Wood Degradation by Fomitiporia mediterranea M. Fischer: Exploring Fungal Adaptation Using Metabolomic Networking.

Fomitiporia mediterranea M. Fischer (Fmed) is a white-rot wood-decaying fungus associated with one of the most important and challenging diseases in vineyards: Esca. To relieve microbial degradation, woody plants, including Vitis vinifera, use structural and chemical weapons. Lignin is the most recalcitrant of the wood cell wall structural compounds and contributes to wood durability. Extractives are constitutive or de novo synthesized specialized metabolites that are not covalently bound to wood cell walls and are often associated with antimicrobial properties. Fmed is able to mineralize lignin and detoxify toxic wood extractives, thanks to enzymes such as laccases and peroxidases. Grapevine wood's chemical composition could be involved in Fmed's adaptation to its substrate. This study aimed at deciphering if Fmed uses specific mechanisms to degrade grapevine wood structure and extractives. Three different wood species, grapevine, beech, and oak. were exposed to fungal degradation by two Fmed strains. The well-studied white-rot fungus Trametes versicolor (Tver) was used as a comparison model. A simultaneous degradation pattern was shown for Fmed in the three degraded wood species. Wood mass loss after 7 months for the two fungal species was the highest with low-density oak wood. For the latter wood species, radical differences in initial wood density were observed. No differences between grapevine or beech wood degradation rates were observed after degradation by Fmed or by Tver. Contrary to the Tver secretome, one manganese peroxidase isoform (MnP2l, jgi protein ID 145801) was the most abundant in the Fmed secretome on grapevine wood only. Non-targeted metabolomic analysis was conducted on wood and mycelium samples, using metabolomic networking and public databases (GNPS, MS-DIAL) for metabolite annotations. Chemical differences between non-degraded and degraded woods, and between mycelia grown on different wood species, are discussed. This study highlights Fmed physiological, proteomic and metabolomic traits during wood degradation and thus contributes to a better understanding of its wood degradation mechanisms.

Modes-of-action of antifungal compounds: Stressors and (target-site-specific) toxins, toxicants, or toxin-stressors.

Fungi and antifungal compounds are relevant to the United Nation's Sustainable Development Goals. However, the modes-of-action of antifungals-whether they are naturally occurring substances or anthropogenic fungicides-are often unknown or are misallocated in terms of their mechanistic category. Here, we consider the most effective approaches to identifying whether antifungal substances are cellular stressors, toxins/toxicants (that are target-site-specific), or have a hybrid mode-of-action as toxin-stressors (that induce cellular stress yet are target-site-specific). This newly described 'toxin-stressor' category includes some photosensitisers that target the cell membrane and, once activated by light or ultraviolet radiation, cause oxidative damage. We provide a glossary of terms and a diagrammatic representation of diverse types of stressors, toxic substances, and toxin-stressors, a classification that is pertinent to inhibitory substances not only for fungi but for all types of cellular life. A decision-tree approach can also be used to help differentiate toxic substances from cellular stressors (Curr Opin Biotechnol 2015 33: 228-259). For compounds that target specific sites in the cell, we evaluate the relative merits of using metabolite analyses, chemical genetics, chemoproteomics, transcriptomics, and the target-based drug-discovery approach (based on that used in pharmaceutical research), focusing on both ascomycete models and the less-studied basidiomycete fungi. Chemical genetic methods to elucidate modes-of-action currently have limited application for fungi where molecular tools are not yet available; we discuss ways to circumvent this bottleneck. We also discuss ecologically commonplace scenarios in which multiple substances act to limit the functionality of the fungal cell and a number of as-yet-unresolved questions about the modes-of-action of antifungal compounds pertaining to the Sustainable Development Goals.

First Description of Non-Enzymatic Radical-Generating Mechanisms Adopted by Fomitiporia mediterranea: An Unexplored Pathway of the White Rot Agent of the Esca Complex of Diseases.

Fomitiporia mediterranea (Fmed) is the primary Basidiomycota species causing white rot in European vineyards affected by the Esca complex of diseases (ECD). In the last few years, an increasing number of studies have highlighted the importance of reconsidering the role of Fmed in ECD etiology, justifying an increase in research interest related to Fmed's biomolecular pathogenetic mechanisms. In the context of the current re-evaluation of the binary distinction (brown vs. white rot) between biomolecular decay pathways induced by Basidiomycota species, our research aims to investigate the potential for non-enzymatic mechanisms adopted by Fmed, which is typically described as a white rot fungus. Our results demonstrate how, in liquid culture reproducing nutrient restriction conditions often found in wood, Fmed can produce low molecular weight compounds, the hallmark of the non-enzymatic "chelator-mediated Fenton" (CMF) reaction, originally described for brown rot fungi. CMF reactions can redox cycle with ferric iron, generating hydrogen peroxide and ferrous iron, necessary reactants leading to hydroxyl radical (•OH) production. These observations led to the conclusion that a non-enzymatic radical-generating CMF-like mechanism may be utilized by Fmed, potentially together with an enzymatic pool, to contribute to degrading wood constituents; moreover, indicating significant variability between strains.

Structural insights into the interactions of glutathione transferases with a nitric oxide carrier and sodium nitroprusside.

Glutathione transferases are detoxification enzymes with multifaceted roles, including a role in the metabolism and scavenging of nitric oxide (NO) compounds in cells. Here, we explored the ability of Trametes versicolor glutathione transferases (GSTs) from the Omega class (TvGSTOs) to bind metal-nitrosyl compounds. TvGSTOs have been studied previously for their ligandin role and are interesting models to study protein‒ligand interactions. First, we determined the X-ray structure of the TvGSTO3S isoform bound to the dinitrosyl glutathionyl iron complex (DNGIC), a physiological compound involved in the storage of nitric oxide. Our results suggested a different binding mode compared to the one previously described in human GST Pi 1 (GSTP1). Then, we investigated the manner in which TvGSTO3S binds three nonphysiological metal-nitrosyl compounds with different metal cores (iron, ruthenium and osmium). We assayed sodium nitroprusside, a well-studied vasodilator used in cases of hypertensive crises or heart failure. Our results showed that the tested GST can bind metal-nitrosyls at two distinct binding sites. Thermal shift analysis with six isoforms of TvGSTOs identified TvGSTO6S as the best interactant. Using the Griess method, TvGSTO6S was found to improve the release of nitric oxide from sodium nitroprusside in vitro, whereas the effects of human GST alpha 1 (GSTA1) and GSTP1 were moderate. Our results open new structural perspectives for understanding the interactions of glutathione transferases with metal-nitrosyl compounds associated with the biochemical mechanisms of NO uptake/release in biological systems.

Wood degradation by Fomitiporia mediterranea M. Fischer: Physiologic, metabolomic and proteomic approaches.

Fomitiporia mediterranea (Fmed) is one of the main fungal species found in grapevine wood rot, also called "amadou," one of the most typical symptoms of grapevine trunk disease Esca. This fungus is functionally classified as a white-rot, able to degrade all wood structure polymers, i.e., hemicelluloses, cellulose, and the most recalcitrant component, lignin. Specific enzymes are secreted by the fungus to degrade those components, namely carbohydrate active enzymes for hemicelluloses and cellulose, which can be highly specific for given polysaccharide, and peroxidases, which enable white-rot to degrade lignin, with specificities relating to lignin composition as well. Furthermore, besides polymers, a highly diverse set of metabolites often associated with antifungal activities is found in wood, this set differing among the various wood species. Wood decayers possess the ability to detoxify these specific extractives and this ability could reflect the adaptation of these fungi to their specific environment. The aim of this study is to better understand the molecular mechanisms used by Fmed to degrade wood structure, and in particular its potential adaptation to grapevine wood. To do so, Fmed was cultivated on sawdust from different origins: grapevine, beech, and spruce. Carbon mineralization rate, mass loss, wood structure polymers contents, targeted metabolites (extractives) and secreted proteins were measured. We used the well-known white-rot model Trametes versicolor for comparison. Whereas no significant degradation was observed with spruce, a higher mass loss was measured on Fmed grapevine culture compared to beech culture. Moreover, on both substrates, a simultaneous degradation pattern was demonstrated, and proteomic analysis identified a relative overproduction of oxidoreductases involved in lignin and extractive degradation on grapevine cultures, and only few differences in carbohydrate active enzymes. These results could explain at least partially the adaptation of Fmed to grapevine wood structural composition compared to other wood species, and suggest that other biotic and abiotic factors should be considered to fully understand the potential adaptation of Fmed to its ecological niche. Proteomics data are available via ProteomeXchange with identifier PXD036889.

Oxygen Radical-Generating Metabolites Secreted by Eutypa and Esca Fungal Consortia: Understanding the Mechanisms Behind Grapevine Wood Deterioration and Pathogenesis.

Eutypa dieback and Esca complex are fungal diseases of grape that cause large economic losses in vineyards. These diseases require, or are enhanced by, fungal consortia growth which leads to the deterioration of the wood tissue in the grapevine trunk; however, pathogenesis and the underlying mechanisms involved in the woody tissue degradation are not understood. We examined the role that the consortia fungal metabolome have in generating oxygen radicals that could potentially play a role in trunk decay and pathogenesis. Unique metabolites were isolated from the consortia fungi with some metabolites preferentially reducing iron whereas others were involved in redox cycling to generate hydrogen peroxide. Metabolite suites with different functions were produced when fungi were grown separately vs. when grown in consortia. Chelator-mediated Fenton (CMF) chemistry promoted by metabolites from these fungi allowed for the generation of highly reactive hydroxyl radicals. We hypothesize that this mechanism may be involved in pathogenicity in grapevine tissue as a causal mechanism associated with trunk wood deterioration/necrosis in these two diseases of grape.

Grapevine Wood-Degrading Activity of Fomitiporia mediterranea M. Fisch.: A Focus on the Enzymatic Pathway Regulation.

Fomitiporia mediterranea is a Basidiomycetes fungus associated with some of the Esca complex diseases and responsible for decay in grapevine wood. Its role in the onset of foliar symptoms has recently been reconsidered, mainly after evidence showing a reduction in foliar symptom expression after removal of rotten wood. The study of its degradation pathways has already been approached by other authors, and with this study much information is consolidated. A microscopic observation of degraded wood provides a first approach to the characterization of F. mediterranea modalities of wood cellular structure degradation. The decay of grapevine wood was reproduced in vitro, and the measurement of each wood-forming polymer loss highlighted characteristics of F. mediterranea common to selective white rot and showed how fungal strain and vine variety are factors determining the wood degradation. All these observations were supported by the analysis of the laccase and manganese peroxidase enzyme activity, as well as by the expression of the genes coding 6 putative laccase isoforms and 3 manganese peroxidase isoforms, thereby highlighting substantial intraspecific variability.

C-STABILITY an innovative modeling framework to leverage the continuous representation of organic matter.

The understanding of soil organic matter (SOM) dynamics has considerably advanced in recent years. It was previously assumed that most SOM consisted of recalcitrant compounds, whereas the emerging view considers SOM as a range of polymers continuously processed into smaller molecules by decomposer enzymes. Mainstreaming this new paradigm in current models is challenging because of their ill-adapted framework. We propose the C-STABILITY model to resolve this issue. Its innovative framework combines compartmental and continuous modeling approaches to accurately reproduce SOM cycling processes. C-STABILITY emphasizes the influence of substrate accessibility on SOM turnover and makes enzymatic and microbial biotransformations of substrate explicit. Theoretical simulations provide new insights on how depolymerization and decomposers ecology impact organic matter chemistry and amount during decomposition and at steady state. The flexible mathematical structure of C-STABILITY offers a promising foundation for exploring new mechanistic hypotheses and supporting the design of future experiments.

Diversity of Omega Glutathione Transferases in mushroom-forming fungi revealed by phylogenetic, transcriptomic, biochemical and structural approaches.

The Omega class of glutathione transferases (GSTs) forms a distinct class within the cytosolic GST superfamily because most of them possess a catalytic cysteine residue. The human GST Omega 1 isoform was first characterized twenty years ago, but it took years of work to clarify the roles of the human isoforms. Concerning the kingdom of fungi, little is known about the cellular functions of Omega glutathione transferases (GSTOs), although they are widely represented in some of these organisms. In this study, we re-assess the phylogeny and the classification of GSTOs based on 240 genomes of mushroom-forming fungi (Agaricomycetes). We observe that the number of GSTOs is not only extended in the order of Polyporales but also in other orders such as Boletales. Our analysis leads to a new classification in which the fungal GSTOs are divided into two Types A and B. The catalytic residue of Type-A is either cysteine or serine, while that of Type-B is cysteine. The present study focuses on Trametes versicolor GSTO isoforms that possess a catalytic cysteine residue. Transcriptomic data show that Type-A GSTOs are constitutive enzymes while Type-B are inducible ones. The crystallographic analysis reveals substantial structural differences between the two types while they have similar biochemical profiles in the tested conditions. Additionally, these enzymes have the ability to bind antioxidant molecules such as wood polyphenols in two possible binding sites as observed from X-ray structures. The multiplication of GSTOs could allow fungal organisms to adapt more easily to new environments.

OSIP1 is a self-assembling DUF3129 protein required to protect fungal cells from toxins and stressors.

Secreted proteins are key players in fungal physiology and cell protection against external stressing agents and antifungals. Oak stress-induced protein 1 (OSIP1) is a fungal-specific protein with unknown function. By using Podospora anserina and Phanerochaete chrysosporium as models, we combined both in vivo functional approaches and biophysical characterization of OSIP1 recombinant protein. The P. anserina OSIP1Δ mutant showed an increased sensitivity to the antifungal caspofungin compared to the wild type. This correlated with the production of a weakened extracellular exopolysaccharide/protein matrix (ECM). Since the recombinant OSIP1 from P. chrysosporium self-assembled as fibers and was capable of gelation, it is likely that OSIP1 is linked to ECM formation that acts as a physical barrier preventing drug toxicity. Moreover, compared to the wild type, the OSIP1Δ mutant was more sensitive to oak extractives including chaotropic phenols and benzenes. It exhibited a strongly modified secretome pattern and an increased production of proteins associated to the cell-wall integrity signalling pathway, when grown on oak sawdust. This demonstrates that OSIP1 has also an important role in fungal resistance to extractive-induced stress.

Elicitation of Antimicrobial Active Compounds by Streptomyces-Fungus Co-Cultures.

The bacteria of the genus Streptomyces and Basidiomycete fungi harbor many biosynthetic gene clusters (BGCs) that are at the origin of many bioactive molecules with medical or industrial interests. Nevertheless, most BGCs do not express in standard lab growth conditions, preventing the full metabolic potential of these organisms from being exploited. Because it generates biotic cues encountered during natural growth conditions, co-culture is a means to elicit such cryptic compounds. In this study, we explored 72 different Streptomyces-fungus interaction zones (SFIZs) generated during the co-culture of eight Streptomyces and nine fungi. Two SFIZs were selected because they showed an elicitation of anti-bacterial activity compared to mono-cultures. The study of these SFIZs showed that co-culture had a strong impact on the metabolic expression of each partner and enabled the expression of specific compounds. These results show that mimicking the biotic interactions present in this ecological niche is a promising avenue of research to explore the metabolic capacities of Streptomyces and fungi.

Oxidized glutathione promotes association between eukaryotic translation elongation factor 1Bγ and Ure2p glutathione transferase from Phanerochaete chrysosporium.

The eukaryotic translation elongation factor 1Bγ (eEF1Bγ) is an atypical member of the glutathione transferase (GST) superfamily. Contrary to more classical GSTs having a role in toxic compound detoxification, eEF1Bγ is suggested to act as a scaffold protein, anchoring the elongation factor complex EF1B to the endoplasmic reticulum. In this study, we show that eEF1Bγ from the basidiomycete Phanerochaete chrysosporium is fully active as a glutathione transferase in vitro and undergoes conformational changes upon binding of oxidized glutathione. Using real-time analyses of biomolecular interactions, we show that GSSG allows eEF1Bγ to physically interact with other GSTs from the Ure2p class, opening new perspectives for a better understanding of the role of eEF1Bγ in cellular oxidative stress response.

Glutathione Transferases: Surrogate Targets for Discovering Biologically Active Compounds.

Glutathione transferases comprise a large class of multifunctional enzymes, some involved in detoxification pathways. Since these enzymes are able to interact with potentially toxic molecules, they could be used as targets to screen for compounds with biological activity. To test this hypothesis, glutathione transferases (GSTs) from the white-rot fungus Trametes versicolor have been used to screen for antifungal molecules from a library of tropical wood extracts. The interactions between a set of six GSTs from the omega class and 116 extracts from 21 tropical species were quantified using a high-throughput thermal shift assay. A correlation between these interactions and the antifungal properties of the tested extracts was demonstrated. This approach has been extended to the fractionation of an Andira coriacea extract and led to the detection of maackiain and lapachol in this wood. Altogether, the present results supported the hypothesis that such detoxification enzymes could be used to detect biologically active molecules.

Genome Sequences of Five Streptomyces Strains Isolated at Microscale.

The genomes of five Streptomyces strains belonging to the same soil community were sequenced and assembled. The strains, which were isolated at microscale, belonged to different Streptomyces species. This sample provides access to understand the functioning of a Streptomyces community in an ecological context.

Mining the Biosynthetic Potential for Specialized Metabolism of a Streptomyces Soil Community.

The diversity and distribution of specialized metabolite gene clusters within a community of bacteria living in the same soil habitat are poorly documented. Here we analyzed the genomes of 8 Streptomyces isolated at micro-scale from a forest soil that belong to the same species or to different species. The results reveal high levels of diversity, with a total of 261 biosynthesis gene clusters (BGCs) encoding metabolites such as terpenes, polyketides (PKs), non-ribosomal peptides (NRPs) and ribosomally synthesized and post-translationally modified peptides (RiPPs) with potential bioactivities. A significant part of these BGCs (n = 53) were unique to only one strain when only 5 were common to all strains. The metabolites belong to very diverse chemical families and revealed that a large diversity of metabolites can potentially be produced in the community. Although that analysis of the global metabolome using GC-MS revealed that most of the metabolites were shared between the strains, they exhibited a specific metabolic pattern. We also observed that the presence of these accessory pathways might result from frequent loss and gain of genes (horizontal transfer), showing that the potential of metabolite production is a dynamic phenomenon in the community. Sampling Streptomyces at the community level constitutes a good frame to discover new biosynthetic pathways and it appears as a promising reservoir for the discovery of new bioactive compounds.

A reverse chemical ecology approach to explore wood natural durability.

The natural durability of wood species, defined as their inherent resistance to wood-destroying agents, is a complex phenomenon depending on many biotic and abiotic factors. Besides the presence of recalcitrant polymers, the presence of compounds with antimicrobial properties is known to be important to explain wood durability. Based on the advancement in our understanding of fungal detoxification systems, a reverse chemical ecology approach was proposed to explore wood natural durability using fungal glutathione transferases. A set of six glutathione transferases from the white-rot Trametes versicolor were used as targets to test wood extracts from seventeen French Guiana neotropical species. Fluorescent thermal shift assays quantified interactions between fungal glutathione transferases and these extracts. From these data, a model combining this approach and wood density significantly predicts the wood natural durability of the species tested previously using long-term soil bed tests. Overall, our findings confirm that detoxification systems could be used to explore the chemical environment encountered by wood-decaying fungi and also wood natural durability.

Target Of Rapamycin pathway in the white-rot fungus Phanerochaete chrysosporium.

The Target Of Rapamycin (TOR) signaling pathway is known to regulate growth in response to nutrient availability and stress in eukaryotic cells. In the present study, we have investigated the TOR pathway in the white-rot fungus Phanerochaete chrysosporium. Inhibition of TOR activity by rapamycin affects conidia germination and hyphal growth highlighting the conserved mechanism of susceptibility to rapamycin. Interestingly, the secreted protein content is also affected by the rapamycin treatment. Finally, homologs of the components of TOR pathway can be identified in P. chrysosporium. Altogether, those results indicate that the TOR pathway of P. chrysosporium plays a central role in this fungus.

The structure of Trametes versicolor glutathione transferase Omega 3S bound to its conjugation product glutathionyl-phenethylthiocarbamate reveals plasticity of its active site.

Trametes versicolor glutathione transferase Omega 3S (TvGSTO3S) catalyzes the conjugation of isothiocyanates (ITC) with glutathione (GSH). Previously, this isoform was investigated in depth both biochemically and structurally. Structural analysis of complexes revealed the presence of a GSH binding site (G site) and a deep hydrophobic binding site (H site) able to bind plant polyphenols. In the present study, crystals of apo TvGSTO3S were soaked with glutathionyl-phenethylthiocarbamate, the product of the reaction between GSH and phenethyl isothiocyanate (PEITC). On the basis of this crystal structure, we show that the phenethyl moiety binds in a new site at loop ß2 -α2 while the glutathionyl part exhibits a particular conformation that occupies both the G site and the entrance to the H site. This binding mode is allowed by a conformational change of the loop ß2 -α2 at the enzyme active site. It forms a hydrophobic slit that stabilizes the phenethyl group at a distinct site from the previously described H site. Structural comparison of TvGSTO3S with drosophila DmGSTD2 suggests that this flexible loop could be the region that binds PEITC for both isoforms. These structural features are discussed in a catalytic context.

Oak extractive-induced stress reveals the involvement of new enzymes in the early detoxification response of Phanerochaete chrysosporium.

Extensive evidence showed that the efficiency of fungal wood degradation is closely dependent on their ability to cope with the myriad of putative toxic compounds called extractives released during this process. By analysing global gene expression of Phanerochaete chrysosporium after short oak extractive treatment (1, 3 and 6 h), we show that the early molecular response of the fungus concerns first mitochondrial stress rescue followed by the oxidation and finally conjugation of the compounds. During these early responses, the lignolytic degradative system is not induced, rather some small secreted proteins could play an important role in cell protection or signaling. By focusing on the functional characterization of an hitherto uncharacterized glutathione transferase, we show that this enzyme interacts with wood molecules suggesting that it could be involved in the detoxification of some of them, or act as a scavenger to prevent their cytosolic toxicity and favour their transport.

Trametes versicolor glutathione transferase Xi 3, a dual Cys-GST with catalytic specificities of both Xi and Omega classes.

Glutathione transferases (GSTs) from the Xi and Omega classes have a catalytic cysteine residue, which gives them reductase activities. Until now, they have been assigned distinct substrates. While Xi GSTs specifically reduce glutathionyl-(hydro)quinones, Omega GSTs are specialized in the reduction of glutathionyl-acetophenones. Here, we present the biochemical and structural analysis of TvGSTX1 and TvGSTX3 isoforms from the wood-degrading fungus Trametes versicolor. TvGSTX1 reduces GS-menadione as expected, while TvGSTX3 reduces both Xi and Omega substrates. An in-depth structural analysis indicates a broader active site for TvGSTX3 due to specific differences in the nature of the residues situated in the C-terminal helix α9. This feature could explain the catalytic duality of TvGSTX3. Based on phylogenetic analysis, we propose that this duality might exist in saprophytic fungi and ascomycetes.