Genome, transcriptome, and secretome analysis of wood decay fungus Postia placenta supports unique mechanisms of lignocellulose conversion

Abstract

Brown-rot fungi such as Postia placenta are common inhabitants of forest ecosystems and are also largely responsible for the destructive decay of wooden structures. Rapid depolymerization of cellulose is a distinguishing feature of brown-rot, but the biochemical mechanisms and underlying genetics are poorly understood. Systematic examination of the P. placenta genome, transcriptome, and secretome revealed unique extracellular enzyme systems, including an unusual repertoire of extracellular glycoside hydrolases. Genes encoding exocellobiohydrolases and cellulose-binding domains, typical of cellulolytic microbes, are absent in this efficient cellulose-degrading fungus. When P. placenta was grown in medium containing cellulose as sole carbon source, transcripts corresponding to many hemicellulases and to a single putative beta-1-4 endoglucanase were expressed at high levels relative to glucose-grown cultures. These transcript profiles were confirmed by direct identification of peptides by liquid chromatography-tandem mass spectrometry (LC-MS/MS). Also up-regulated during growth on cellulose medium were putative iron reductases, quinone reductase, and structurally divergent oxidases potentially involved in extracellular generation of Fe(II) and H(2)O(2). These observations are consistent with a biodegradative role for Fenton chemistry in which Fe(II) and H(2)O(2) react to form hydroxyl radicals, highly reactive oxidants capable of depolymerizing cellulose. The P. placenta genome resources provide unparalleled opportunities for investigating such unusual mechanisms of cellulose conversion. More broadly, the genome offers insight into the diversification of lignocellulose degrading mechanisms in fungi. Comparisons with the closely related white-rot fungus Phanerochaete chrysosporium support an evolutionary shift from white-rot to brown-rot during which the capacity for efficient depolymerization of lignin was lost.

Fig. 1.

Distribution of various CAZymes in P. placenta (inner ring), T. reesei (middle ring), and P. chrysosporium (outer ring). Proteins not found in P. placenta are underlined. Comparisons with additional species are listed in Table S1. CBM1, family 1 carbohydrate binding modules; GH#, modules within individual glycoside hydrolase families; GH5 (CBM1), glycoside hydrolase family 5 modules associated with family 1 carbohydrate binding modules; GT, glycosyl transferases; CE, carbohydrate esterases; PL, polysaccharide lyases; EXPN, expansin-related proteins.

Fig. 2.

Phylogenies of glycoside hydrolase (GH 61, GH10), glyoxal oxidase/copper radical oxidase (GLOX), laccase (LAC) and related multicopper oxidase, and low redox peroxidase (LRP) and related class II fungal peroxidases from complete genomes of P. placenta (Pospl1), P. chrysosporium (Phchr1), C. cinerea (CC1G), L. bicolor (Lacbi1), C. neoformans (CNAG), U. maydis (UM), M. grisea (MGG), Stagonospora nodorum (SNOG), T. reesei (Trire2), and Pichia stipitis (Picst3). Datasets were assembled by using BLAST (with qUniProtKB query sequences Q5XXE5, O60206, P36218, Q00023, O14405, Q01772 and Q12718), with a cut-off value of E-06. Parsimony (PAUP* 4.0; 10,000 heuristic searches, 1000 bootstrap replicates), maximum likelihood (RAxML; 1000 bootstrap replicates, with models suggested by ProtTest), and Bayesian (MrBayes v3.1.2; 2 runs of 4 chains, 10 million generations each, with mixed protein models) support values are indicated in the order MP/PP/ML. Topologies shown are from Bayesian phylogenetic analyses. An alternative topology from parsimony analysis is shown for part of the GH10 phylogeny. Inferred gene losses, duplication events (paralogy), and speciation events (orthology) are indicated within Postia and Phanerochaete only.

Fig. 3.

Regulation of CAZY-encoding genes. (A) Expression profile of 144 glycoside hydrolase-encoding genes in media containing glucose versus microcrystalline cellulose as sole carbon sources. In B, a cluster of 24 of highly expressed genes is expanded and the color scale recalibrated to illustrate differences in transcript accumulation. Expression ratios were derived from comparisons of glucose-grown versus cellulose-grown mycelia. Analysis is based on 3 biological replicates per culture medium. Quantile normalization and robust multiarray averaging (RMA) were applied to the entire dataset. ANOVA showed 120 GH-encoding genes, including all 24 above, were significantly regulated (P < 0.01). Reciprocals of ratios <1.0 are multiplied by −1. Asterisks indicate proteins identified by LC-MS/MS. A detailed listing of CAZYs with statistical analyses of expression data are presented in SI Appendix and GEO accession GSE12540 si_table_1.xls.