Establishment of in vivo proximity labeling with biotin using TurboID in the filamentous fungus Sordaria macrospora.

Proximity-dependent biotin identification (BioID) has emerged as a powerful methodology to identify proteins co-localizing with a given bait protein in vivo. The approach has been established in animal cells, plants and yeast but not yet in filamentous fungi. BioID relies on promiscuous biotin ligases fused to bait proteins to covalently label neighboring proteins with biotin. Biotinylated proteins are specifically enriched through biotin affinity capture from denatured cell lysates and subsequently identified and quantified with liquid chromatography-mass spectrometry (LC-MS). In contrast to many other affinity capture approaches for studying protein-protein interactions, BioID does not rely on physical protein-protein binding within native cell lysates. This feature allows the identification of protein proximities of weak or transient and dynamic nature. Here, we demonstrate the application of BioID for the fungal model organism Sordaria macrospora (Sm) using the example of the STRIPAK complex interactor 1 (SCI1) of the well-characterized striatin-interacting phosphatase and kinase (SmSTRIPAK) complex as proof of concept. For the establishment of BioID in S. macrospora, a codon-optimized TurboID biotin ligase was fused to SCI1. Biotin capture of the known SmSTRIPAK components PRO11, SmMOB3, PRO22 and SmPP2Ac1 demonstrates the successful BioID application in S. macrospora. BioID proximity labeling approaches will provide a powerful proteomics tool for fungal biologists.

The vacuolar morphology protein VAC14 plays an important role in sexual development in the filamentous ascomycete Sordaria macrospora.

The multiprotein Fab1p/PIKfyve-complex regulating the abundance of the phospholipid phosphatidylinositol 3,5-bisphosphate (PtdIns(3,5)P2) is highly conserved among eukaryotes. In yeast/mammals, it is composed of the phosphatidylinositol 3-phosphate 5-kinase Fab1p/PIKfyve, the PtdIns(3,5)P2 phosphatase Fig4p/Sac3 and the scaffolding subunit Vac14p/ArPIKfyve. The complex is located to vacuolar membranes in yeast and to endosomal membranes in mammals, where it controls the synthesis and turnover of PtdIns(3,5)P2. In this study, we analyzed the role and function of the Fab1p/PIKfyve-complex scaffold protein SmVAC14 in the filamentous ascomycete Sordaria macrospora (Sm). We generated the Smvac14 deletion strain ∆vac14 and performed phenotypic analysis of the mutant. Furthermore, we conducted fluorescence microscopic localization studies of fluorescently labeled SmVAC14 with vacuolar and late endosomal marker proteins. Our results revealed that SmVAC14 is important for maintaining vacuolar size and appearance as well as proper sexual development in S. macrospora. In addition, SmVAC14 plays an important role in starvation stress response. Accordingly, our results propose that the turnover of PtdIns(3,5)P2 is of great significance for developmental processes in filamentous fungi.

Secondary metabolites of Hülle cells mediate protection of fungal reproductive and overwintering structures against fungivorous animals.

Fungal Hülle cells with nuclear storage and developmental backup functions are reminiscent of multipotent stem cells. In the soil, Hülle cells nurse the overwintering fruiting bodies of Aspergillus nidulans. The genome of A. nidulans harbors genes for the biosynthesis of xanthones. We show that enzymes and metabolites of this biosynthetic pathway accumulate in Hülle cells under the control of the regulatory velvet complex, which coordinates development and secondary metabolism. Deletion strains blocked in the conversion of anthraquinones to xanthones accumulate emodins and are delayed in maturation and growth of fruiting bodies. Emodin represses fruiting body and resting structure formation in other fungi. Xanthones are not required for sexual development but exert antifeedant effects on fungivorous animals such as springtails and woodlice. Our findings reveal a novel role of Hülle cells in establishing secure niches for A. nidulans by accumulating metabolites with antifeedant activity that protect reproductive structures from animal predators.

Analysis of the Putative Nucleoporin POM33 in the Filamentous Fungus Sordaria macrospora.

In the filamentous fungus Sordaria macrospora (Sm), the STRIPAK complex is required for vegetative growth, fruiting-body development and hyphal fusion. The SmSTRIPAK core consists of the striatin homolog PRO11, the scaffolding subunit of phosphatase PP2A, SmPP2AA, and its catalytic subunit SmPP2Ac1. Among other STRIPAK proteins, the recently identified coiled-coil protein SCI1 was demonstrated to co-localize around the nucleus. Pulldown experiments with SCI identified the transmembrane nucleoporin (TM Nup) SmPOM33 as a potential nuclear-anchor of SmSTRIPAK. Localization studies revealed that SmPOM33 partially localizes to the nuclear envelope (NE), but mainly to the endoplasmic reticulum (ER). We succeeded to generate a Δpom33 deletion mutant by homologous recombination in a new S. macrospora Δku80 recipient strain, which is defective in non-homologous end joining. Deletion of Smpom33 did neither impair vegetative growth nor sexual development. In pulldown experiments of SmPOM33 followed by LC/MS analysis, ER-membrane proteins involved in ER morphology, protein translocation, glycosylation, sterol biosynthesis and Ca2+-transport were significantly enriched. Data are available via ProteomeXchange with identifier PXD026253. Although no SmSTRIPAK components were identified as putative interaction partners, it cannot be excluded that SmPOM33 is involved in temporarily anchoring the SmSTRIPAK to the NE or other sites in the cell.

Tracking Fungal Growth: Establishment of Arp1 as a Marker for Polarity Establishment and Active Hyphal Growth in Filamentous Ascomycetes.

Polar growth is a key characteristic of all filamentous fungi. It allows these eukaryotes to not only effectively explore organic matter but also interact within its own colony, mating partners, and hosts. Therefore, a detailed understanding of the dynamics in polar growth establishment and maintenance is crucial for several fields of fungal research. We developed a new marker protein, the actin-related protein 1 (Arp1) fused to red and green fluorescent proteins, which allows for the tracking of polar axis establishment and active hyphal growth in microscopy approaches. To exclude a probable redundancy with known polarity markers, we compared the localizations of the Spitzenkörper (SPK) and Arp1 using an FM4-64 staining approach. As we show in applications with the coprophilous fungus Sordaria macrospora and the hemibiotrophic plant pathogen Colletotrichum graminicola, the monitoring of Arp1 can be used for detailed studies of hyphal growth dynamics and ascospore germination, the interpretation of chemotropic growth processes, and the tracking of elongating penetration pegs into plant material. Since the Arp1 marker showed the same dynamics in both fungi tested, we believe this marker can be broadly applied in fungal research to study the manifold polar growth processes determining fungal life.

A 20-kb lineage-specific genomic region tames virulence in pathogenic amphidiploid Verticillium longisporum.

Amphidiploid fungal Verticillium longisporum strains Vl43 and Vl32 colonize the plant host Brassica napus but differ in their ability to cause disease symptoms. These strains represent two V. longisporum lineages derived from different hybridization events of haploid parental Verticillium strains. Vl32 and Vl43 carry same-sex mating-type genes derived from both parental lineages. Vl32 and Vl43 similarly colonize and penetrate plant roots, but asymptomatic Vl32 proliferation in planta is lower than virulent Vl43. The highly conserved Vl43 and Vl32 genomes include less than 1% unique genes, and the karyotypes of 15 or 16 chromosomes display changed genetic synteny due to substantial genomic reshuffling. A 20 kb Vl43 lineage-specific (LS) region apparently originating from the Verticillium dahliae-related ancestor is specific for symptomatic Vl43 and encodes seven genes, including two putative transcription factors. Either partial or complete deletion of this LS region in Vl43 did not reduce virulence but led to induction of even more severe disease symptoms in rapeseed. This suggests that the LS insertion in the genome of symptomatic V. longisporum Vl43 mediates virulence-reducing functions, limits damage on the host plant, and therefore tames Vl43 from being even more virulent.

The Glyoxysomal Protease LON2 Is Involved in Fruiting-Body Development, Ascosporogenesis and Stress Resistance in Sordaria macrospora.

Microbodies, including peroxisomes, glyoxysomes and Woronin bodies, are ubiquitous dynamic organelles that play important roles in fungal development. The ATP-dependent chaperone and protease family Lon that maintain protein quality control within the organelle significantly regulate the functionality of microbodies. The filamentous ascomycete Sordaria macrospora is a model organism for studying fruiting-body development. The genome of S. macrospora encodes one Lon protease with the C-terminal peroxisomal targeting signal (PTS1) serine-arginine-leucine (SRL) for import into microbodies. Here, we investigated the function of the protease SmLON2 in sexual development and during growth under stress conditions. Localization studies revealed a predominant localization of SmLON2 in glyoxysomes. This localization depends on PTS1, since a variant without the C-terminal SRL motif was localized in the cytoplasm. A ΔSmlon2 mutant displayed a massive production of aerial hyphae, and produced a reduced number of fruiting bodies and ascospores. In addition, the growth of the ΔSmlon2 mutant was completely blocked under mild oxidative stress conditions. Most of the defects could be complemented with both variants of SmLON2, with and without PTS1, suggesting a dual function of SmLON2, not only in microbody, but also in cytosolic protein quality control.

Establishment of the monomeric yellow-green fluorescent protein mNeonGreen for life cell imaging in mycelial fungi.

The engineered monomeric version of the lancelet Branchiostoma lanceolatum fluorescent protein, mNeonGreen (mNG), has several positive characteristics, such as a very bright fluorescence, high photostability and fast maturation. These features make it a good candidate for the utilization as fluorescent tool for cell biology and biochemical applications in filamentous fungi. We report the generation of plasmids for the expression of the heterologous mNG gene under the control of an inducible and a constitutive promoter in the filamentous ascomycete Sordaria macrospora and display a stable expression of mNG in the cytoplasm. To demonstrate its usefulness for labeling of organelles, the peroxisomal targeting sequence serine-lysine-leucine (SKL) was fused to mNG. Expression of this tagged version led to protein import of mNG into peroxisomes and their bright fluorescence in life cell imaging.

A 3D Printed Device for Easy and Reliable Quantification of Fungal Chemotropic Growth.

Chemical gradients are surrounding living organisms in all habitats of life. Microorganisms, plants and animals have developed specific mechanisms to sense such gradients. Upon perception, chemical gradients can be categorized either as favorable, like nutrients or hormones, or as disadvantageous, resulting in a clear orientation toward the gradient and avoiding strategies, respectively. Being sessile organisms, fungi use chemical gradients for their orientation in the environment. Integration of this data enables them to successfully explore nutrient sources, identify probable plant or animal hosts, and to communicate during sexual reproduction or early colony development. We have developed a 3D printed device allowing a highly standardized, rapid and low-cost investigation of chemotropic growth processes in fungi. Since the 3D printed device is placed on a microscope slide, detailed microscopic investigations and documentation of the chemotropic process is possible. Using this device, we provide evidence that germlings derived from oval conidia of the hemibiotrophic plant pathogen Colletotrichum graminicola can sense gradients of glucose and reorient their growth toward the nutrient source. We describe in detail the method establishment, probable pitfalls, and provide the original program files for 3D printing to enable broad application of the 3D device in basic, agricultural, medical, and applied fungal science.

Anion Inhibition Studies of the ß-Class Carbonic Anhydrase CAS3 from the Filamentous Ascomycete Sordaria macrospora.

CAS3 is a newly cloned cytosolic ß-class carbonic anhydrase (CA, EC 4.2.1.1) from the filamentous ascomycete Sordaria macrospora. This enzyme has a high catalytic activity for the physiological CO2 hydration reaction and herein, we report the inhibition profile of CAS3 with anions and small molecules. The most effective CAS3 anions/small molecule inhibitors were diethyl-dithiocarbamate, sulfamide, sulfamate, phenyl boronic and phenyl arsonic acids, with KIs in the range of 0.89 mM-97 µM. Anions such as iodide, the pseudohalides, bicarbonate, carbonate, nitrate, nitrite, hydrogensulfide, stannate, selenate, tellurate, tetraborate, perrhenate, perruthenate, selenocyanide and trithiocarbonate were low millimolar CAS3 inhibitors. The light halides, sulfate, hydrogensulfite, peroxydisulfate, diphosphate, divanadate, perchlorate, tetrafluoroborate, fluorosulfonate and iminodisulfonate did not significantly inhibit this enzyme. These data may be useful for developing antifungals based on CA inhibition, considering the fact that many of the inhibitors reported here may be used as lead molecules and, by incorporating the appropriate organic scaffolds, potent nanomolar inhibitors could be developed.

Sordaria macrospora: 25 years as a model organism for studying the molecular mechanisms of fruiting body development.

Fruiting bodies are among the most complex multicellular structures formed by fungi, and the molecular mechanisms that regulate their development are far from understood. However, studies with a number of fungal model organisms have started to shed light on this developmental process. One of these model organisms is Sordaria macrospora, a filamentous ascomycete from the order Sordariales. This fungus has been a genetic model organism since the 1950s, but its career as a model organism for molecular genetics really took off in the 1990s, when the establishment of a transformation protocol, a mutant collection, and an indexed cosmid library provided the methods and resources to start revealing the molecular mechanisms of fruiting body development. In the 2000s, "omics" methods were added to the S. macrospora tool box, and by 2020, 58 developmental genes have been identified in this fungus. This review gives a brief overview of major method developments for S. macrospora, and then focuses on recent results characterizing different processes involved in regulating development including several regulatory protein complexes, autophagy, transcriptional and chromatin regulation, and RNA editing. KEY POINTS: •Sordaria macrospora is a model system for analyzing fungal fruiting body development. •More than 100 developmental mutants are available for S. macrospora. •More than 50 developmental genes have been characterized in S. macrospora.

Sulfonamide Inhibition Studies of the ß-Class Carbonic Anhydrase CAS3 from the Filamentous Ascomycete Sordaria macrospora.

A new ß-class carbonic anhydrase was cloned and purified from the filamentous ascomycete Sordaria macrospora, CAS3. This enzyme has a higher catalytic activity compared to the other two such enzymes from this fungus, CAS1 and CAS2, which were reported earlier, with the following kinetic parameters: kcat of (7.9 ± 0.2) × 105 s-1, and kcat/Km of (9.5 ± 0.12) × 107 M-1∙s-1. An inhibition study with a panel of sulfonamides and one sulfamate was also performed. The most effective CAS3 inhibitors were benzolamide, brinzolamide, dichlorophnamide, methazolamide, acetazolamide, ethoxzolamide, sulfanilamide, methanilamide, and benzene-1,3-disulfonamide, with KIs in the range of 54-95 nM. CAS3 generally shows a higher affinity for this class of inhibitors compared to CAS1 and CAS2. As S. macrospora is a model organism for the study of fruiting body development in fungi, these data may be useful for developing antifungal compounds based on CA inhibition.

Comparative Genomics and Transcriptomics To Analyze Fruiting Body Development in Filamentous Ascomycetes.

Many filamentous ascomycetes develop three-dimensional fruiting bodies for production and dispersal of sexual spores. Fruiting bodies are among the most complex structures differentiated by ascomycetes; however, the molecular mechanisms underlying this process are insufficiently understood. Previous comparative transcriptomics analyses of fruiting body development in different ascomycetes suggested that there might be a core set of genes that are transcriptionally regulated in a similar manner across species. Conserved patterns of gene expression can be indicative of functional relevance, and therefore such a set of genes might constitute promising candidates for functional analyses. In this study, we have sequenced the genome of the Pezizomycete Ascodesmis nigricans, and performed comparative transcriptomics of developing fruiting bodies of this fungus, the Pezizomycete Pyronema confluens, and the Sordariomycete Sordaria macrospora With only 27 Mb, the A. nigricans genome is the smallest Pezizomycete genome sequenced to date. Comparative transcriptomics indicated that gene expression patterns in developing fruiting bodies of the three species are more similar to each other than to nonsexual hyphae of the same species. An analysis of 83 genes that are upregulated only during fruiting body development in all three species revealed 23 genes encoding proteins with predicted roles in vesicle transport, the endomembrane system, or transport across membranes, and 13 genes encoding proteins with predicted roles in chromatin organization or the regulation of gene expression. Among four genes chosen for functional analysis by deletion in S. macrospora, three were shown to be involved in fruiting body formation, including two predicted chromatin modifier genes.

Specialized infection strategies of falcate and oval conidia of Colletotrichum graminicola.

For many filamentous fungi with pathogenic lifestyles, the presence of distinct asexual conidia has been described. However, the role of these spore types remains mostly obscure. Colletotrichum graminicola is a hemibiotrophic filamentous fungus, causing anthracnose on maize plants with a high potential of epidemic disease spreading. C. graminicola generates two types of conidia. Falcate shaped conidia formed in necrotic lesions on maize tissues are able to generate appressoria with high efficiency and are considered key disease spreading propagules. The second conidia type, the smaller oval conidia, is formed in the vascular system of the infected plant, probably causing the distribution of the disease in planta. Barely any knowledge exists about how these conidia are able to exhibit their specific functions in the life cycle and pathogenicity of C. graminicola. Here, we show that germlings derived from both falcate and oval conidia differ in the secretion of a germination inhibitor and signals for germling fusion. Germination experiments combined with HPLC and mass spectrometry analyses revealed that germination of falcate conidia is regulated by the self-inhibitor mycosporine-glutamine, whereas this compound is absent from oval conidia cultures. Additionally, germlings derived from oval conidia undergo germling fusions at high frequencies and are able to induce such a fusion when co-incubated with falcate conidia. Falcate conidia germlings alone, however, were never observed to fuse. Plant infection experiments showed a positive correlation between germling fusions and efficient leaf infection by oval conidia. However, this correlation was not observed for infection by falcate conidia. Together, our findings reveal significant differences of two types of conidia derived from the same pathogenic fungus with distinct roles in pathogenesis.

NBR1 is involved in selective pexophagy in filamentous ascomycetes and can be functionally replaced by a tagged version of its human homolog.

Macroautophagy/autophagy is a conserved degradation process in eukaryotic cells involving the sequestration of proteins and organelles within double-membrane vesicles termed autophagosomes. In filamentous fungi, its main purposes are the regulation of starvation adaptation and developmental processes. In contrast to nonselective bulk autophagy, selective autophagy is characterized by cargo receptors, which bind specific cargos such as superfluous organelles, damaged or harmful proteins, or microbes, and target them for autophagic degradation. Herein, using the core autophagy protein ATG8 as bait, GFP-Trap analysis followed by liquid chromatography mass spectrometry (LC/MS) identified a putative homolog of the human autophagy cargo receptor NBR1 (NBR1, autophagy cargo receptor) in the filamentous ascomycete Sordaria macrospora (Sm). Fluorescence microscopy revealed that SmNBR1 colocalizes with SmATG8 at autophagosome-like structures and in the lumen of vacuoles. Delivery of SmNBR1 to the vacuoles requires SmATG8. Both proteins interact in an LC3 interacting region (LIR)-dependent manner. Deletion of Smnbr1 leads to impaired vegetative growth under starvation conditions and reduced sexual spore production under non-starvation conditions. The human NBR1 homolog partially rescues the phenotypic defects of the fungal Smnbr1 deletion mutant. The Smnbr1 mutant can neither use fatty acids as a sole carbon source nor form fruiting bodies under oxidative stress conditions. Fluorescence microscopy revealed that degradation of a peroxisomal reporter protein is impaired in the Smnbr1 deletion mutant. Thus, SmNBR1 is a cargo receptor for pexophagy in filamentous ascomycetes.

A novel STRIPAK complex component mediates hyphal fusion and fruiting-body development in filamentous fungi.

The STRIPAK complex is involved in growth, cell fusion, development and signaling pathways, and thus malfunctions in the human STRIPAK complex often result in severe neuronal diseases and cancer. Despite the high degree of general conservation throughout the complex, several STRIPAK complex-associated small coiled-coil proteins of animals and yeasts are not conserved across species. As there are no data for filamentous ascomycetes, we addressed this through affinity purification with HA-tagged striatin ortholog PRO11 in Sordaria macrospora. Combining the method with liquid chromatography-mass spectrometry, we were able to co-purify STRIPAK complex interactor 1 (SCI1), the first STRIPAK-associated small coiled-coil protein in filamentous ascomycetes. Using yeast two-hybrid experiments, we identified SCI1 protein regions required for SCI1-PRO11 interaction, dimerization of SCI1 and interaction with other STRIPAK components. Further, both proteins PRO11 and SCI1 co-localize with the nuclear basket protein SmPOM152 at the nuclear envelope. Expression of the gene sci1 occurs during early developmental stages of S. macrospora, and the protein SCI1 in combination with PRO11 is required for cell fusion, vegetative growth and sexual development. The results of the present study will help to understand the underlying molecular mechanisms of STRIPAK signaling and function in cellular development and diseases in higher eukaryotes.

Genome Sequencing and analyses of Two Marine Fungi from the North Sea Unraveled a Plethora of Novel Biosynthetic Gene Clusters.

Marine Fungi are potent secondary metabolite producers. However, limited genetic information are available their biosynthetic gene clusters (BGCs) and their biotechnological applications. To overcome this lack of information, herein, we used next-generation sequencing methods for genome sequencing of two marine fungi, isolated from the German Wadden Sea, namely Calcarisporium sp. KF525 and Pestalotiopsis sp. KF079. The assembled genome size of the marine isolate Calcarisporium sp. KF525 is about 36.8 Mb with 60 BGCs, while Pestalotiopsis sp. KF079 has a genome size of 47.5 Mb harboring 67 BGCs. Of all BGCs, 98% and 97% are novel clusters of Calcarisporium sp. and Pestalotiopsis sp., respectively. Only few of the BGCs were found to be expressed under laboratory conditions by RNA-seq analysis. The vast majority of all BGCs were found to be novel and unique for these two marine fungi. Along with a description of the identified gene clusters, we furthermore present important genomic features and life-style properties of these two fungi. The two novel fungal genomes provide a plethora of new BGCs, which may have biotechnological applications in the future, for example as novel drugs. The genomic characterizations will provide assistance in future genetics and genomic analyses of marine fungi.

A Hippo Pathway-Related GCK Controls Both Sexual and Vegetative Developmental Processes in the Fungus Sordaria macrospora.

The supramolecular striatin-interacting phosphatases and kinases (STRIPAK) complex is conserved from yeast to human, and regulates a variety of key biological processes. In animals, this complex consists of the scaffold protein striatin, the protein phosphatase 2A, and kinases, such as germinal center kinase (GCK) III and GCKIV family members, as well as other associated proteins. The STRIPAK complex was identified as a negative regulator of the Hippo pathway, a large eukaryotic signaling network with a core composed of a GCK and a nuclear Dbf2-related kinase. The signaling architecture of the Hippo core resembles the fungal septation initiation network (SIN) that regulates cytokinesis in fission yeast as well as septation in filamentous fungi. In the filamentous model fungus Sordaria macrospora, core components of the STRIPAK complex have been functionally described and the striatin homolog PRO11 has been shown to interact with the GCK SmKIN3. However, the exact role of SmKIN3 in fungal development has not yet been fully elucidated. Here, we provide comprehensive genetic and functional analysis of SmKIN3 from S. macrospora Using deletion mutants and site-directed mutagenesis, along with phenotypic and phylogenetic analysis, we provide compelling evidence that SmKIN3 is involved in fruiting body formation, hyphal fusion, and septation. Strains carrying the ATP-binding mutant SmKIN3K39R, as well as a double-deletion strain lacking SmKIN3 and the core STRIPAK subunit PRO11, also revealed severe developmental defects. Collectively, this study suggests that SmKIN3 links both the SIN and STRIPAK complex, thereby regulating multiple key cellular processes.

Sulfonamide inhibition studies of two ß-carbonic anhydrases from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2.

The two ß-carbonic anhydrases (CAs, EC 4.2.1.1) recently cloned and purified from the ascomycete fungus Sordaria macrospora, CAS1 and CAS2, were investigated for their inhibition with a panel of 39 aromatic, heterocyclic, and aliphatic sulfonamides and one sulfamate, many of which are clinically used agents. CAS1 was efficiently inhibited by tosylamide, 3-fluorosulfanilamide, and 3-chlorosulfanilamide (KIs in the range of 43.2-79.6 nM), whereas acetazolamide, methazolamide, topiramate, ethoxzolamide, dorzolamide, and brinzolamide were medium potency inhibitors (KIs in the range of 360-445 nM). CAS2 was less sensitive to sulfonamide inhibitors. The best CAS2 inhibitors were 5-amino-1,3,4-thiadiazole-2-sulfonamide (the deacetylated acetazolamide precursor) and 4-hydroxymethyl-benzenesulfonamide, with KIs in the range of 48.1-92.5 nM. Acetazolamide, dorzolamide, ethoxzolamide, topiramate, sulpiride, indisulam, celecoxib, and sulthiame were medium potency CAS2 inhibitors (KIs of 143-857 nM). Many other sulfonamides showed affinities in the high micromolar range or were ineffective as CAS1/2 inhibitors. Small changes in the structure of the inhibitor led to important differences of the activity. As these enzymes may show applications for the removal of anthropically generated polluting gases, finding modulators of their activity may be crucial for designing environmental-friendly CO2 capture processes.

Ectomycorrhizal ecology is imprinted in the genome of the dominant symbiotic fungus Cenococcum geophilum.

The most frequently encountered symbiont on tree roots is the ascomycete Cenococcum geophilum, the only mycorrhizal species within the largest fungal class Dothideomycetes, a class known for devastating plant pathogens. Here we show that the symbiotic genomic idiosyncrasies of ectomycorrhizal basidiomycetes are also present in C. geophilum with symbiosis-induced, taxon-specific genes of unknown function and reduced numbers of plant cell wall-degrading enzymes. C. geophilum still holds a significant set of genes in categories known to be involved in pathogenesis and shows an increased genome size due to transposable elements proliferation. Transcript profiling revealed a striking upregulation of membrane transporters, including aquaporin water channels and sugar transporters, and mycorrhiza-induced small secreted proteins (MiSSPs) in ectomycorrhiza compared with free-living mycelium. The frequency with which this symbiont is found on tree roots and its possible role in water and nutrient transport in symbiosis calls for further studies on mechanisms of host and environmental adaptation.