Transcriptome and secretome analyses of Phanerochaete chrysosporium reveal complex patterns of gene expression

Abstract

The wood decay basidiomycete Phanerochaete chrysosporium was grown under standard ligninolytic or cellulolytic conditions and subjected to whole-genome expression microarray analysis and liquid chromatography-tandem mass spectrometry of extracellular proteins. A total of 545 genes were flagged on the basis of significant changes in transcript accumulation and/or peptide sequences of the secreted proteins. Under nitrogen or carbon limitation, lignin and manganese peroxidase expression increased relative to nutrient replete medium. Various extracellular oxidases were also secreted in these media, supporting a physiological connection based on peroxide generation. Numerous genes presumed to be involved in mobilizing and recycling nitrogen were expressed under nitrogen limitation, and among these were several secreted glutamic acid proteases not previously observed. In medium containing microcrystalline cellulose as the sole carbon source, numerous genes encoding carbohydrate-active enzymes were upregulated. Among these were six members of the glycoside hydrolase family 61, as well as several polysaccharide lyases and carbohydrate esterases. Presenting a daunting challenge for future research, more than 190 upregulated genes are predicted to encode proteins of unknown function. Of these hypothetical proteins, approximately one-third featured predicted secretion signals, and 54 encoded proteins detected in extracellular filtrates. Our results affirm the importance of certain oxidative enzymes and, underscoring the complexity of lignocellulose degradation, also support an important role for many new proteins of unknown function.

FIG. 1.

Venn diagrams illustrating the distribution of regulated genes (A and B) and MS-identified proteins (C). (A) Genes with transcript levels that change ≥2-fold in comparisons of nitrogen-limited and nutrient-replete media (NLB versus RB), carbon-limited and nutrient-replete media (CLB versus RB), and cellulose- and glucose-containing media (HBA versus HBG). (B) Genes with transcripts that are upregulated ≥2-fold in these same comparisons. (C) Genes encoding proteins identified by LC-MS/MS in NLB, CLB, and HBA. “Upregulation” is arbitrarily assigned to genes whose transcripts accumulate in NLB relative to RB, in CLB relative to RB, and in HBA relative to HBG. The corresponding “downregulated” genes are not enumerated in this figure but are listed in Table S3 in the supplemental material. In all cases, values within overlapping regions have not been subtracted from totals. For example, peptides corresponding to a total of 37 protein models were identified in HBA medium. Of the 37, 8 were also present in carbon-limited medium, and 4 of these were common to all three media. An additional 61 proteins were identified in Wood's cellulose-containing medium (WBC), and these are listed in Table S3 in the supplemental material.

FIG. 2.

Distribution of P. chrysosporium genes encoding upregulated transcripts in carbon limited B3 medium (CLB, inner ring), nitrogen-limited B3 medium (NLB, center ring), and Highley's basal medium containing avicel (HBA, outer ring). Parenthetical values following each category refer to the number of genes in CLB, NLB, and HBA.

FIG. 3.

Organization and expression of scaffold 5 cluster of family G1 proteases. Purple filled arrows denote G1 family peptidases. Protein models Pchr31964 and Pchr43144 (indicated by asterisks) are truncated at the 5′ termini, but alternative ab initio models Pchr3963 and Pchr4008, respectively, reveal complete sequence with predicted secretion signals (green fill). Vertical colored bars indicate microarray-derived expression results from nutrient-limited cultures versus nutrient-replete medium and from cellulose-containing medium versus glucose-containing medium. The lower right scale indicates the log₂ signal strength. Peptides corresponding to models Pchr131262, Pchr43144, and Pchr121400 were identified by LC-MS/MS, and the corresponding Mascot scores are listed.

FIG. 4.

Transcript levels of copper radical oxidase (cro2), CDH (cdh1), and copper radical oxidase (glx) as determined by qRT-PCR (A) and microarray (B).