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 β-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.

Fungi exposed to toxic substances including heavy metals, xenobiotics, or secondary metabolites formed by co-occurring plants or other microorganisms require a detoxification system provided by exporters of several classes of transmembrane proteins. In case of mycorrhiza, plant metabolites need to be exported at the plant interface, while the extraradical hyphae may prevent heavy metal uptake, thus acting as a biofilter to the host plant at high environmental concentrations. One major family of such transporter proteins is the multidrug and toxic compound extrusion (MATE) class, a member of which, Mte1, was studied in the ectomycorrhizal fungus Tricholoma vaccinum. Phylogenetic analyses placed the protein in a subgroup of basidiomycete MATE sequences. The gene mte1 was found to be induced during symbiotic interaction. It mediated detoxification of xenobiotics and metal ions such as Cu, Li, Al, and Ni, as well as secondary plant metabolites if heterologously expressed in Saccharomyces cerevisiae.

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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.

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.