Analysis of the Phlebiopsis gigantea Genome, Transcriptome and Secretome Provides Insight into Its Pioneer Colonization Strategies of Wood

Collectively classified as white-rot fungi, certain basidiomycetes efficiently degrade the major structural polymers of wood cell walls. A small subset of these Agaricomycetes, exemplified by Phlebiopsis gigantea, is capable of colonizing freshly exposed conifer sapwood despite its high content of extractives, which retards the establishment of other fungal species. The mechanism(s) by which P. gigantea tolerates and metabolizes resinous compounds have not been explored. Here, we report the annotated P. gigantea genome and compare profiles of its transcriptome and secretome when cultured on fresh-cut versus solvent-extracted loblolly pine wood. The P. gigantea genome contains a conventional repertoire of hydrolase genes involved in cellulose/hemicellulose degradation, whose patterns of expression were relatively unperturbed by the absence of extractives. The expression of genes typically ascribed to lignin degradation was also largely unaffected. In contrast, genes likely involved in the transformation and detoxification of wood extractives were highly induced in its presence. Their products included an ABC transporter, lipases, cytochrome P450s, glutathione S-transferase and aldehyde dehydrogenase. Other regulated genes of unknown function and several constitutively expressed genes are also likely involved in P. gigantea's extractives metabolism. These results contribute to our fundamental understanding of pioneer colonization of conifer wood and provide insight into the diverse chemistries employed by fungi in carbon cycling processes.

[1]  Hong-Wei Sun,et al.  Progressive sequence alignment and molecular evolution of the Zn-containing alcohol dehydrogenase family , 1992, Journal of Molecular Evolution.

[2]  P. Kersten Glyoxal oxidase of Phanerochaete chrysosporium: its characterization and activation by lignin peroxidase. , 1990, Proceedings of the National Academy of Sciences of the United States of America.

[3]  Thomas L. Madden,et al.  Gapped BLAST and PSI-BLAST: a new generation of protein database search programs. , 1997, Nucleic acids research.

[4]  Ichael,et al.  Analysis of Character Correlations Among Wood Decay Mechanisms , Mating Systems , and Substrate Ranges in Homobasidiomycetes , 2001 .

[5]  Andrey Rzhetsky,et al.  Statistical properties of the ordinary least-squares, generalized least-squares, and minimum-evolution methods of phylogenetic inference , 1992, Journal of Molecular Evolution.

[6]  R. Blanchette,et al.  Structure, Organization, and Transcriptional Regulation of a Family of Copper Radical Oxidase Genes in the Lignin-Degrading Basidiomycete Phanerochaete chrysosporium , 2006, Applied and Environmental Microbiology.

[7]  A. Salamov,et al.  Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis , 2012, Proceedings of the National Academy of Sciences.

[8]  Inna Dubchak,et al.  The Genome Portal of the Department of Energy Joint Genome Institute , 2011, Nucleic Acids Res..

[9]  Katherine H. Huang,et al.  Genome sequence of the lignocellulose degrading fungus Phanerochaete chrysosporium strain RP78 , 2004, Nature Biotechnology.

[10]  Julie Dawn Thompson,et al.  Detection of dsRNA-binding domains in RNA helicase A and Drosophila maleless: implications for monomeric RNA helicases , 1994, Nucleic Acids Res..

[11]  S. Bastian,et al.  Engineering of pyranose 2-oxidase from Peniophora gigantea towards improved thermostability and catalytic efficiency , 2005, Applied Microbiology and Biotechnology.

[12]  D. Hibbett,et al.  Molecular Evolution and Diversity of Lignin Degrading Heme Peroxidases in the Agaricomycetes , 2008, Journal of Molecular Evolution.

[13]  Motoyuki Shimizu,et al.  Metabolic regulation at the tricarboxylic acid and glyoxylate cycles of the lignin‐degrading basidiomycete Phanerochaete chrysosporium against exogenous addition of vanillin , 2005, Proteomics.

[14]  Motoyuki Shimizu,et al.  Proteomic and metabolomic analyses of the white-rot fungus Phanerochaete chrysosporium exposed to exogenous benzoic acid. , 2008, Journal of proteome research.

[15]  P. Baldrian,et al.  Degradation of cellulose by basidiomycetous fungi. , 2008, FEMS microbiology reviews.

[16]  Albee Y. Ling,et al.  The Paleozoic Origin of Enzymatic Lignin Decomposition Reconstructed from 31 Fungal Genomes , 2012, Science.

[17]  A. Salamov,et al.  Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi , 2014, Proceedings of the National Academy of Sciences.

[18]  J. Yadav,et al.  P450 monooxygenases (P450ome) of the model white rot fungus Phanerochaete chrysosporium , 2012, Critical reviews in microbiology.

[19]  B. Henrissat,et al.  Expansion of the enzymatic repertoire of the CAZy database to integrate auxiliary redox enzymes , 2013, Biotechnology for Biofuels.

[20]  T. V. van Beek,et al.  Elimination and detoxification of softwood extractives by white-rot fungi. , 2000, Journal of biotechnology.

[21]  Igor Grigoriev,et al.  Comparative Transcriptome and Secretome Analysis of Wood Decay Fungi Postia placenta and Phanerochaete chrysosporium , 2010, Applied and Environmental Microbiology.

[22]  M. Nei,et al.  MEGA4: Molecular Evolutionary Genetics Analysis (MEGA) software version 4.0. , 2007, Molecular biology and evolution.

[23]  K. Eriksson,et al.  Pyranose 2-oxidase from Phanerochaete chrysosporium , 1988 .

[24]  T. V. van Beek,et al.  Fungal biotransformation products of dehydroabietic acid. , 2007, Journal of natural products.

[25]  F. Giffhorn Fungal pyranose oxidases: occurrence, properties and biotechnical applications in carbohydrate chemistry , 2000, Applied Microbiology and Biotechnology.

[26]  T. Annesi,et al.  Biological control of Heterobasidion annosum on Pinus pinea by Phlebiopsis gigantea , 2005 .

[27]  A. Gutiérrez,et al.  Microbial and enzymatic control of pitch in the pulp and paper industry , 2009, Applied Microbiology and Biotechnology.

[28]  E. Panisko,et al.  Proteomic and Functional Analysis of the Cellulase System Expressed by Postia placenta during Brown Rot of Solid Wood , 2011, Applied and Environmental Microbiology.

[29]  L. Pauling,et al.  Evolutionary Divergence and Convergence in Proteins , 1965 .

[30]  Gordon Robertson,et al.  A specialized ABC efflux transporter GcABC-G1 confers monoterpene resistance to Grosmannia clavigera, a bark beetle-associated fungal pathogen of pine trees. , 2013, The New phytologist.

[31]  Alex L. Shigo,et al.  Successions of Organisms in Discoloration and Decay of Wood , 1967 .

[32]  R. Bourbonnais,et al.  Reactivities of various mediators and laccases with kraft pulp and lignin model compounds , 1997, Applied and environmental microbiology.

[33]  R. Aebersold,et al.  A statistical model for identifying proteins by tandem mass spectrometry. , 2003, Analytical chemistry.

[34]  Feng Xu,et al.  Oxidoreductive Cellulose Depolymerization by the Enzymes Cellobiose Dehydrogenase and Glycoside Hydrolase 61 , 2011, Applied and Environmental Microbiology.

[35]  N. Saitou,et al.  The neighbor-joining method: a new method for reconstructing phylogenetic trees. , 1987, Molecular biology and evolution.

[36]  Francisco Melo,et al.  A Novel Extracellular Multicopper Oxidase from Phanerochaete chrysosporium with Ferroxidase Activity , 2003, Applied and Environmental Microbiology.

[37]  A. Salamov,et al.  Erratum: Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis (Proceedings of the National Academy of Sciences (2012) 109 (5458-5463) DOI: 10.1073/pnas. 1119912109) , 2012 .

[38]  B. Henrissat,et al.  Evolution, substrate specificity and subfamily classification of glycoside hydrolase family 5 (GH5) , 2012, BMC Evolutionary Biology.

[39]  Pramod P Wangikar,et al.  Combined sequence and structure analysis of the fungal laccase family , 2003, Biotechnology and bioengineering.

[40]  A. Salamov,et al.  Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion , 2009, Proceedings of the National Academy of Sciences.

[41]  R. Blanchette,et al.  Biological Processing of Pine Logs for Pulp and Paper Production with Phlebiopsis gigantea , 1997, Applied and environmental microbiology.

[42]  A. Salamov,et al.  Computational analysis of the Phanerochaete chrysosporium v2.0 genome database and mass spectrometry identification of peptides in ligninolytic cultures reveal complex mixtures of secreted proteins. , 2006, Fungal genetics and biology : FG & B.

[43]  M. Garbelotto,et al.  Population genetic analyses provide insights on the introduction pathway and spread patterns of the North American forest pathogen Heterobasidion irregulare in Italy , 2013, Molecular ecology.

[44]  V. Eijsink,et al.  An Oxidative Enzyme Boosting the Enzymatic Conversion of Recalcitrant Polysaccharides , 2010, Science.

[45]  D. Cullen,et al.  Recent Advances on the Genomics of Litter- and Soil-Inhabiting Agaricomycetes , 2013 .

[46]  R. Blanchette Delignification by wood-decay fungi , 1991 .

[47]  L. Lo Leggio,et al.  Insights into the oxidative degradation of cellulose by a copper metalloenzyme that exploits biomass components , 2011, Proceedings of the National Academy of Sciences.

[48]  Brandi L. Cantarel,et al.  The Carbohydrate-Active EnZymes database (CAZy): an expert resource for Glycogenomics , 2008, Nucleic Acids Res..

[49]  W. V. van Berkel,et al.  Site‐directed mutagenesis of selected residues at the active site of aryl‐alcohol oxidase, an H2O2‐producing ligninolytic enzyme , 2006, The FEBS journal.

[50]  Robert A. Blanchette,et al.  Microbial and Enzymatic Degradation of Wood and Wood Components , 2012, Springer Series in Wood Science.

[51]  Keith Bradnam,et al.  CEGMA: a pipeline to accurately annotate core genes in eukaryotic genomes , 2007, Bioinform..

[52]  D. Cullen,et al.  Isolation and Purification of Pyranose 2-Oxidase from Phanerochaete chrysosporium and Characterization of Gene Structure and Regulation , 2004, Applied and Environmental Microbiology.

[53]  C. Divne,et al.  Mechanism of the Reductive Half-reaction in Cellobiose Dehydrogenase* , 2003, The Journal of Biological Chemistry.

[54]  J. Thompson,et al.  CLUSTAL W: improving the sensitivity of progressive multiple sequence alignment through sequence weighting, position-specific gap penalties and weight matrix choice. , 1994, Nucleic acids research.

[55]  A. Gutiérrez,et al.  Biodegradability of Extractives in Sapwood and Heartwood from Scots Pine by Sapstain and White-Rot Fungi , 1999 .

[56]  D. Hibbett,et al.  Genomewide analysis of polysaccharides degrading enzymes in 11 white- and brown-rot Polyporales provides insight into mechanisms of wood decay , 2013, Mycologia.

[57]  J. Weiner,et al.  Fundamentals and applications , 2003 .

[58]  M. Hofrichter,et al.  New and classic families of secreted fungal heme peroxidases , 2010, Applied Microbiology and Biotechnology.

[59]  G. Suen,et al.  Mountain Pine Beetles Colonizing Historical and Naïve Host Trees Are Associated with a Bacterial Community Highly Enriched in Genes Contributing to Terpene Metabolism , 2013, Applied and Environmental Microbiology.

[60]  R. Blanchette,et al.  An Antarctic Hot Spot for Fungi at Shackleton's Historic Hut on Cape Royds , 2010, Microbial Ecology.

[61]  J. Ståhlberg,et al.  The Putative Endoglucanase PcGH61D from Phanerochaete chrysosporium Is a Metal-Dependent Oxidative Enzyme that Cleaves Cellulose , 2011, PloS one.

[62]  M. Mann,et al.  Exponentially Modified Protein Abundance Index (emPAI) for Estimation of Absolute Protein Amount in Proteomics by the Number of Sequenced Peptides per Protein*S , 2005, Molecular & Cellular Proteomics.

[63]  R. Blanchette,et al.  Biological Control of Blue Stain in Pulpwood: Mechanisms of Control used by Phlebiopsis gigantea , 2001 .

[64]  D. Sheehan,et al.  Glutathione S-transferases from the white-rot fungus, Phanerochaete chrysosporium. , 1997, The Biochemical journal.

[65]  Silvia Vidal‐Melgosa,et al.  Discovery of LPMO activity on hemicelluloses shows the importance of oxidative processes in plant cell wall degradation , 2014, Proceedings of the National Academy of Sciences.

[66]  Timothy Y James,et al.  Phylogenetic comparison and classification of laccase and related multicopper oxidase protein sequences , 2006, The FEBS journal.

[67]  U. Kües,et al.  Lcc1 and Lcc5 are the main laccases secreted in liquid cultures of Coprinopsis cinerea strains , 2013, Antonie van Leeuwenhoek.

[68]  A. Salamov,et al.  Insight into trade-off between wood decay and parasitism from the genome of a fungal forest pathogen. , 2012, The New phytologist.

[69]  C. Evans,et al.  Anisaldehyde and Veratraldehyde Acting as Redox Cycling Agents for H2O2 Production by Pleurotus eryngii , 1994, Applied and environmental microbiology.

[70]  U. Temp,et al.  Laccase is essential for lignin degradation by the white‐rot fungus Pycnoporus cinnabarinus , 1997, FEBS letters.

[71]  A. Salamov,et al.  The Plant Cell Wall–Decomposing Machinery Underlies the Functional Diversity of Forest Fungi , 2011, Science.

[72]  A. Gutiérrez,et al.  Analysis of lipophilic extractives from wood and pitch deposits by solid-phase extraction and gas chromatography , 1998 .

[73]  B. Henrissat,et al.  Cello-Oligosaccharide Oxidation Reveals Differences between Two Lytic Polysaccharide Monooxygenases (Family GH61) from Podospora anserina , 2012, Applied and Environmental Microbiology.

[74]  L. Birolo,et al.  Identification of a new member of Pleurotus ostreatus laccase family from mature fruiting body. , 2010, Fungal biology.

[75]  A. Käärik 5 – Decomposition of Wood , 1974 .

[76]  Lynne Boddy,et al.  Fungal decomposition of wood. Its biology and ecology. , 1988 .

[77]  M. Nei,et al.  MEGA5: molecular evolutionary genetics analysis using maximum likelihood, evolutionary distance, and maximum parsimony methods. , 2011, Molecular biology and evolution.

[78]  J. Reiser,et al.  Aryl-alcohol dehydrogenase from the white-rot fungus Phanerochaete chrysosporium. Gene cloning, sequence analysis, expression, and purification of the recombinant enzyme. , 1994, The Journal of biological chemistry.

[79]  M. Soellner,et al.  Tandem cytochrome P450 monooxygenase genes and splice variants in the white rot fungus Phanerochaete chrysosporium: cloning, sequence analysis, and regulation of differential expression. , 2003, Fungal genetics and biology : FG & B.

[80]  M. Hofrichter,et al.  Substrate oxidation by dye-decolorizing peroxidases (DyPs) from wood- and litter-degrading agaricomycetes compared to other fungal and plant heme-peroxidases , 2012, Applied Microbiology and Biotechnology.

[81]  D. Cavener,et al.  GMC oxidoreductases. A newly defined family of homologous proteins with diverse catalytic activities. , 1992, Journal of molecular biology.

[82]  J. Rishbeth Stump protection against Fomes annosus. III. Inoculation with Peniophora gigantea. , 1963 .

[83]  N. Blom,et al.  Feature-based prediction of non-classical and leaderless protein secretion. , 2004, Protein engineering, design & selection : PEDS.

[84]  Asaf Salamov,et al.  Fungal Genomic Annotation , 2006 .

[85]  Jamie H. D. Cate,et al.  Cellobiose dehydrogenase and a copper-dependent polysaccharide monooxygenase potentiate cellulose degradation by Neurospora crassa. , 2011, ACS chemical biology.

[86]  T. Tokimatsu,et al.  A physiological role for oxalic acid biosynthesis in the wood-rotting basidiomycete Fomitopsis palustris , 2001, Proceedings of the National Academy of Sciences of the United States of America.

[87]  S. Kilaru,et al.  The laccase multi-gene family in Coprinopsis cinerea has seventeen different members that divide into two distinct subfamilies , 2006, Current Genetics.

[88]  P. Halada,et al.  Characteristics of Gloeophyllum trabeum Alcohol Oxidase, an Extracellular Source of H2O2 in Brown Rot Decay of Wood , 2007, Applied and Environmental Microbiology.

[89]  Aleksey A. Porollo,et al.  CYP63A2, a Catalytically Versatile Fungal P450 Monooxygenase Capable of Oxidizing Higher-Molecular-Weight Polycyclic Aromatic Hydrocarbons, Alkylphenols, and Alkanes , 2013, Applied and Environmental Microbiology.

[90]  B. Horwitz,et al.  Genomics of Soil- and Plant-Associated Fungi , 2013, Soil Biology.

[91]  Jill Gaskell,et al.  Transcriptome and Secretome Analyses of Phanerochaete chrysosporium Reveal Complex Patterns of Gene Expression , 2009, Applied and Environmental Microbiology.

[92]  Jacqueline MacDonald,et al.  Transcriptomic Responses of the Softwood-Degrading White-Rot Fungus Phanerochaete carnosa during Growth on Coniferous and Deciduous Wood , 2011, Applied and Environmental Microbiology.

[93]  K. Messner,et al.  Reduction of Resin Content in Wood Chips during Experimental Biological Pulping Processes , 1994 .

[94]  C. Martius Decomposition of Wood , 1997 .

[95]  E. Sjöström,et al.  Wood Chemistry: Fundamentals and Applications , 1981 .

[96]  V. Bryson,et al.  Evolving Genes and Proteins. , 1965, Science.

[97]  U. Kües,et al.  Multiple Multi-Copper Oxidase Gene Families in Basidiomycetes – What for? , 2011, Current genomics.

[98]  S. Kawai,et al.  Degradation of a non-phenolic β-O-4 substructure and of polymeric lignin model compounds by laccase of Coriolus versicolor in the presence of 1-hydroxybenzotriazole , 1999 .

[99]  F. Asiegbu,et al.  Identification and analysis of differentially expressed cDNAs during nonself-competitive interaction between Phlebiopsis gigantea and Heterobasidion parviporum. , 2006, FEMS microbiology ecology.