Pre-symbiotic response of the compatible host spruce and low-compatibility host pine to the ectomycorrhizal fungus Tricholoma vaccinum.

Mutualistic ectomycorrhizal symbiosis requires the exchange of signals even before direct contact of the partners. Volatiles, and specifically volatile terpenoids, can be detected at a distance and may trigger downstream signaling and reprogramming of metabolic responses. The late-stage ectomycorrhizal fungus Tricholoma vaccinum shows high host specificity with its main host spruce, Picea abies, while rarely associations can be found with pine, Pinus sylvestris. Hence, a comparison of the host and the low-compatibility host's responses can untangle differences in early signaling during mycorrhiza formation. We investigated sesquiterpenes and identified different patterns of phytohormone responses with spruce and pine. To test the specific role of volatiles, trees were exposed to the complete volatilome of the fungus versus volatiles present when terpene synthases were inhibited by rosuvastatin. The pleiotropic response in spruce included three non-identified products, a pyridine derivative as well as two diterpenes. In pine, other terpenoids responded to the fungal signal. Using exposure to the fungal volatilome with or without terpene synthesis inhibited, we could find a molecular explanation for the longer time needed to establish the low-compatibility interaction.

Ectomycorrhizal Community Shifts at a Former Uranium Mining Site.

Ectomycorrhizal communities at young oak, pine, and birch stands in a former uranium mining site showed a low diversity of morphotypes with a preference for contact and short-distance exploration strategies formed by the fungi Russulaceae, Inocybaceae, Cortinariaceae, Thelephoraceae, Rhizopogonaceae, Tricholomataceae, as well as abundant Meliniomyces bicolor. In order to have better control over abiotic conditions, we established pot experiments with re-potted trees taken from the sites of direct investigation. This more standardized cultivation resulted in a lower diversity and decreased prominence of M. bicolor. In addition, the exploration strategies shifted to include long-distance exploration types. To mimic secondary succession with a high prevalence of fungal propagules present in the soil, inoculation of re-potted trees observed under standardized conditions for two years was used. The super-inoculation increased the effect of lower abundance and diversity of morphotypes. The contact morphotypes correlated with high Al, Cu, Fe, Sr, and U soil contents, the dark-colored short-distance exploration type did not show a specific preference for soil characteristics, and the medium fringe type with rhizomorphs on oaks correlated with total nitrogen. Thus, we could demonstrate that field trees, in a species-dependent manner, selected for ectomycorrhizal fungi with exploration types are likely to improve the plant's tolerance to specific abiotic conditions.

Ectomycorrhizal Influence on the Dynamics of Sesquiterpene Release by Tricholoma vaccinum.

Tricholoma vaccinum is an ectomycorrhizal basidiomycete with high host specificity. The slow-growing fungus is able to produce twenty sesquiterpenes, including α-barbatene, sativene, isocaryophyllene, α-cuprenene, ß-cedrene, ß-copaene, 4-epi-α-acoradiene, and chamigrene in axenic culture. For the three major compounds, Δ6-protoilludene, ß-barbatene, and an unidentified oxygenated sesquiterpene (m/z 218.18), changed production during co-cultivation with the ectomycorrhizal partner tree, Picea abies, could be shown with distinct dynamics. During the mycorrhizal growth of T. vaccinum-P. abies, Δ6-protoilludene and the oxygenated sesquiterpene appeared at similar times, which warranted further studies of potential biosynthesis genes. In silico analyses identified a putative protoilludene synthesis gene, pie1, as being up-regulated in the mycorrhizal stage, in addition to the previously identified, co-regulated geosmin synthase, ges1. We therefore hypothesize that the sesquiterpene synthase pie1 has an important role during mycorrhization, through Δ6-protoilludene and/or its accompanied oxygenated sesquiterpene production.

Improving consistency at testing cementitious materials in the Dynamic Surface Leaching Test on the basis of the European technical specification CEN/TS 16637-2 - Results of a round robin test.

The environmental impact assessment of materials is usually based on laboratory tests, mostly in combination with models describing the longterm fate of the substances of interest in the targeted environmental compartment. Thus, laboratory tests are the fundamental link to achieve appropriate assessment conclusions which makes it essential to generate consistent results. This just as applies to the leaching of cementitious materials. In Europe, the leaching behavior of monolithic building materials is tested in the Dynamic Surface Leaching Test following the specification CEN/TS 16637-2. An interlaboratory comparison on European level regarding this technical specification showed relatively high intra- and interlaboratory variations for the tested materials (monolithic copper slag and cement stabilized coal fly ash). Therefore the German Committee for Structural Concrete (DAfStb) framed a guideline to specify additional testing conditions for cementitious materials. To assess the possible improvement by this guidelines measures, a round robin test with 11 participants from Germany and the Netherlands was conducted. This work aims to provide insight into the factors to be considered in the testing of alkaline materials, including sample preparation, and highlights crucial procedures and their manifestation in the results. All evaluated parameters showed improved results compared to the earlier round robin test. The relative standard deviations for repeatability (RSDr) and reproducibility (RSDR) of the elements calcium, barium, antimony, chromium, molybdenum and vanadium, which are the parameters evaluated in both round robin tests, were RSDr = 4%, 4%, 2%, 5%, 5%, and 5% respectively (4% in average) for this work, in comparison to the European round robin test with an average RSDr of 29% (17%, 17%, 20%, 40%, 36%, and 42%). The RSDR improved from 41% (30%, 36%, 29%, 57%, 40%, and 56%) to 14% (12%, 8%, 6%, 28%, 15%, and 12%). CO2 ingress during testing and the inaccuracy of eluate analytics for concentrations close to the determination limits were identified as the main sources of error.

Metal adaptation and transport in hyphae of the wood-rot fungus Schizophyllum commune.

Fungi living in heavy metals and radionuclides contaminated environments, namely the Chernobyl Exclusion Zone need to be able to cope with these pollutants. In this study, the wood-rot fungus Schizophyllum commune was investigated for its metal tolerance mechanisms, and for its ability to transport such metals through its hyphae. Effects of temperature and pH on tolerance of Cs, Sr, Cd, and Zn were tested. At concentrations allowing for half-maximal growth, adapted strains were raised. The strontium-adapted strain, S. commune 12-43 Sr, showed transport of specifically Sr over distances on a cm-scale using split plates. The adaptation did not yield changes in cell or colony morphology. Intracellular metal localization was not changed, and gene expression profiles under metal stress growing on soil versus artificial medium showed a higher impact of a structured surface for growth on soil than with different metal concentrations. In the transcriptome, transporter genes were mostly down-regulated, while up-regulation was seen for genes involved in the secretory pathway under metal stress. A comparison of wildtype and adapted strains could confirm lower cellular stress levels leading to lack of glutathione S-transferase up-regulation in the adapted strain. Thus, we could show metal transport as well as specific mechanisms in metal stress avoidance.

Geosmin synthase ges1 knock-down by siRNA in the dikaryotic fungus Tricholoma vaccinum.

Genetic manipulation for generating knock-out experiments is essential in deciphering the precise function of a gene. However, dikaryotic fungi pose the inherent challenge of having two allelic versions of each gene, one in each nucleus. In addition, they often are slow-growing and do not withstand protoplasting, which is why Agrobacterium tumefaciens-mediated transformation has been adapted. To obtain knock-out strains, however, is not feasible with a mere deletion construct transformation and screening for deletions in both nuclear copies. Hence, a convenient method using chemically synthesized dicer substrate interfering RNA (DsiRNA) for posttranscriptional interference of targeted mRNA was developed, based on the fungal dicer/argonaute system inherent in fungi for sequence recognition and degradation. A proof-of-principle using this newly established method for knock-down of the volatile geosmin is presented in the dikaryotic fungus Tricholoma vaccinum that is forming ectomycorrhizal symbiosis with spruce trees. The gene ges1, a terpene synthase, was transcribed with a 50-fold reduction in transcript levels in the knockdown strain. The volatile geosmin was slightly reduced, but not absent in the fungus carrying the knockdown construct pointing at low specificity in other terpene synthases known for that class of enzymes.

Inositol Signaling in the Basidiomycete Fungus Schizophyllum commune.

Intracellular signaling is conserved in eukaryotes to allow for response to extracellular signals and to regulate development and cellular functions. In fungi, inositol phosphate signaling has been shown to be involved in growth, sexual reproduction, and metabolic adaptation. However, reports on mushroom-forming fungi are lacking so far. In Schizophyllum commune, an inositol monophosphatase has been found up-regulated during sexual development. The enzyme is crucial for inositol cycling, where it catalyzes the last step of inositol phosphate metabolism, restoring the inositol pool from the monophosphorylated inositol monophosphate. We overexpressed the gene in this model basidiomycete and verified its involvement in cell wall integrity and intracellular trafficking. Strong phenotypes in mushroom formation and cell metabolism were evidenced by proteome analyses. In addition, altered inositol signaling was shown to be involved in tolerance towards cesium and zinc, and increased metal tolerance towards cadmium, associated with induced expression of kinases and repression of phosphatases within the inositol cycle. The presence of the heavy metals Sr, Cs, Cd, and Zn lowered intracellular calcium levels. We could develop a model integrating inositol signaling in the known signal transduction pathways governed by Ras, G-protein coupled receptors, and cAMP, and elucidate their different roles in development.

What Role Might Non-Mating Receptors Play in Schizophyllum commune?

The B mating-type locus of the tetrapolar basidiomycete Schizophyllum commune encodes pheromones and pheromone receptors in multiple allelic specificities. This work adds substantial new evidence into the organization of the B mating-type loci of distantly related S. commune strains showing a high level of synteny in gene order and neighboring genes. Four pheromone receptor-like genes were found in the genome of S. commune with brl1, brl2 and brl3 located at the B mating-type locus, whereas brl4 is located separately. Expression analysis of brl genes in different developmental stages indicates a function in filamentous growth and mating. Based on the extensive sequence analysis and functional characterization of brl-overexpression mutants, a function of Brl1 in mating is proposed, while Brl3, Brl4 and Brl2 (to a lower extent) have a role in vegetative growth, possible determination of growth direction. The brl3 and brl4 overexpression mutants had a dikaryon-like, irregular and feathery phenotype, and they avoided the formation of same-clone colonies on solid medium, which points towards enhanced detection of self-signals. These data are supported by localization of Brl fusion proteins in tips, at septa and in not-yet-fused clamps of a dikaryon, confirming their importance for growth and development in S. commune.

Function of sesquiterpenes from Schizophyllum commune in interspecific interactions.

Wood is a habitat for a variety of organisms, including saprophytic fungi and bacteria, playing an important role in wood decomposition. Wood inhabiting fungi release a diversity of volatiles used as signaling compounds to attract or repel other organisms. Here, we show that volatiles of Schizophyllum commune are active against wood-decay fungi and bacteria found in its mycosphere. We identified sesquiterpenes as the biologically active compounds, that inhibit fungal growth and modify bacterial motility. The low number of cultivable wood inhabiting bacteria prompted us to analyze the microbial community in the mycosphere of S. commune using a culture-independent approach. Most bacteria belong to Actinobacteria and Proteobacteria, including Pseudomonadaceae, Sphingomonadaceae, Erwiniaceae, Yersiniaceae and Mariprofundacea as the dominating families. In the fungal community, the phyla of ascomycetes and basidiomycetes were well represented. We propose that fungal volatiles might have an important function in the wood mycosphere and could meditate interactions between microorganisms across domains and within the fungal kingdom.

DutaFabs are engineered therapeutic Fab fragments that can bind two targets simultaneously.

We report the development of a platform of dual targeting Fab (DutaFab) molecules, which comprise two spatially separated and independent binding sites within the human antibody CDR loops: the so-called H-side paratope encompassing HCDR1, HCDR3 and LCDR2, and the L-side paratope encompassing LCDR1, LCDR3 and HCDR2. Both paratopes can be independently selected and combined into the desired bispecific DutaFabs in a modular manner. X-ray crystal structures illustrate that DutaFabs are able to bind two target molecules simultaneously at the same Fv region comprising a VH-VL heterodimer. In the present study, this platform is applied to generate DutaFabs specific for VEGFA and PDGF-BB, which show high affinities, physico-chemical stability and solubility, as well as superior efficacy over anti-VEGF monotherapy in vivo. These molecules exemplify the usefulness of DutaFabs as a distinct class of antibody therapeutics, which is currently being evaluated in patients.

Phytohormones and volatile organic compounds, like geosmin, in the ectomycorrhiza of Tricholoma vaccinum and Norway spruce (Picea abies).

The ectomycorrhizospheric habitat contains a diverse pool of organisms, including the host plant, mycorrhizal fungi, and other rhizospheric microorganisms. Different signaling molecules may influence the ectomycorrhizal symbiosis. Here, we investigated the potential of the basidiomycete Tricholoma vaccinum to produce communication molecules for the interaction with its coniferous host, Norway spruce (Picea abies). We focused on the production of volatile organic compounds and phytohormones in axenic T. vaccinum cultures, identified the potential biosynthesis genes, and investigated their expression by RNA-Seq analyses. T. vaccinum released volatiles not usually associated with fungi, like limonene and ß-barbatene, and geosmin. Using stable isotope labeling, the biosynthesis of geosmin was elucidated. The geosmin biosynthesis gene ges1 of T. vaccinum was identified, and up-regulation was scored during mycorrhiza, while a different regulation was seen with mycorrhizosphere bacteria. The fungus also released the volatile phytohormone ethylene and excreted salicylic and abscisic acid as well as jasmonates into the medium. The tree excreted the auxin, indole-3-acetic acid, and its biosynthesis intermediate, indole-3-acetamide, as well as salicylic acid with its root exudates. These compounds could be shown for the first time in exudates as well as in soil of a natural ectomycorrhizospheric habitat. The effects of phytohormones present in the mycorrhizosphere on hyphal branching of T. vaccinum were assessed. Salicylic and abscisic acid changed hyphal branching in a concentration-dependent manner. Since extensive branching is important for mycorrhiza establishment, a well-balanced level of mycorrhizospheric phytohormones is necessary. The regulation thus can be expected to contribute to an interkingdom language.

Response of the wood-decay fungus Schizophyllum commune to co-occurring microorganisms.

Microorganisms are constantly interacting in a given environment by a constant exchange of signaling molecules. In timber, wood-decay fungi will come into contact with other fungi and bacteria. In naturally bleached wood, dark, pigmented lines arising from confrontation of two fungi often hint at such interactions. The metabolites (and pigment) exchange was investigated using the lignicolous basidiomycete Schizophyllum commune, and co-occurring fungi and bacteria inoculated directly on sterilized wood, or on media. In interactions with competitive wood degrading fungi, yeasts or bacteria, different competition strategies and communication types were observed, and stress reactions, as well as competitor-induced enzymes or pigments were analyzed. Melanin, indole, flavonoids and carotenoids were shown to be induced in S. commune interactions. The induced genes included multi-copper oxidases lcc1, lcc2, mco1, mco2, mco3 and mco4, possibly involved in both pigment production and lignin degradation typical for wood bleaching by wood-decay fungi.

Organic acids, siderophores, enzymes and mechanical pressure for black slate bioweathering with the basidiomycete Schizophyllum commune.

Although many fungi are known to be able to perform bioweathering of rocks and minerals, little information is available concerning the role of basidiomycetes in this process. The wood-rotting basidiomycete Schizophyllum commune was investigated for its ability to degrade black slate, a rock rich in organic carbon. Mechanical pressure of hyphae and extracellular polymeric substances was investigated for biophysical weathering. A mixed ß1-3/ß1-6 glucan, likely schizophyllan that is well known from S. commune, could be identified on black slate surfaces. Secretion of siderophores and organic acids as biochemical weathering agents was shown. Both may contribute to biochemical weathering in addition to enzymatic functions. Previously, the exoenzyme laccase was believed to attack organic the matter within the black slate, thereby releasing metals from the rock. Here, overexpression of laccase showed enhanced dissolution of quartz phases by etching and pitting. At the same time, the formation of a new secondary mineral phase, whewellite, could be demonstrated. Hence, a more comprehensive understanding of biophysical as well as biochemical weathering by S. commune could be reached and unexpected mechanisms like quartz dissolution linked to shale degradation.

The Ectomycorrhizospheric Habitat of Norway Spruce and Tricholoma vaccinum: Promotion of Plant Growth and Fitness by a Rich Microorganismic Community.

The contribution of the mycorrhizospheric microbes in a stand of ectomycorrhizal Norway spruce (Picea abies) featuring mycorrhiza with the basidiomycete Tricholoma vaccinum was addressed by microbiome analysis and in vitro reconstruction of microbial as well as plant-microbe interactions. The protective role of the mycorrhizal fungus with respect to pathogen attack could be validated against Botrytis cinerea and Heterobasidion annosum in co-cultures revealing reduced pathogen growth, higher survival rate of the spruce trees and reduced symptoms on needles upon symbiosis with T. vaccinum. The community structure was shown to yield a high diversity in ECM forming basidiomycetes of Thelephorales and Agaricales associated with a rich bacterial diversity dominated by Rhizobiales with the most abundant Nitrobacter winogradski (3.9%). Isolated bacteria were then used to address plant growth promoting abilities, which included production of the phytohormone indole-3-acetic acid (performed by 74% of the bacterial isolates), siderophores (22%), and phosphate mobilization (23%). Among the isolates, mycorrhiza helper bacteria (MHB) were identified, with Bacillus cereus MRZ-1 inducing hyperbranching in T. vaccinum, supporting tree germination, shoot elongation, and root formation as well as higher mycorrhization rates. Thus, a huge pool of potential MHB and fungal community with widely distributed auxin-production potential extended the ability of T. vaccinum to form ectomycorrhiza. The forest community profited from the mycorrhizal fungus T. vaccinum, with spruce survival enhanced by 33% in microcosms using soil from the native habitat. A higher fungal abundance and diversity in cases where the tree had died during the experiment, showing that decomposition of plant litter from a dead tree supported a different community. T. vaccinum thus actively structured the community of microorganisms in its habitat.

Crosstalk between Ras and inositol phosphate signaling revealed by lithium action on inositol monophosphatase in Schizophyllum commune.

Mushroom forming basidiomycete Schizophyllum commune has been used as a tractable model organism to study fungal sexual development. Ras signaling activation via G-protein-coupled receptors (GPCRs) has been postulated to play a significant role in the mating and development of S. commune. In this study, a crosstalk between Ras signaling and inositol phosphate signaling by inositol monophosphatase (IMPase) is revealed. Constitutively active Ras1 leads to the repression of IMPase transcription and lithium action on IMPase activity is compensated by the induction of IMPase at transcriptome level. Astonishingly, in S. commune lithium induces a considerable shift to inositol phosphate metabolism leading to a massive increase in the level of higher phosphorylated inositol species up to the inositol pyrophosphates. The lithium induced metabolic changes are not observable in a constitutively active Ras1 mutant. In addition to that, proteome profile helps us to elucidate an overview of lithium action to the broad aspect of fungal metabolism and cellular signaling. Taken together, these findings imply a crosstalk between Ras and inositol phosphate signaling.

Metal release and sequestration from black slate mediated by a laccase of Schizophyllum commune.

Schizophyllum commune is a filamentous basidiomycete which can degrade complex organic macromolecules like lignin by the secretion of a large repertoire of enzymes. One of these white rot enzymes, laccase, exhibits a broad substrate specificity and is able to oxidize a variety of substances including carbonaceous rocks. To investigate the role of laccase in bioweathering, laccase gene lcc2 was overexpressed, and the influence on weathering of black slate, originating from a former alum mine in Schmiedefeld, Germany, was examined. The metal release from the rock material was enhanced, associated with a partial metal accumulation into the mycelium. A sequestration of metals could be shown with fluorescent staining methods, and an accumulation of Zn, Cd, and Pb was visualized in different cell organelles. Additionally, we could show an increased metal resistance of the laccase overexpressing strain.

Enzymatic Bioweathering and Metal Mobilization From Black Slate by the Basidiomycete Schizophyllum commune.

Schizophyllum commune is a filamentous basidiomycete causing white-rot in many wood species with the help of a broad range of enzymes including multicopper oxidases such as laccases and laccase-like oxidases. Since these enzymes exhibit a broad substrate range, their ability to oxidatively degrade black slate was investigated. Both haploid monokaryotic, and mated dikaryotic strains were able to grow on black slate rich in organic carbon as sole carbon source. On defined media, only the monokaryon showed growth promotion by addition of slate. At the same time, metals were released from the slate and, after reaching a threshold concentration, inhibited further growth of the fungus. The proteome during decomposition of the black slate showed induction of proteins potentially involved in rock degradation and stress resistance, and the gene for laccase-like oxidase mco2 was up-regulated. Specifically in the dikaryon, the laccase gene lcc1 was induced, while lcc2 as well as mco1, mco3, and mco4 expression levels remained similar. Spectrophotometric analysis revealed that both life forms were able to degrade the rock and produce smaller particles.

The regulator of G-protein signalling Thn1 links pheromone response to volatile production in Schizophyllum commune.

The regulator of G-protein signalling, Thn1, is involved in sexual development through pheromone signalling in the mushroom forming basidiomycete Schizophyllum commune affecting hyphal morphology and mating interactions. Thn1 plays a key role in coordinating sesquiterpene production, pheromone response and sexual development. The gene thn1 is transcriptionally regulated in response to mating with a role in clamp cell development and hydrophobin gene transcription. Further, it negatively regulates cAMP signalling and secondary metabolism. Disruption of thn1 affects dikaryotization by reducing clamp fusion and development with predominant non-fused pseudoclamps. Enhanced protein kinase A (PKA) activities in Δthn1 strains indicate that Thn1 regulates pheromone signalling by de-activating G-protein α subunits, which control cAMP-dependent PKA. The repressed formation of aerial hyphae could be linked to a reduced metabolic activity and to a transcriptional down-regulation of hyd6 and sc3 hydrophobin genes. Thn1 was also shown to be necessary for the biosynthesis of sesquiterpenes and an altered spectrum of sesquiterpenes in Δthn1 is linked to transcriptional up-regulation of biosynthesis genes. Proteome analysis indicated changes in cytoskeletal structure affecting actin localization, linking the major regulator Thn1 to growth and development of S. commune. The results support a role for Thn1 in G-protein signalling connecting development and secondary metabolism.

Smelling the difference: Transcriptome, proteome and volatilome changes after mating.

Mushrooms, such as Schizophyllum commune, have a specific odor. Whether this is linked to mating, prerequisite for mushroom formation, or also found in monokaryotic, unmated strains, was investigated with a comprehensive study on the transcriptome and proteome of this model organism. Mating interactions were investigated using a complete, cytosolic proteome map for unmated, monokaryotic, as well as for mated, dikaryotic mycelia. The regulations of the proteome were compared to transcriptional changes upon mating and to changes in smell by volatilome studies. We could show a good overlap between proteome and transcriptome data, but extensive posttranslational regulation was identified for more than 80% of transcripts. This suggests down-stream regulation upon interaction of mating partners and formation of the dikaryon that is competent to form fruiting bodies. The volatilome was shown to respond to mating by a broader spectrum of volatiles and increased emission of the mushroom smell molecules 3-octanone and 1-octen-3-ol, as well as ethanol and ß-bisabolol in the dikaryon. Putatively involved biosynthetic proteins like alcohol dehydrogenases, Ppo-like oxygenases, or sesquiterpene synthases showed correlating transcriptional regulation depending on either mono- or dikaryotic stages.

Stone-Eating Fungi: Mechanisms in Bioweathering and the Potential Role of Laccases in Black Slate Degradation With the Basidiomycete Schizophyllum commune.

Many enzymes, such as laccases, are involved in the saprotrophic lifestyle of fungi and the effects of those may be linked to enhanced bioweathering on stone surfaces. To test this hypothesis, we studied the decomposition of kerogen-enriched lithologies, especially with black slate containing up to 20% of Corg. Indeed, a formation of ditches with attached hyphal material could be observed. To address enzymes involved, proteomics was performed and one group of enzymes, the multicopper oxidase family members of laccases, was specifically investigated. A role in bioweathering of rocks containing high contents of organic carbon in the form of kerogen could be shown using the basidiomycete Schizophyllum commune, a white rot fungus that has been used as a model organism to study the role of filamentous basidiomycete fungi in bioweathering of black slate.