-Q (MQ) water, placed in 100 ml autoclaved MQ water with 100 μl 0.2 g ml−1 glucose solution, and incubated for 5 days. Samples were then harvested and frozen in liquid nitrogen followed by RNA extraction. Growth on pine bark, sapwood and heartwood The Scots pine wood chips (bark, sapwood and heartwood) were homogenized to a powder. Eight grams of each wood material were autoclaved, mixed with 8 ml of low nitrogen media (NH4NO3 0.6 g l−1, K2HPO4 0.4 g l−1, KH2PO4 0.5 g l−1, MgSO4·7H2O 0.4 g l−1) followed by the addition of 8 ml of sterile distilled water. Three small agar blocks pre-colonized with H. annosum were placed in contact with each wood material and incubated for 3 months in the dark at 20°C. Samples were then harvested and frozen in liquid nitrogen followed by RNA extraction. cDNA preparation for microarray experiments The RNA samples from each condition were processed as follows: 2 μg of total RNA was treated with DNase I (Promega, Finland), and incubated for 30 min at 37°C

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

1656 T. Raffaello, H. Chen, A. Kohler and F. O. Asiegbu followed by DNase I inactivation at 65°C for 10 min. The treated RNA was then purified using RNeasy MinElute Cleanup Kit according to manufacturer’s instruction (Qiagen, Finland) and eluted in 10 μl Nuclease Free water. RNA quality and integrity was assessed using Agilent RNA 6000 Nano Kit and Agilent 2100 Bioanalyzer following manufacturer’s instruction (Agilent Technologies, Germany). Total RNA (100 ng) was subjected to reverse transcription and amplification using whole transcriptome amplification (WTA) kit according to manufacturer’s instruction (Sigma-Aldrich, Finland). In order to avoid transcript abundance alteration in the sample, the minimum number of 17 amplification cycles was used. The cDNA generated with the WTA kit was purified with GenElute PCR Clean-Up kit (Sigma-Aldrich, Finland) and eluted in 50 μl nuclease free water. The cDNA was run on 1.5% agarose gel to assess the integrity and the range of fragment length obtained after the amplification with the WTA kit. Microarray hybridization and data analysis Due to the high level of correlation in the gene expression and sequence conservation among the various Heterobasidion species [H. parviporum, H. abietinum, H. annosum s.s.; (Lunden et al., 2008); H. irregulare and H. annosum s.s.; (Raffaello and Asiegbu, 2013)] as documented in previous study, it was concluded that the cDNA array of one species can be reasonably used to study gene expression in the others. Therefore, in this study, 5 μg of cDNA from H. annosum s.s. for each condition was sent to NimbleGen (Roche, Iceland) for the hybridization on H. irregulare customized microarray. The microarray was based on the H. irregulare gene predictions from the DOE Joint Genome Institute (JGI) sequencing project. The microarray composed of 74 000 probes 60 nucleotides long (five probes per gene model). Non-specific oligos on the array were removed from the analysis if they shared more than 90% homology with a gene model different from the one it was made for. After the non-specific oligos filtering, the raw file was composed of 11 578 gene models with expression data. The NimbleGen microarray raw data was normalized with ArraySTAR (DNASTAR, France). The microarray data are available in the Gene Expression Omnibus (GEO, http:// www.ncbi.nlm.nih.gov/geo/) database (accession number GSE39805). Cluster analysis and differential expression The normalized transcript expression values for each condition were analysed using the Gene Pattern online platform to discover the upregulated and downregulated genes [http://www.broadinstitute.org/cancer/software/

genepattern/, Broad Institute, (Reich et al., 2006)]. The gene expression dataset was filtered with the Preprocess Dataset module with the following settings: floor threshold 20, ceiling threshold 60 000, min fold change 1, and leaving the rest as default. The pre-processed dataset was used in the Comparative Marker Selection module to reveal the upregulated and downregulated genes for each condition. The asymptotic P-values calculation in the standard independent two-sample t-test and the default settings were applied to calculate the statistical significance of the differentially expressed genes. Finally, the total list of differentially expressed genes was then filtered to obtain a statistically significant list of upregulated and downregulated genes according to the following criteria: false discovery rate (FDR) < 0.05 for all samples except for salts (FDR < 0.1) and H2O2 (FDR < 0.25), fold change (FC) > 2 cut-off for all the conditions. The list of glycosyl hydrolase, Cu-oxidase and peroxidase genes was retrieved by searching with the terms ‘Glyco_hydro’, ‘Cu-oxidase’ and ‘peroxidase’ respectively in the pre-processed dataset. The cluster analysis was performed on the Preprocess Dataset using the Hierarchical Clustering module with the following settings: column (samples) and row (genes) distance measure set on Pearson Correlation and leaving the rest as default. The Venn diagrams were prepared with the Venn Diagram module in the Gene Pattern online tool. Quantitative real-time polymerase chain reaction (qPCR) and data analysis Transcript abundance of a total of 51 genes selected based on functional relevance and expression profile was further analysed using quantitative real time polymerase chain reaction (qPCR) with the aid of Light Cycler 480 II (Roche, Finland). Transcript sequences were retrieved from the JGI H. irregulare genome browser (http://genome.jgi-psf .org/Hetan2/Hetan2.home.html). Primers were designed using the Roche Universal ProbeLibrary Assay Design Center (http://www.roche-applied-science.com). Each qPCR reaction was carried out in a total volume of 15 μl as follows: 5.5 μl cDNA (5 ng μl−1), 1 μl Forward primer (10 μM), 1 μl Reverse primer (10 μM), and 7.5 μl MasterMix using 384-well plates (Roche, Finland). The amplification cycle was as follows: pre-incubation at 95°C for 5 min, denaturation 94°C for 10 s (4.8°C s−1), annealing at 60°C for 10 s (2.5°C s−1), extension at 72°C for 10 s (4.8°C s−1), 45 cycles of amplification and final extension at 72°C for 3 min. A melting curve analysis was also performed to assess primers specificity. Primers’ information is summarized in Table S1. Quantitative PCR data were analysed with the Light Cycler 480 II Software 1.5.1. The standard curves and

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

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Fig. 1. Venn diagrams relative to the number of upregulated genes in H. annosum s.s. in different conditions (FDR < 0.05 for all samples except for salts (FDR < 0.1) and H2O2 (FDR < 0.25), Fold Change > 2 cut-off for all the conditions).

the primers’ efficiency (E) were calculated using the Absolute Quantification/2nd Derivative Max method. Transcript levels for each gene were calculated by the Advanced Relative Quantification method using both Tryp Met (Tryptophan metabolism) and RNA Pol3 TF (RNA Polymerase 3 Transcription Factor) as reference genes. The stability of the two reference genes has been investigated and found to be generally stable under the conditions studied by qPCR (Raffaello and Asiegbu, 2013). The primers’ specificity for each gene was analysed by the Melt Curve Genotyping method in the Light Cycler 480 II Software 1.5.1. Statistical significance of the qPCR data was assessed with one-way analysis of variance (ANOVA) statistical test using GraphPad Prism software (GraphPad Software, Inc., USA). Results The pre-filtered data relative to the transcriptomic profiles of all the conditions studied (Tables S2–S10) was subjected to hierarchical cluster analysis (Fig. S1). All the biological replicates cluster together, indicating reproducibility and a consistent transcriptional pattern within the biological replicates. The general hierarchical clustering analysis revealed a first level of separation between the saprotrophic growth

on pine wood (heartwood, sapwood and bark) and the abiotic stressors (Control, 27°C, 8°C, CaCl2, NaCl, H2O2 and Nutrient Starvation), which indicates a different fungal response and adaptation to these diverse conditions. There were also separate clades for control and temperature stress, saline stress and oxidative stress and nutrient starvation (Fig. S1). The comparative transcriptome analysis revealed that compared to the control, 13 genes were upregulated in both 27°C and 8°C (Fig. 1A), 39 in both CaCl2 and NaCl (Fig. 1B) and 57 in both H2O2 and nutrient starvation (Fig. 1C). In pine wood, 529 genes were induced in all wood compartments. Bark and heartwood material induced almost the same number of specific transcripts in H. annosum (148 and 174 respectively, Fig. 1D). However, a total of 448 transcripts were specifically induced during the fungal growth on sapwood (Fig. 1D). The 10 most induced genes with predicted function in each condition are listed in the Table S11. Temperature stress Several genes involved in lipid metabolism were affected during temperature stress. Compared to the control, a phospholipid methyltransferase (ID 117248) was strongly induced at 8°C (Fig. 2A) and several predicted cytochrome

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

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Fig. 2. Expression of different genes involved in response to (A) temperature, (B) osmotic stress and (C) H2O2 and starvation in H. annosum s.s. quantified by qPCR. The expression of each gene was normalized using two reference genes (Tryptophane Metabolism and RNA Polymerase 3 Transcription Factor). One-way ANOVA, followed by Dunnet post-test, *P < 0.05, **P < 0.01, ***P < 0.001 compared to control.

P450s were upregulated in both 8°C and 27°C. In particular, two P450s (ID 103794 and ID 37362) were strongly induced at 8°C (Fig. 2A). Interestingly, one transcription factor (ID 57899) was upregulated specifically at 27°C, one

DNA ligase (ID 101918) at 8°C and up to 17 predicted genes of the major facilitator superfamily 1 (MFS-1) were found upregulated in temperature stress of which one (ID 63463) was extremely induced at 8°C (Fig. 2A).

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

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Fig. 3. Number of different classes of upregulated genes in H. annosum s.s. in different conditions (FDR < 0.05 for all samples except for salts (FDR < 0.1) and H2O2 (FDR < 0.25), Fold Change > 2 cut-off for all the conditions).

Salt stress The H. annosum s.s. response to salt stress was investigated by the exposure of the fungal mycelia to high concentration of NaCl and CaCl2. CaCl2 condition was characterized by a significantly higher amount of upregulated genes (415) compared to NaCl (89) (Tables S4 and S5). Twenty predicted protein kinases were induced in CaCl2 (Table S4), one of which was highly induced in CaCl2 and to a less extent in NaCl (Fig. 2B). Interestingly, a predicted tyrosinase (ID 146209) and a predicted Na+/Ca2+ exchanger (ID 145724) were induced only in CaCl2 but not in all the other conditions (Fig. 2B). Among the downregulated genes in CaCl2, two predicted pheromone receptors which are orthologous of the Saccharomyces cerevisiae Ste2p protein were found to be repressed compared to the control, especially for ID 181123 (Fig. 2B). A predicted gene (ID 147759) similar to the S. cerevisiae glycerol-3-phosphatase gene (GPP2) involved in glycerol metabolism and osmotic stress response was induced in both conditions compared to the control but with a stronger induction in CaCl2 than NaCl (Fig. 2B).

Oxidative stress and starvation Several genes involved in glyoxylate metabolism [malate synthase (ID 150533), citrate synthase (ID 154376) and isocitrate lyase (ID 65122)], one protein kinase (ID 26194), two predicted phosphatidylinositol-4-phosphate 5-kinases (PIP5K, ID 40533 and ID 148659) and at least two fungal specific transcription factors (ID 174016 and ID 149265) were specifically induced during starvation (Fig. 2C). Although the amount of upregulated transcripts compared to the control in H. annosum s.s. exposed to H2O2 was lower than other conditions (Tables S2–S10), we were able

to identify genes putatively involved in oxidative stress response. Several predicted cytochrome P450 transcripts were induced in hydrogen peroxide (Table S6). In particular, one specific cytochrome P450 (ID 174745) was induced both in oxidative and temperature conditions but its level remained low in all the other conditions in this study (Fig. 2C). We also confirmed a specific induction of a flavin oxidoreductase (ID 122807) involved in intracellular redox activity in hydrogen peroxide (Fig. 2C). Pine bark, sapwood and heartwood Three large gene families were generally induced in different wood compartments: GH, cytochrome P450 and MFS-1 transcripts (Fig. 3). The growth of H. annosum s.s. on pine wood compartments stimulated the production of different enzymes for wood degradation. The carbohydrate active enzymes (CAZy) specifically induced in the different wood materials are listed in Table S12. A total of 31 predicted GH genes were found upregulated in heartwood, 20 in sapwood and 23 in bark compared to the control (Fig. 3). Some were specifically induced in pine heartwood and bark (Supporting Information Fig. 2A), while others were induced only in heartwood (Fig. S2B). Nine out of 10 predicted GH61 genes (Fig. S2C), two predicted GH12 family genes, and some members of the GH1, GH5 and GH10 family were specifically induced during growth on heartwood (Fig. 4). Three predicted GH28 family genes and several other GH transcripts (GH15, GH18, GH30, GH35, GH53 and GH88) were induced in all wood constituents, with a stronger induction in heartwood and bark compared to sapwood, except GH15 and GH18 which conversely showed a higher expression level in sapwood compared to bark and heartwood (Fig. 4). Interestingly, none of the GH displayed a selective expression only in bark or only in sapwood.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

1660 T. Raffaello, H. Chen, A. Kohler and F. O. Asiegbu Fig. 4. Expression levels of GH genes during saprotrophic growth of H. annosum s.s. on pine wood material quantified by qPCR. The expression of each gene was normalized using two reference genes (Tryptophane Metabolism and RNA Polymerase 3 Transcription Factor). One-way ANOVA, followed by Dunnet post-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

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Fig. 5. Cluster analysis of H. annosum s.s. (A) multicopper oxidases and (B) oxidoreductases with expression value in the microarray. Indicated with ‘*’ and in red are genes specifically induced in wood and validated by qPCR. (C) Expression levels of selected laccase and oxidoreductase genes (indicated by ‘*’ in the cluster analysis) during saprotrophic growth of H. annosum s.s. on pine wood material quantified by qPCR. The expression of each gene was normalized using two reference genes (Tryptophane Metabolism and RNA Polymerase 3 Transcription Factor). One-way ANOVA, followed by Dunnet post-test, *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001 compared to control.

Besides GH genes, five manganese peroxidases (MnP), one cytosolic oxidoreductase and three MCOs were found specifically induced only during saprotrophic growth (Fig. 5). In particular, MCO transcripts (163392, 165788 and 181063) displayed stronger expression in bark, sapwood and heartwood compared to the other wood materials, respectively (Fig. 5). On the other hand, four MnPs (ID 108376, ID 106089, ID 181068 and ID 101580) had higher expressions in heartwood than bark and sapwood, while another one (ID 127157) had higher expressions only in bark (Fig. 5). Moreover, two MnPs (ID 181068 and ID 106089) in heartwood, one MCO (ID

181063) and one MnP (101580) in sapwood and one MCO (ID 181063) and one MnP (181068) in bark were among the top 10 upregulated genes compared to the control (Table S11). Discussion The ability of fungi to sense and survive in diverse range of environmental stress as well as actively engage in nutrient acquisition has always been a central issue in fungal biology research. However, finding a balance between natural field conditions and laboratory studies in understanding fungal life style is not without drawbacks.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

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Several scholars have used artificial media to study the interactions and mechanisms of fungal competition (Carruthers and Rayner, 1979; Magan and Lacey, 1984a,b). Other authors have used secreted metabolites from liquid cultures of Bacillus amyloliquefaciens to test its antifungal properties (Alfonzo et al., 2012). Although impressive results have been obtained from such laboratory studies, some authors have expressed doubts about extrapolating results from such studies to field situations (Dowding, 1978). However, Magan and Lacey (1984a,b) have shown that such methods could offer alternative approaches for understanding fungal life styles. Interesting correlations have however been reported on tests carried out on the behaviour of fungi in artificial media and natural conditions and their ecological roles (Rayner, 1978; Boddy and Rayner, 1983). In this study, we used the global transcriptomic approach to monitor the response of H. annosum s.s. to several abiotic stresses and during saprotrophic growth on specific wood compartments. H. annosum s.s. response to environmental stressors The evident separation in the cluster analysis between the transcriptome profiles in saprotrophic conditions compared to the abiotic stresses suggests specialized adaptation to these extremely diverse conditions. The presence of sub-clusters represented by the oxidative-starvation, temperature, and salt stress indicates that H. annosum s.s. can use common pathways to adapt in similar type of stressors. The few numbers of transcripts recorded at 27°C probably suggest that a small increase of temperature was not sufficient to cause a major perturbation in the transcriptome profile since the optimal growth temperature of H. annosum s.s. is in the range of 22°C–26°C (Cowling and Kelman, 1964). However, a particular transcription factor that was upregulated specifically at 27°C could be an indication of a temperature-specific response. The general physiological adaptation of fungi to low temperature is characterized by the accumulation of trehalose and cryoprotectant sugars like glycerol and mannitol, changes in the lipid membrane composition with an increase of unsaturated fatty acids and the production of anti-freeze proteins (Robinson, 2001). When H. annosum s.s. was exposed to low temperature, a phospholipid methyltransferase similar to the yeast PEM2 was strongly induced (Kodaki and Yamashita, 1987). Phospholipid methyltransferases are involved in the synthesis of phosphatidylcholine from phosphatidylethanolamine (PE) in yeast. From this point of view, the enrichment of the fungal membrane in phosphatidylcholine may be important in cold stress adaptation. Also, the expression of a predicted gene similar to S. cerevisiae SNF7, which is

involved in the formation of the endosomal sorting complexes, required for transport III (ESCRT-III) which are crucial for the endocytic pathway, was increased at 8°C compared to the control (Babst et al., 2002). The change of lipid and protein composition in plasma membrane to allow cold stress adaptation requires a constant turnover of the plasma membrane components. The upregulation of a predicted SNF7 orthologous gene may strongly suggest the involvement of multivesicular body (MVB) structures in low temperature adaptation for H. annosum s.s. Several predicted cytochrome P450s were induced in both 8°C and 27°C. The cytochrome P450 is a superfamily of monooxygenases which are capable of oxidizing a variety of endogenous or exogenous substrates (Meunier et al., 2004). In this study, several cytochrome P450 transcripts were induced when the temperature was the only changing parameter. Besides cytochrome P450s, members of the MFS-1 family were also induced by the temperature shift especially in low temperature condition. MFS-1 genes are predicted to be transmembrane transporters (antiporters, symporters and uniporters) of a wide range of substrates (Marger and Saier, 1993; Law et al., 2008). The induction of both cytochrome P450 and MFS-1 genes indicate that these monooxygenases may take part in intracellular pathways required for temperature adaptation or are involved in detoxification of molecules. The high concentration of CaCl2 and NaCl has activated the salt stress regulation and ion homeostasis in H. annosum s.s. Some genes are induced in both CaCl2 and NaCl, such as an orthologue of the Cryptococcus neoformans calcium/calmodulin-dependent protein kinase I CAMK1 and a predicted protein similar to the C. neoformans glycerol-1-phosphatase and S. cerevisiae glycerol-3-phosphatase (GPP2). The accumulation of glycerol as an osmolyte has been well characterized in S.cerevisiae, and the GPP2 gene induction under osmotic stress is triggered by the Hog1p phosphorylation (Hohmann, 2002). We have already demonstrated the activation of the H. annosum s.s. HaHog1p under NaCl stress condition (Raffaello et al., 2012). While the role of glycerol in osmotic stress tolerance has been investigated in the Ascomycota phylum, fewer studies have been carried out in the Basidiomycota. For example, the level of polyols was quantified in the freshly harvested Agaricus bisporus where glycerol was found initially only in the gills followed by a significant increase in concentration in the inner cap tissue after 5 days storage (Beecher et al., 2001). Despite the substantially lack of knowledge about the role of glycerol in basidiomycetes, the induction of a predicted glycerol phosphatase may indicate the role of this polyol as an osmoprotectant in H. annosum s.s. to help the fungus to counteract severe environmental osmotic stress.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

Heterobasidion annosum On the other hand, other gene transcripts were induced specifically by certain ions, such as a predicted tyrosinase and a predicted vacuolar Ca2+/H+ antiporter similar to the S. cerevisiae Vcx1 which are upregulated only in CaCl2 (Miseta et al., 1999). The type-3 copper tyrosinase proteins are key components in melanin biosynthesis, which, together with the melanization of fungal cell wall, has been associated to stress response to harsh environmental conditions (like UV-radiation, temperature, dehydration) (Bell and Wheeler, 1986; Halaouli et al., 2006). The strong induction of a tyrosinase in CaCl2 can then indicate the activation of the melanin biosynthesis pathway in H. annosum s.s. to respond to osmotic stress by reinforcing the fungal cell wall. Melanization could also be important to respond to the high level of Ca2+ that accumulates in decayed wood and reaction zone in infected trees (Oliva et al., 2011; Nagy et al., 2012). The induction of the predicted vacuolar Ca2+/H+ antiporter also suggests a general fungal response to keep the intracellular Ca2+ concentration at low level by sequestration into the vacuolar compartment. In H. annosum, we have previously shown how the MAPK Hog1p is activated by phosphorylation in oxidative stress within 60 min after 5 mM of H2O2 is added to the liquid media (Raffaello et al., 2012). Although the amount of upregulated transcripts in H. annosum s.s. exposed to H2O2 was lower compared to other conditions, we were able to identify genes possibly involved in oxidative stress response. The phosphorylation of the Hog1p kinase is generally associated with the induction of stress response elements (STREs) which in turn activate specific genes involved in stress tolerance (Schüller et al., 1994; Bahn et al., 2007). The presence among the most 10 induced genes of two transcripts like UvrD/REP helicase and Rad1 suggests the activation of pathways involved in DNA repair from damages caused by high level of oxidants represented by the hydrogen peroxide. Also, the strong and specific induction of a cytochrome P450 could also indicate a stress response to high level of oxidant. The induction of many predicted kinases, transcription factors and several components of the phosphatidylinositol-signalling pathway and the glyoxylate cycle indicates an intracellular response to the change of nutrient availability in the environment. In eukaryotic cells, activation of autophagy is a way for the cell to respond to nutrient deprivation. In yeast, the formation of autophagosomes is activated by phosphatydilinositol-3kinase (PtdIns3K), which are used to degrade cellular components in order to recycle macromolecules (He and Klionsky, 2009). Candida albicans has been shown to overexpress genes involved in the glyoxylate cycle like isocitrate lyase (ICL1) and malate synthase (MLS1) to survive in starving condition inside the phagosomes of macrophage cells (Lorenz and Fink, 2001). All of these

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suggest the autophagy could be a way of H. annosum s.s. to overcome at least temporarily the lack of nutrients in the surrounding environment. H. annosum s.s. response during saprotrophic growth in pine bark, sapwood and heartwood The analysis of the induced transcripts specific for each wood material revealed that sapwood is characterized by nearly three times more number of genes with increased transcript levels (448) compared to bark (148) and heartwood (174). Probably the high content of easily utilizable sugars and polysaccharides in the sapwood (Terziev et al., 1997) facilitates the induction of an optimal set of genes to allow the fungus to utilize these carbon sources effectively. This may indicate an evolutionary adaptation of H. annosum s.s. for pine sapwood degradation compared to heartwood and bark. However, the results equally revealed potential mechanisms for pine heartwood degradation during saprotrophic growth. In this study, the glycosyl hydrolase (GH) is one of the largest gene families induced during wood colonization. GH genes encode diverse enzymes that hydrolyse glycosidic bonds which characterize cellulose, hemicellulose and pectin. Some genes of these GH families (GH1, GH3, GH15, GH18, GH28, GH30, GH35, GH53 and GH88) showed general induction in all wood compartments, while some members of other GH families (GH5, GH10, GH12, GH45 and GH61) displayed strong induction only in heartwood. GH28 family is important for pectin degradation which is one of the major constituents of plant cell wall (Sprockett et al., 2011). The strong upregulation of some members of the GH28 gene family indicates the importance of these enzymes in H. annosum s.s. during saprotrophic growth. GH5 is one of the largest GH families and comprises a vast variety of carbohydrate active enzymes, including more than 240 different endoglucanases. It is a well-studied family, and for many members the catalytic mechanism has been experimentally characterized (Aspeborg et al., 2012). The GH5 of H. annosum s.s. which is induced in pine heartwood has a high level of similarity with a mannanase from several other wood degrading basidiomycetes like Armillaria tabescens (GenBank: ABB88954.1), the dry rot Serpula lacrymans var. lacrymans S7.9 (GenBank: EGO25412.1) and the white-rot Phanerochaete chrysosporium (GenBank: ABG79370.1). GH10 family members possess xylanase activity and are able to hydrolyse decorated xylans which are xylan polysaccharides carrying for example acetyl and arabinofuranosyl units (Pell et al., 2004). GH12 family comprises endoglucanases, xyloglucan hydrolases, β-1,3-1,4-glucanases, xyloglucan endotransglycosylases. The xyloglucanase activity is particularly important in degrading hemicellulose

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1664 T. Raffaello, H. Chen, A. Kohler and F. O. Asiegbu which is composed by xyloglucan polymers. The induction of specific GH genes in different part of the pine wood during saprotrophic growth highlights the fine regulation of distinct gene families which are pivotal for wood degradation in H. annosum s.s. In pine heartwood, we reported the induction of several transcripts encoding GH61 enzymes which do not have the typical glycosyl hydrolase mechanism of action since they are enzymes characterized by a copper-binding site [http://www.cazy.org/Auxiliary-Activities.html, under the new ‘Auxiliary activities’ category (Levasseur et al., 2013)]. GH61s support the activity of the other GH enzymes through an oxidative degradation of cellulose (Harris et al., 2010; Phillips et al., 2011; Quinlan et al., 2011). Interestingly, in a recent study, several GH61 genes in H. irregulare were extremely induced on spruce heartwood, and only one of them was moderately expressed on pine heartwood (Yakovlev et al., 2012). These results are in contrast to the high expression levels of all the GH61 genes in H. annosum s.s. in pine heartwood documented in our study. However, the differences between our results and that of Yakovlev and colleagues (2012) could be due to variations in the incubation time for the decay experiments which in their study was only 21 days. During saprotrophic growth on pine wood, the induction of MCOs and MnPs was also documented. These enzymes together with lignin peroxidases (LiPs) are the main enzymes involved in lignin degradation in white-rot fungi (Aro et al., 2005). Besides the hydrolysis of cellulose and its associates, lignin degradation is another essential part of wood degradation. Lignin is a heterogeneous aromatic polymer, which protects cellulose and hemicellulose from microbial attack (Floudas et al., 2012). Thus, to gain access to cellulose or hemicellulose, wood-decaying fungi must overcome or circumvent lignin (Floudas et al., 2012). As a white-rot species, H. irregulare possesses eight MnPs and 17 MCOs (Floudas et al., 2012; Olson et al., 2012). In this study, only five MnPs and three MCOs were specifically upregulated in saprotrophic growth indicating that not all of the MnPs or MCOs were simultaneously activated during wood degradation. In the earlier study (Olson et al., 2012), only one MnP and one MCO were upregulated during wood degradation. The differences could be attributed to variations in the experimental design, where the fungus was grown on whole pine wood shaving instead of separated pine bark, heartwood and sapwood. Unlike the earlier study, the current results provide additional details on the gene expression pattern when the fungus navigates specific wood compartments during saprotrophic wood decay process. The expressions of MCOs and MnPs have been reported to be regulated by environmental signals, such as concentration of carbon and nitrogen, metal ions and xenobiotics pres-

ence, temperature shock and lengths of day light (Janusz et al., 2013). Besides the general induction during saprotrophic growth, the stronger induction of three MCOs in pine sapwood, four MnPs in heartwood and one MnP in bark suggests that the chemical compositions of different wood compartments might lead to selectively induced expression of ligninolytic enzymes. In addition to selective induction, the co-expression of the ligninolytic enzymes are also reported in Phlebia radiata when wood was used as substrate, which indicates potential synergy among ligninolytic enzymes as well as the relevance of oxidoreductase enzymes (Hilden et al., 2006; Makela et al., 2006; Lundell et al., 2010). These results have confirmed the specific roles and importance of paralogous genes encoding MCOs and MnPs in pine sapwood and heartwood degradation. Conclusions In this study, we investigated the transcriptional response of the basidiomycete H. annosum s.s. to several abiotic stresses and during saprotrophic growth on specific pine wood compartments. The balance between sensing and survival in abiotic stress and nutrient uptake during saprotrophic growth for H. annosum s.s. is summarized in Fig. 6. The possible regulatory mechanisms in H. annosum s.s. during saprotrophic growth and in the presence of abiotic stressors might be associated with the change of lipid and protein composition in plasma membrane and the induction of cytochrome P450s and MFS-1 families for cold stress adaptation. The synthesis and accumulation of glycerol as osmoprotectant controlled by MAPK Hog1p, the melanization of fungal cell wall and use of vacuolar Ca2+/H+ antiporter could be relevant for survival under osmotic stress. Additionally, the activation of the MAPK HOG pathway and DNA repairing pathway will be important for oxidative stress. Furthermore, the activation of the glycoxylate cycle and autophagy might be involved in starvation and induction of a specific set of enzymes (GHs, MCOs, MnPs, cytosolic oxidoreductases, cytochrome P450 and MFS-1 members) will be highly relevant for pine heartwood degradation. Finally, the independent qPCR of 51 genes further confirmed these results and strengthened our conclusions. Acknowledgement This work was supported by the Academy of Finland and the University of Helsinki research grant. Support from Viikki Doctoral Programme in Molecular Biosciences (VGSB) and Helsinki Graduate Program in Biotechnology and Molecular Biology (GPBM) to TR and HC respectively is gratefully acknowledged. We thank Dr. Francis Martin for facilitating the mutual collaborative work with members of his research group.

© 2013 Society for Applied Microbiology and John Wiley & Sons Ltd, Environmental Microbiology, 16, 1654–1667

Heterobasidion annosum

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Fig. 6. Model of the balance between survival and growth for H. annosum s.s. during stump colonization (H = heartwood, S = sapwood, B = bark, GH = glycosyl hydrolase, CE = carboxylesterase, MCO = multicopper oxidase, MnP = manganese peroxidase).

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Fig. S1. Representative portion of the general hierarchical cluster analysis. The normalized absolute gene expression levels on the microarray were filtered (floor threshold 20, ceiling threshold 60000, min fold change (1) and the cluster analysis was performed for samples and genes with Pearson Correlation distance statistical test. Fig. S2. (A and B) Subset of glycosyl hydrolases (GHs) genes induced in H. annosum in different conditions. (C) Members of the glycosyl hydrolase family 61 (GH61) genes induced in H. annosum in different conditions. Specific upregulation in heartwood and/or bark is indicated by arrows (FDR < 0.05 for all samples except for salts (FDR < 0.1) and H2O2 (FDR < 0.25), Fold Change > 2 cut-off for all the conditions). Gene validated by qPCR are marked with ‘*’. Table S1. List of the primer sequences used in this study (E = efficiency). Table S2. Upregulated genes in 27°C. Table S3. Downregulated genes in 8°C. Table S4. Upregulated genes in CaCl2. Table S5. Upregulated genes in NaCl. Table S6. Downregulated genes in H2O2. Table S7. Downregulated genes in starvation. Table S8. Downregulated genes in Pine Bark. Table S9. Downregulated genes in Pine Sapwood. Table S10. Downregulated genes in Pine Heartwood. Table S11. List of the 10 most induced genes with predicted function in each condition (FDR < 0.05 for all samples except for salts (FDR < 0.1) and H2O2 (FDR < 0.25), Fold Change > 2 cut-off for all the conditions). Table S12. List of the carbohydrate active enzymes (CAZYs) selectively induced in each wood compartment (FDR < 0.05 for all samples except for salts (FDR < 0.1) and H2O2 (FDR < 0.25), Fold Change > 2 cut-off for all the conditions).

Supporting information Additional Supporting Information may be found in the online version of this article at the publisher’s web-site:

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