Distinct Growth and Secretome Strategies for Two Taxonomically Divergent Brown Rot Fungi

ABSTRACT Brown rot fungi are wood-degrading fungi that employ both oxidative and hydrolytic mechanisms to degrade wood. Hydroxyl radicals that facilitate the oxidative component are powerful nonselective oxidants and are incompatible with hydrolytic enzymes unless they are spatially segregated in wood. Differential gene expression has been implicated in the segregation of these reactions in Postia placenta, but it is unclear if this two-step mechanism varies in other brown rot fungi with different traits and life history strategies that occupy different niches in nature. We employed proteomics to analyze a progression of wood decay on thin wafers, using brown rot fungi with significant taxonomic and niche distances: Serpula lacrymans (Boletales; “dry rot” lumber decay) and Gloeophyllum trabeum (order Gloeophyllales; slash, downed wood). Both fungi produced greater oxidoreductase diversity upon wood colonization and greater glycoside hydrolase activity later, consistent with a two-step mechanism. The two fungi invested very differently, however, in terms of growth (infrastructure) versus protein secretion (resource capture), with the ergosterol/extracted protein ratio being 7-fold higher with S. lacrymans than with G. trabeum. In line with the native substrate associations of these fungi, hemicellulase-specific activities were dominated by mannanase in S. lacrymans and by xylanase in G. trabeum. Consistent with previous observations, S. lacrymans did not produce glycoside hydrolase 6 (GH6) cellobiohydrolases (CBHs) in this study, despite taxonomically belonging to the order Boletales, which is distinguished among brown rot fungi by having CBH genes. This work suggests that distantly related brown rot fungi employ staggered mechanisms to degrade wood, but the underlying strategies vary among taxa. IMPORTANCE Wood-degrading fungi are important in forest nutrient cycling and offer promise in biotechnological applications. Brown rot fungi are unique among these fungi in that they use a nonenzymatic oxidative pretreatment before enzymatic carbohydrate hydrolysis, enabling selective removal of carbohydrates from lignin. This capacity has independently evolved multiple times, but it is unclear if different mechanisms underpin similar outcomes. Here, we grew fungi directionally on wood wafers and we found similar two-step mechanisms in taxonomically divergent brown rot fungi. The results, however, revealed strikingly different growth strategies, with S. lacrymans investing more in biomass production than secretion of proteins and G. trabeum showing the opposite pattern, with a high diversity of uncharacterized proteins. The “simplified” S. lacrymans secretomic system could help narrow gene targets central to oxidative brown rot pretreatments, and a comparison of its distinctions with G. trabeum and other brown rot fungi (e.g., Postia placenta) might offer similar traction in noncatabolic genes.

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Localizing gene regulation reveals a staggered wood decay mechanism for the brown rot fungusPostia placenta

Proceedings of the National Academy of Sciences ◽

10.1073/pnas.1608454113 ◽

2016 ◽

Vol 113 (39) ◽

pp. 10968-10973 ◽

Cited By ~ 73

Author(s):

Jiwei Zhang ◽

Gerald N. Presley ◽

Kenneth E. Hammel ◽

Jae-San Ryu ◽

Jon R. Menke ◽

...

Keyword(s):

Cell Walls ◽

Plant Biomass ◽

Wood Decay ◽

Brown Rot ◽

Glycoside Hydrolases ◽

Ros Generation ◽

Rna Seq ◽

Postia Placenta ◽

Brown Rot Fungi ◽

Whole Transcriptome

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 (LOX) by reactive oxygen species (ROS) and polysaccharide hydrolysis by a limited set of glycoside hydrolases (GHs). Given that ROS are strongly oxidizing and nonselective, 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 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 inPostia placenta, sectioning thin wood wafers colonized directionally. Among sections, we measured gene expression by whole-transcriptome shotgun sequencing (RNA-seq) and assayed relevant enzyme activities. We found a marked pattern of LOX up-regulation in a narrow (5-mm, 48-h) zone at the hyphal front, which included many genes likely involved in ROS generation. Up-regulation of GH5 endoglucanases and many other GHs clearly occurred later, behind the hyphal front, with the 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.

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Comparative Morphology of Early Stages of Brown-Rot Wood Decay

IAWA Journal ◽

10.1163/22941932-90001306 ◽

1993 ◽

Vol 14 (2) ◽

pp. 127-138 ◽

Cited By ~ 14

Author(s):

W. Wayne Wilcox

Keyword(s):

Wood Decay ◽

Brown Rot ◽

Comparative Morphology ◽

Diagnostic Feature ◽

Postia Placenta ◽

Brown Rot Fungi ◽

Early Stages ◽

Poria Placenta ◽

White Fir ◽

Early Progression

Early stages of decay by two brown-rot fungi in two woods were studied by light and scanning electron microscopy. The earliest diagnostic feature to appear was hyphae in the earlywood lumina. The earliest effect on cell walls was the loss of birefringence in the earlywood; Poria placenta (syn. Postia placenta) caused this loss at the earliest stage of decay observed, in both Douglas-fir and white fir, while Gloeophyllum trabeum caused significant weight loss before loss of birefringence was visible. Attack on the latewood progressed from the earlywood, and was different in pattern among the wood/fungus combinations. Hyphal and bore hole diameter increased throughout the early progression of decay and would be useful in evaluating the stage of decay, if the starting diameter of hyphae could be determined. Separation between cells was not observed until moderate stages of decay and, therefore, was not useful in diagnosing early stages of decay.

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Samanlarda Biyolojik Muamelelerle Lignoselüloz Kompleksin Sindirilebilirliğinin Artırılması

Turkish Journal of Agriculture - Food Science and Technology ◽

10.24925/turjaf.v5i13.1704-1709.1522 ◽

2017 ◽

Vol 5 (13) ◽

pp. 1704

Author(s):

Aydan Atalar ◽

Nurcan Çetinkaya

Keyword(s):

Biological Treatment ◽

Hydrolytic Enzymes ◽

White Rot Fungi ◽

Ligninolytic Enzymes ◽

Brown Rot ◽

Soft Rot ◽

White Rot ◽

Enzyme Treatment ◽

Environmental Friendliness ◽

Brown Rot Fungi

The efforts to break down the lignocellulosic complex found in the cell wall of straws, besides digestible cellulose and hemicellulose by rumen fermentation, improvement of straw digestibility by the degradation of indigestible lignin fraction of complex by using of biotechnological methods is one of the focus areas of animal nutritionists in recent years. Biological method sare prefer redover other methods due to the environmental friendliness. In the biological treatment methods of lignocellulosic complex, biodiversity of bacteria, enzymes and fungi gives opportunity to select lignin degrading species. Mycobacterium, Arthrobacter and Flavobacterium genre bacteria are used to degrade lignin by bacterial treatment. Lignocellulolytic enzymes isolated from different varieties of fungi are used in enzyme treatment. There are 3 genres of fungus that are white, Brown and soft rot in fungal treatments. Brown rot fungi prefer ably attack cellulose and hemicelluloses, but not lignin. White rot fungi attack the lignin and break up lignol bonds and aromatic ring. White rot fungi break down polysaccharides with hydrolytic enzymes such as cellulase, xylanase, and lignin with oxidative ligninolytic enzymes such as lignin peroxidase and laccase. Because of the fact that the microorganisms that can break down the lignocellulosic materials are the fungi and the cost is low, the application of white rot fungi is possible. In this paper, improvement the lignocellulosic comlex digestibility of straw by biological treatment with the advantage of biodiversity is discussed.

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Initial Rhodonia placenta Gene Expression in Acetylated Wood: Group-Wise Upregulation of Non-Enzymatic Oxidative Wood Degradation Genes Depending on the Treatment Level

Forests ◽

10.3390/f10121117 ◽

2019 ◽

Vol 10 (12) ◽

pp. 1117 ◽

Cited By ~ 4

Author(s):

Martina Kölle ◽

Rebecka Ringman ◽

Annica Pilgård

Keyword(s):

Hydrolytic Enzymes ◽

Oxidative Degradation ◽

Brown Rot ◽

Treatment Level ◽

Wood Degradation ◽

Weight Percentage ◽

Brown Rot Fungi ◽

Number Of Genes ◽

Brown Rot Decay ◽

Underlying Mechanisms

Acetylation has been shown to delay fungal decay, but the underlying mechanisms are poorly understood. Brown-rot fungi, such as Rhodonia placenta (Fr.) Niemelä, K.H. Larss. & Schigel, degrade wood in two steps, i.e., oxidative depolymerization followed by secretion of hydrolytic enzymes. Since separating the two degradation steps has been proven challenging, a new sample design was applied to the task. The aim of this study was to compare the expression of 10 genes during the initial decay phase in wood and wood acetylated to three different weight percentage gains (WPG). The results showed that not all genes thought to play a role in initiating brown-rot decay are upregulated. Furthermore, the results indicate that R. placenta upregulates an increasing number of genes involved in the oxidative degradation phase with increasing WPG.

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Influence of carbon source on wood decay-associated gene expression in sequential hyphal zones of the brown rot fungus Gloeophyllum trabeum

Bioscience Biotechnology and Biochemistry ◽

10.1093/bbb/zbab080 ◽

2021 ◽

Author(s):

Kiwamu Umezawa ◽

Shuji Itakura

Keyword(s):

Gene Expression ◽

Gene Regulation ◽

Carbon Source ◽

Glycoside Hydrolase ◽

Degradation Mechanism ◽

Enzymatic Saccharification ◽

Brown Rot ◽

Gloeophyllum Trabeum ◽

Brown Rot Fungi ◽

Brown Rot Fungus

Abstract Brown rot fungi show a two-step wood degradation mechanism comprising oxidative radical-based and enzymatic saccharification systems. Recent studies have demonstrated that the brown rot fungus Rhodonia placenta expresses oxidoreductase genes ahead of glycoside hydrolase genes and spatially protects the saccharification enzymes from oxidative damage of the oxidoreductase reactions. This study aimed to assess the generality of the spatial gene regulation of these genes in other brown rot fungi and examine the effects of carbon source on the gene regulation. Gene expression analysis was performed on 14 oxidoreductase and glycoside hydrolase genes in the brown rot fungus Gloeophyllum trabeum, directionally grown on wood, sawdust-agar, and glucose-agar wafers. In G. trabeum, both oxidoreductase and glycoside hydrolase genes were expressed at higher levels in sections behind the wafers. The upregulation of glycoside hydrolase genes was significantly higher in woody substrates than in glucose, whereas the oxidoreductase gene expression was not affected by substrates.

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Samanlarda Biyolojik Muamelelerle Lignoselüloz Kompleksin Sindirilebilirliğinin Artırılması

Turkish Journal of Agriculture - Food Science and Technology ◽

10.24925/turjaf.v5i13.1720-1725.1522 ◽

2017 ◽

Vol 5 (13) ◽

pp. 1720 ◽

Cited By ~ 1

Author(s):

Aydan Atalar ◽

Nurcan Çetinkaya

Keyword(s):

Biological Treatment ◽

Hydrolytic Enzymes ◽

White Rot Fungi ◽

Ligninolytic Enzymes ◽

Brown Rot ◽

Soft Rot ◽

White Rot ◽

Enzyme Treatment ◽

Environmental Friendliness ◽

Brown Rot Fungi

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Brown Rot-Type Fungal Decomposition of Sorghum Bagasse: Variable Success and Mechanistic Implications

International Journal of Microbiology ◽

10.1155/2018/4961726 ◽

2018 ◽

Vol 2018 ◽

pp. 1-7 ◽

Cited By ~ 2

Author(s):

Gerald N. Presley ◽

Bongani K. Ndimba ◽

Jonathan S. Schilling

Keyword(s):

Plant Biomass ◽

Specific Activity ◽

Biomass Conversion ◽

Xylanase Activity ◽

Brown Rot ◽

Fomitopsis Pinicola ◽

Postia Placenta ◽

Brown Rot Fungi ◽

Protein Levels ◽

Serpula Lacrymans

Sweet sorghum is a promising crop for a warming, drying African climate, and basic information is lacking on conversion pathways for its lignocellulosic residues (bagasse). Brown rot wood-decomposer fungi use carbohydrate-selective pathways that, when assessed on sorghum, a grass substrate, can yield information relevant to both plant biomass conversion and fungal biology. In testing sorghum decomposition by brown rot fungi (Gloeophyllum trabeum,Serpula lacrymans), we found thatG. trabeumreadily degraded sorghum, removing xylan prior to removing glucan.Serpula lacrymans, conversely, caused little decomposition. Ergosterol (fungal biomarker) and protein levels were similar for both fungi, butS. lacrymansproduced nearly 4x lower polysaccharide-degrading enzyme specific activity on sorghum thanG. trabeum, perhaps a symptom of starvation. Linking this information to genome comparisons including other brown rot fungi known to have a similar issue regarding decomposing grasses(Postia placenta, Fomitopsis pinicola)suggested that a lack of CE 1 feruloyl esterases as well as low xylanase activity inS. lacrymans(3x lower than inG. trabeum) may hinderS. lacrymans,P. placenta,andF. pinicolawhen degrading grass substrates. These results indicate variability in brown rot mechanisms, which may stem from a differing ability to degrade certain lignin-carbohydrate complexes.

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Evidence from Serpula lacrymans that 2,5-Dimethoxyhydroquinone Is a Lignocellulolytic Agent of Divergent Brown Rot Basidiomycetes

Applied and Environmental Microbiology ◽

10.1128/aem.03880-12 ◽

2013 ◽

Vol 79 (7) ◽

pp. 2377-2383 ◽

Cited By ~ 33

Author(s):

Premsagar Korripally ◽

Vitaliy I. Timokhin ◽

Carl J. Houtman ◽

Michael D. Mozuch ◽

Kenneth E. Hammel

Keyword(s):

Coniferous Forest ◽

White Rot Fungi ◽

Brown Rot ◽

White Rot ◽

Fenton Reagent ◽

Fenton Chemistry ◽

Content Type ◽

Brown Rot Fungi ◽

Serpula Lacrymans

ABSTRACTBasidiomycetes that cause brown rot of wood are essential biomass recyclers in coniferous forest ecosystems and a major cause of failure in wooden structures. Recent work indicates that distinct lineages of brown rot fungi have arisen independently from ligninolytic white rot ancestors via loss of lignocellulolytic enzymes. Brown rot thus proceeds without significant lignin removal, apparently beginning instead with oxidative attack on wood polymers by Fenton reagent produced when fungal hydroquinones or catechols reduce Fe3+in colonized wood. Since there is little evidence that white rot fungi produce these metabolites, one question is the extent to which independent lineages of brown rot fungi may have evolved different Fe3+reductants. Recently, the catechol variegatic acid was proposed to drive Fenton chemistry inSerpula lacrymans, a brown rot member of the Boletales (D. C. Eastwood et al., Science 333:762-765, 2011). We found no variegatic acid in wood undergoing decay byS. lacrymans. We found also that variegatic acid failed to reducein vitrothe Fe3+oxalate chelates that predominate in brown-rotting wood and that it did not drive Fenton chemistryin vitrounder physiological conditions. Instead, the decaying wood contained physiologically significant levels of 2,5-dimethoxyhydroquinone, a reductant with a demonstrated biodegradative role when wood is attacked by certain brown rot fungi in two other divergent lineages, the Gloeophyllales and Polyporales. Our results suggest that the pathway for 2,5-dimethoxyhydroquinone biosynthesis may have been present in ancestral white rot basidiomycetes but do not rule out the possibility that it appeared multiple times via convergent evolution.

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Coupling Secretomics with Enzyme Activities To Compare the Temporal Processes of Wood Metabolism among White and Brown Rot Fungi

Applied and Environmental Microbiology ◽

10.1128/aem.00159-18 ◽

2018 ◽

Vol 84 (16) ◽

Cited By ~ 14

Author(s):

Gerald N. Presley ◽

Ellen Panisko ◽

Samuel O. Purvine ◽

Jonathan S. Schilling

Keyword(s):

Enzyme Activities ◽

Temporal Trends ◽

White Rot Fungi ◽

Brown Rot ◽

Glycoside Hydrolases ◽

White Rot ◽

Directional Growth ◽

Content Type ◽

Brown Rot Fungi ◽

Lignocellulose Conversion

ABSTRACTWood-degrading fungi use a sequence of oxidative and hydrolytic mechanisms to loosen lignocellulose and then release and metabolize embedded sugars. These temporal sequences have recently been mapped at high resolution using directional growth on wood wafers, revealing previously obscured dynamics as fungi progressively colonize wood. Here, we applied secretomics in the same wafer design to track temporal trends on aspen decayed by fungi with distinct nutritional modes: two brown rot (BR) fungi (Postia placentaandGloeophyllum trabeum) and two white rot (WR) fungi (Stereum hirsutumandTrametes versicolor). We matched secretomic data from three zones of decay (early, middle, and late) with enzyme activities in these zones, and we included measures of total protein and ergosterol as measures of fungal biomass. In line with previous transcriptomics data, the fungi tested showed an initial investment in pectinases and a delayed investment in glycoside hydrolases (GHs). Brown rot fungi also staggered the abundance of some oxidoreductases ahead of GHs to produce a familiar two-step mechanism. White rot fungi, however, showed late-stage investment in pectinases as well, unlike brown rot fungi. Ligninolytic enzyme activities and abundances were also different between the two white rot fungi. Specifically,S. hirsutumligninolytic activity was delayed, which was explained almost entirely by the activity and abundance of five atypical manganese peroxidases, unlike more varied peroxidases and laccases inT. versicolor. These secretomic analyses support brown rot patterns generated via transcriptomics, they reveal distinct patterns among and within rot types, and they link spectral counts with activities to help functionalize these multistrain secretomic data.IMPORTANCEWood decay, driven primarily by wood-degrading basidiomycetes, is an essential component of global carbon cycles, and decay mechanisms are essential for understanding forest ecosystem function. These fungi efficiently consolidate pretreatment and saccharification of wood under mild conditions, making them promising templates for low-cost lignocellulose conversion. Species are categorized as ligninolytic white rots and polysaccharide-selective brown rots, with considerable undescribed variability in decay mechanism that may manifest in the sequential variation in protein secretion over the progression of decay. Here we resolved spatially a temporal progression of decay on intact wood wafers and compared secretome dynamics in two white and two brown rot fungi. We identified several universal mechanistic components among decay types, including early pectinolytic “pretreatment” and later-stage glycoside hydrolase-mediated saccharification. Interspecific comparisons also identified considerable mechanistic diversity within rot types, indicating that there are multiple avenues to facilitate white and brown rots.

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Oxidative Damage Control during Decay of Wood by Brown Rot Fungus Using Oxygen Radicals

Applied and Environmental Microbiology ◽

10.1128/aem.01937-18 ◽

2018 ◽

Vol 84 (22) ◽

Cited By ~ 3

Author(s):

Jesus D. Castaño ◽

Jiwei Zhang ◽

Claire E. Anderson ◽

Jonathan S. Schilling

Keyword(s):

Reactive Oxygen Species ◽

Cell Walls ◽

Reactive Oxygen ◽

Brown Rot ◽

White Rot ◽

Ros Generation ◽

Oxygen Species ◽

Content Type ◽

Brown Rot Fungi ◽

Brown Rot Fungus

ABSTRACTBrown rot wood-degrading fungi deploy reactive oxygen species (ROS) to loosen plant cell walls and enable selective polysaccharide extraction. These ROS, including Fenton-generated hydroxyl radicals (HO˙), react with little specificity and risk damaging hyphae and secreted enzymes. Recently, it was shown that brown rot fungi reduce this risk, in part, by differentially expressing genes involved in HO˙ generation ahead of those coding carbohydrate-active enzymes (CAZYs). However, there are notable exceptions to this pattern, and we hypothesized that brown rot fungi would require additional extracellular mechanisms to limit ROS damage. To assess this, we grewPostia placentadirectionally on wood wafers to spatially segregate early from later decay stages. Extracellular HO˙ production (avoidance) and quenching (suppression) capacities among the stages were analyzed, along with the ability of secreted CAZYs to maintain activity postoxidation (tolerance). First, we found that H2O2and Fe2+concentrations in the extracellular environment were conducive to HO˙ production in early (H2O2:Fe2+ratio 2:1) but not later (ratio 1:131) stages of decay. Second, we found that ABTS radical cation quenching (antioxidant capacity) was higher in later decay stages, coincident with higher fungal phenolic concentrations. Third, by surveying enzyme activities before/after exposure to Fenton-generated HO˙, we found that CAZYs secreted early, amid HO˙, were more tolerant of oxidative stress than those expressed later and were more tolerant than homologs in the model CAZY producerTrichoderma reesei. Collectively, this indicates thatP. placentauses avoidance, suppression, and tolerance mechanisms, extracellularly, to complement intracellular differential expression, enabling this brown rot fungus to use ROS to degrade wood.IMPORTANCEWood is one of the largest pools of carbon on Earth, and its decomposition is dominated in most systems by fungi. Wood-degrading fungi specialize in extracting sugars bound within lignin, either by removing lignin first (white rot) or by using Fenton-generated reactive oxygen species (ROS) to “loosen” wood cell walls, enabling selective sugar extraction (brown rot). Although white rot lignin-degrading pathways are well characterized, there are many uncertainties in brown rot fungal mechanisms. Our study addressed a key uncertainty in how brown rot fungi deploy ROS without damaging themselves or the enzymes they secrete. In addition to revealing differentially expressed genes to promote ROS generation only in early decay, our study revealed three spatial control mechanisms to avoid/tolerate ROS: (i) constraining Fenton reactant concentrations (H2O2, Fe2+), (ii) quenching ROS via antioxidants, and (iii) secreting ROS-tolerant enzymes. These results not only offer insight into natural decomposition pathways but also generate targets for biotechnological development.

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