X-Ray Scattering Reveals Two Mechanisms of Cellulose Microfibril Degradation by Filamentous Fungi.

Mushroom-forming fungi (Agaricomycetes) employ enzymatic and nonenzymatic cellulose degradation mechanisms, the latter presumably relying on Fenton-generated radicals. The effects of the two mechanisms on the cellulose microfibrils structure remain poorly understood. We examined cellulose degradation caused by litter decomposers and wood decomposers, including brown-rot and white-rot fungi and one fungus with uncertain wood decay type, by combining small- and wide-angle X-ray scattering. We also examined the effects of commercial enzymes and Fenton-generated radicals on cellulose using the same method. We detected two main degradation or modification mechanisms. The first characterized the mechanism used by most fungi and resembled enzymatic cellulose degradation, causing simultaneous microfibril thinning and decreased crystalline cellulose. The second mechanism was detected in one brown-rot fungus and one litter decomposer and was characterized by patchy amorphogenesis of crystalline cellulose without substantial thinning of the fibers. This pattern did not resemble the effect of Fenton-generated radicals, suggesting a more complex mechanism is involved in the destruction of cellulose crystallinity by fungi. Furthermore, our results showed a mismatch between decay classifications and cellulose degradation patterns and that even within litter decomposers two degradation mechanisms were found, suggesting higher functional diversity under current ecological classifications of fungi. IMPORTANCE Cellulose degradation by fungi plays a fundamental role in terrestrial carbon cycling, but the mechanisms by which fungi cope with the crystallinity of cellulose are not fully understood. We used X-ray scattering to analyze how fungi, a commercial enzyme mix, and a Fenton reaction-generated radical alter the crystalline structure of cellulose. Our data revealed two mechanisms involved in crystalline cellulose degradation by fungi: one that results in the thinning of the cellulose fibers, resembling the enzymatic degradation of cellulose, and one that involves amorphogenesis of crystalline cellulose by yet-unknown pathways, resulting in a patchy-like degradation pattern. These results pave the way to a deeper understanding of cellulose degradation and the development of novel ways to utilize crystalline cellulose.

Comparative Genomics of Early-Diverging Mushroom-Forming Fungi Provides Insights into the Origins of Lignocellulose Decay Capabilities.

Evolution of lignocellulose decomposition was one of the most ecologically important innovations in fungi. White-rot fungi in the Agaricomycetes (mushrooms and relatives) are the most effective microorganisms in degrading both cellulose and lignin components of woody plant cell walls (PCW). However, the precise evolutionary origins of lignocellulose decomposition are poorly understood, largely because certain early-diverging clades of Agaricomycetes and its sister group, the Dacrymycetes, have yet to be sampled, or have been undersampled, in comparative genomic studies. Here, we present new genome sequences of ten saprotrophic fungi, including members of the Dacrymycetes and early-diverging clades of Agaricomycetes (Cantharellales, Sebacinales, Auriculariales, and Trechisporales), which we use to refine the origins and evolutionary history of the enzymatic toolkit of lignocellulose decomposition. We reconstructed the origin of ligninolytic enzymes, focusing on class II peroxidases (AA2), as well as enzymes that attack crystalline cellulose. Despite previous reports of white rot appearing as early as the Dacrymycetes, our results suggest that white-rot fungi evolved later in the Agaricomycetes, with the first class II peroxidases reconstructed in the ancestor of the Auriculariales and residual Agaricomycetes. The exemplars of the most ancient clades of Agaricomycetes that we sampled all lack class II peroxidases, and are thus concluded to use a combination of plesiomorphic and derived PCW degrading enzymes that predate the evolution of white rot.

Extensive sampling of basidiomycete genomes demonstrates inadequacy of the white-rot/brown-rot paradigm for wood decay fungi.

Basidiomycota (basidiomycetes) make up 32% of the described fungi and include most wood-decaying species, as well as pathogens and mutualistic symbionts. Wood-decaying basidiomycetes have typically been classified as either white rot or brown rot, based on the ability (in white rot only) to degrade lignin along with cellulose and hemicellulose. Prior genomic comparisons suggested that the two decay modes can be distinguished based on the presence or absence of ligninolytic class II peroxidases (PODs), as well as the abundance of enzymes acting directly on crystalline cellulose (reduced in brown rot). To assess the generality of the white-rot/brown-rot classification paradigm, we compared the genomes of 33 basidiomycetes, including four newly sequenced wood decayers, and performed phylogenetically informed principal-components analysis (PCA) of a broad range of gene families encoding plant biomass-degrading enzymes. The newly sequenced Botryobasidium botryosum and Jaapia argillacea genomes lack PODs but possess diverse enzymes acting on crystalline cellulose, and they group close to the model white-rot species Phanerochaete chrysosporium in the PCA. Furthermore, laboratory assays showed that both B. botryosum and J. argillacea can degrade all polymeric components of woody plant cell walls, a characteristic of white rot. We also found expansions in reducing polyketide synthase genes specific to the brown-rot fungi. Our results suggest a continuum rather than a dichotomy between the white-rot and brown-rot modes of wood decay. A more nuanced categorization of rot types is needed, based on an improved understanding of the genomics and biochemistry of wood decay.

Ectomycorrhizal fungi decompose soil organic matter using oxidative mechanisms adapted from saprotrophic ancestors.

Ectomycorrhizal fungi are thought to have a key role in mobilizing organic nitrogen that is trapped in soil organic matter (SOM). However, the extent to which ectomycorrhizal fungi decompose SOM and the mechanism by which they do so remain unclear, considering that they have lost many genes encoding lignocellulose-degrading enzymes that are present in their saprotrophic ancestors. Spectroscopic analyses and transcriptome profiling were used to examine the mechanisms by which five species of ectomycorrhizal fungi, representing at least four origins of symbiosis, decompose SOM extracted from forest soils. In the presence of glucose and when acquiring nitrogen, all species converted the organic matter in the SOM extract using oxidative mechanisms. The transcriptome expressed during oxidative decomposition has diverged over evolutionary time. Each species expressed a different set of transcripts encoding proteins associated with oxidation of lignocellulose by saprotrophic fungi. The decomposition 'toolbox' has diverged through differences in the regulation of orthologous genes, the formation of new genes by gene duplications, and the recruitment of genes from diverse but functionally similar enzyme families. The capacity to oxidize SOM appears to be common among ectomycorrhizal fungi. We propose that the ancestral decay mechanisms used primarily to obtain carbon have been adapted in symbiosis to scavenge nutrients instead.

Evolution of novel wood decay mechanisms in Agaricales revealed by the genome sequences of Fistulina hepatica and Cylindrobasidium torrendii.

Wood decay mechanisms in Agaricomycotina have been traditionally separated in two categories termed white and brown rot. Recently the accuracy of such a dichotomy has been questioned. Here, we present the genome sequences of the white-rot fungus Cylindrobasidium torrendii and the brown-rot fungus Fistulina hepatica both members of Agaricales, combining comparative genomics and wood decay experiments. C. torrendii is closely related to the white-rot root pathogen Armillaria mellea, while F. hepatica is related to Schizophyllum commune, which has been reported to cause white rot. Our results suggest that C. torrendii and S. commune are intermediate between white-rot and brown-rot fungi, but at the same time they show characteristics of decay that resembles soft rot. Both species cause weak wood decay and degrade all wood components but leave the middle lamella intact. Their gene content related to lignin degradation is reduced, similar to brown-rot fungi, but both have maintained a rich array of genes related to carbohydrate degradation, similar to white-rot fungi. These characteristics appear to have evolved from white-rot ancestors with stronger ligninolytic ability. F. hepatica shows characteristics of brown rot both in terms of wood decay genes found in its genome and the decay that it causes. However, genes related to cellulose degradation are still present, which is a plesiomorphic characteristic shared with its white-rot ancestors. Four wood degradation-related genes, homologs of which are frequently lost in brown-rot fungi, show signs of pseudogenization in the genome of F. hepatica. These results suggest that transition toward a brown-rot lifestyle could be an ongoing process in F. hepatica. Our results reinforce the idea that wood decay mechanisms are more diverse than initially thought and that the dichotomous separation of wood decay mechanisms in Agaricomycotina into white rot and brown rot should be revisited.

Genome sequence of the button mushroom Agaricus bisporus reveals mechanisms governing adaptation to a humic-rich ecological niche.

Agaricus bisporus is the model fungus for the adaptation, persistence, and growth in the humic-rich leaf-litter environment. Aside from its ecological role, A. bisporus has been an important component of the human diet for over 200 y and worldwide cultivation of the "button mushroom" forms a multibillion dollar industry. We present two A. bisporus genomes, their gene repertoires and transcript profiles on compost and during mushroom formation. The genomes encode a full repertoire of polysaccharide-degrading enzymes similar to that of wood-decayers. Comparative transcriptomics of mycelium grown on defined medium, casing-soil, and compost revealed genes encoding enzymes involved in xylan, cellulose, pectin, and protein degradation are more highly expressed in compost. The striking expansion of heme-thiolate peroxidases and ß-etherases is distinctive from Agaricomycotina wood-decayers and suggests a broad attack on decaying lignin and related metabolites found in humic acid-rich environment. Similarly, up-regulation of these genes together with a lignolytic manganese peroxidase, multiple copper radical oxidases, and cytochrome P450s is consistent with challenges posed by complex humic-rich substrates. The gene repertoire and expression of hydrolytic enzymes in A. bisporus is substantially different from the taxonomically related ectomycorrhizal symbiont Laccaria bicolor. A common promoter motif was also identified in genes very highly expressed in humic-rich substrates. These observations reveal genetic and enzymatic mechanisms governing adaptation to the humic-rich ecological niche formed during plant degradation, further defining the critical role such fungi contribute to soil structure and carbon sequestration in terrestrial ecosystems. Genome sequence will expedite mushroom breeding for improved agronomic characteristics.

Comparative genomics of Ceriporiopsis subvermispora and Phanerochaete chrysosporium provide insight into selective ligninolysis.

Efficient lignin depolymerization is unique to the wood decay basidiomycetes, collectively referred to as white rot fungi. Phanerochaete chrysosporium simultaneously degrades lignin and cellulose, whereas the closely related species, Ceriporiopsis subvermispora, also depolymerizes lignin but may do so with relatively little cellulose degradation. To investigate the basis for selective ligninolysis, we conducted comparative genome analysis of C. subvermispora and P. chrysosporium. Genes encoding manganese peroxidase numbered 13 and five in C. subvermispora and P. chrysosporium, respectively. In addition, the C. subvermispora genome contains at least seven genes predicted to encode laccases, whereas the P. chrysosporium genome contains none. We also observed expansion of the number of C. subvermispora desaturase-encoding genes putatively involved in lipid metabolism. Microarray-based transcriptome analysis showed substantial up-regulation of several desaturase and MnP genes in wood-containing medium. MS identified MnP proteins in C. subvermispora culture filtrates, but none in P. chrysosporium cultures. These results support the importance of MnP and a lignin degradation mechanism whereby cleavage of the dominant nonphenolic structures is mediated by lipid peroxidation products. Two C. subvermispora genes were predicted to encode peroxidases structurally similar to P. chrysosporium lignin peroxidase and, following heterologous expression in Escherichia coli, the enzymes were shown to oxidize high redox potential substrates, but not Mn(2+). Apart from oxidative lignin degradation, we also examined cellulolytic and hemicellulolytic systems in both fungi. In summary, the C. subvermispora genetic inventory and expression patterns exhibit increased oxidoreductase potential and diminished cellulolytic capability relative to P. chrysosporium.

Lignin-degrading peroxidases in Polyporales: an evolutionary survey based on 10 sequenced genomes.

The genomes of three representative Polyporales (Bjerkandera adusta, Phlebia brevispora and a member of the Ganoderma lucidum complex) were sequenced to expand our knowledge on the diversity of ligninolytic and related peroxidase genes in this Basidiomycota order that includes most wood-rotting fungi. The survey was completed by analyzing the heme-peroxidase genes in the already available genomes of seven more Polyporales species representing the antrodia, gelatoporia, core polyporoid and phlebioid clades. The study confirms the absence of ligninolytic peroxidase genes from the manganese peroxidase (MnP), lignin peroxidase (LiP) and versatile peroxidase (VP) families, in the brown-rot fungal genomes (all of them from the antrodia clade), which include only a limited number of predicted low redox-potential generic peroxidase (GP) genes. When members of the heme-thiolate peroxidase (HTP) and dye-decolorizing peroxidase (DyP) superfamilies (up to a total of 64 genes) also are considered, the newly sequenced B. adusta appears as the Polyporales species with the highest number of peroxidase genes due to the high expansion of both the ligninolytic peroxidase and DyP (super)families. The evolutionary relationships of the 111 genes for class-II peroxidases (from the GP, MnP, VP, LiP families) in the 10 Polyporales genomes is discussed including the existence of different MnP subfamilies and of a large and homogeneous LiP cluster, while different VPs mainly cluster with short MnPs. Finally, ancestral state reconstructions showed that a putative MnP gene, derived from a primitive GP that incorporated the Mn(II)-oxidation site, is the precursor of all the class-II ligninolytic peroxidases. Incorporation of an exposed tryptophan residue involved in oxidative degradation of lignin in a short MnP apparently resulted in evolution of the first VP. One of these ancient VPs might have lost the Mn(II)-oxidation site being at the origin of all the LiP enzymes, which are found only in species of the order Polyporales.

Uncovering the hidden diversity of litter-decomposition mechanisms in mushroom-forming fungi.

Litter decomposing Agaricales play key role in terrestrial carbon cycling, but little is known about their decomposition mechanisms. We assembled datasets of 42 gene families involved in plant-cell-wall decomposition from seven newly sequenced litter decomposers and 35 other Agaricomycotina members, mostly white-rot and brown-rot species. Using sequence similarity and phylogenetics, we split the families into phylogroups and compared their gene composition across nutritional strategies. Subsequently, we used Raman spectroscopy to examine the ability of litter decomposers, white-rot fungi, and brown-rot fungi to decompose crystalline cellulose. Both litter decomposers and white-rot fungi share the enzymatic cellulose decomposition, whereas brown-rot fungi possess a distinct mechanism that disrupts cellulose crystallinity. However, litter decomposers and white-rot fungi differ with respect to hemicellulose and lignin degradation phylogroups, suggesting adaptation of the former group to the litter environment. Litter decomposers show high phylogroup diversity, which is indicative of high functional versatility within the group, whereas a set of white-rot species shows adaptation to bulk-wood decomposition. In both groups, we detected species that have unique characteristics associated with hitherto unknown adaptations to diverse wood and litter substrates. Our results suggest that the terms white-rot fungi and litter decomposers mask a much larger functional diversity.

The Paleozoic origin of enzymatic lignin decomposition reconstructed from 31 fungal genomes.

Wood is a major pool of organic carbon that is highly resistant to decay, owing largely to the presence of lignin. The only organisms capable of substantial lignin decay are white rot fungi in the Agaricomycetes, which also contains non-lignin-degrading brown rot and ectomycorrhizal species. Comparative analyses of 31 fungal genomes (12 generated for this study) suggest that lignin-degrading peroxidases expanded in the lineage leading to the ancestor of the Agaricomycetes, which is reconstructed as a white rot species, and then contracted in parallel lineages leading to brown rot and mycorrhizal species. Molecular clock analyses suggest that the origin of lignin degradation might have coincided with the sharp decrease in the rate of organic carbon burial around the end of the Carboniferous period.