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Label-free quantitative proteomic analysis of Peniophora incarnata T-7 during lignocellulose degradation under different substrate conditions

ABSTRACT: Peniophora incarnata T-7 is a white-rot fungus with a strong capability for lignocellulose degradation, particularly lignin-rich substrates. To investigate protein-level responses during lignocellulose degradation, P. incarnata T-7 was cultured on four solid-state conditions, including poplar wood (PW), Chinese fir wood (FW), rice straw (RS), and potato dextrose agar (PDA) as the control. All substrates were cultivated for 30 days under solid-state culture conditions. Label-free quantitative proteomic analysis was performed to characterize substrate-induced protein expression profiles and to identify proteins involved in lignocellulose degradation and metabolic adaptation. Proteins were extracted from fungal mycelia, digested with trypsin, and analyzed using LC-MS/MS mass spectrometry. Raw mass spectrometry data were processed using standard database search workflows for protein identification and label-free quantification.

INSTRUMENT(S):

ORGANISM(S): Fungi Peniophora Incarnata

TISSUE(S): Hyphal Cell, Mycelium

SUBMITTER: Yujiang Qin

LAB HEAD: Jiangshan Ma

PROVIDER: PXD080412 | Pride | 2026-07-01

REPOSITORIES: pride

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Project description:Wood-degrading brown rot fungi are essential recyclers of plant biomass in forest ecosystems. Their efficient cellulolytic systems, which have potential biotechnological applications, apparently depend on a combination of two mechanisms: lignocellulose oxidation by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and non-selective, these two steps are likely segregated. A common hypothesis has been that brown rot fungi use a concentration gradient of chelated metal ions to confine ROS generation inside wood cell walls before enzymes can infiltrate. We examined an alternative: that lignocellulose-oxidation (LOX) components involved in ROS production are differentially expressed by brown rot fungi ahead of GH components. We used spatial mapping to resolve a temporal sequence in Postia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole transcriptome sequencing (RNAseq) and assayed relevant enzyme activities. We found a marked pattern of LOX upregulation in a narrow (5-mm; 48-hr) zone at the hyphal front, which included many genes likely involved in ROS generation. Upregulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with notable exceptions of two likely expansins and a GH28 pectinase. Our results support a staggered mechanism for brown rot that is controlled by differential expression rather than microenvironmental gradients. This mechanism likely results in an oxidative pretreatment of lignocellulose, possibly facilitated by expansin- and pectinase-assisted cell wall swelling, before cellulases and hemicellulases are deployed for polysaccharide depolymerization. We sequenced mRNA from 9 Postia placenta samples taken from 3 wood sections of wafer design, with 3 bioreplicates for each wood section, to compare the gene expression during brown rot processes. Three wood sections of the wafer are representing early to late decay stages.

Project description:The biodegradation of lignocellulose requires the disruption of its lignin, which shields the metabolically assimilable polysaccharides in this recalcitrant natural composite. Although a variety of microorganisms can attack lignocellulose, white rot basidiomycetes are uniquely efficient at this process, cleaving the recalcitrant intermonomer linkages of lignin via extracellular oxidative mechanisms and mineralizing many of the resulting fragments to carbon dioxide via intracellular processes. Considerable progress has been made in understanding this process in the model white rot fungus Phanerochaete chrysosporium, which expresses important components of its ligninolytic system in response to nutrient limitation, as part of its secondary metabolism. Biochemical and genetic evidence point to an important role in P. chrysosporium for secreted lignin peroxidases (LiPs), manganese peroxidases (MnPs), and H2O2-producing oxidases, which are thought to work together to cleave lignin into low molecular weight fragments. However, many aspects of ligninolysis by P. chrysosporium remain poorly understood. Although a definitive picture of the entire ligninolytic system in P. chrysosporium is not yet attainable, transcriptome analyses of the fungus grown on wood can provide useful clues. With the advent of the initial genome assembly and annotations (v1.0 and v2.1), microarray-based transcriptome analysis allowed examination of transcript levels of P. chrysosporium genes when grown in ball-milled wood and in defined growth media. This approach provided useful insights but was limited to 10048 v2.1 targets and complicated by the unpredictable manner in which the fungus responds to unnatural carbon sources in submerged basal salts media. A complete, fully coordinated ligninolytic system is likely not expressed by P. chrysosporium on ball-milled wood, because a potential route for regulatory feedback has been eliminated: the cellulose and hemicellulose in this substrate is readily accessible to enzymes, and thus ligninolysis is not essential for growth. An alternative approach is to compare levels of gene expression just before and after the onset of secondary metabolism and extracellular substrate oxidation by P. chrysosporium as it utilizes solid wood as its carbon source. If this can be done, and decay of the substrate is also confirmed, then the genes undergoing marked changes in expression during the metabolic transition can be identified with greater confidence. Although not all such genes are expected to have roles in biodegradation, this strategy may identify interesting candidates for future investigation. Here we report RNAseq-based transcriptomes to characterize changes in gene expression that occur during the transition to ligninolytic metabolism. Phanerochaete chrysosporium was inoculated onto thin sections of wood. RNA was purified from colonized material after 40 and 96 hours. Single read 100 bp Illumina runs were performed.

			Action	DRS
	Database_search_result.zip	Other
	Sample_annotation_proteomics.xlsx	Xlsx
	checksum.txt	Txt
	raw_data.zip	Other

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Label-free quantitative proteomic analysis of Peniophora incarnata T-7 during lignocellulose degradation under different substrate conditions

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