Matthias Gube

A Consistent Phylogenetic Backbone for the Fungi

The kingdom of fungi provides model organisms for biotechnology, cell biology, genetics, and life sciences in general. Only when their phylogenetic relationships are stably resolved, can individual results from fungal research be integrated into a holistic picture of biology. However, and despite recent progress, many deep relationships within the fungi remain unclear. Here, we present the first phylogenomic study of an entire eukaryotic kingdom that uses a consistency criterion to strengthen phylogenetic conclusions. We reason that branches (splits) recovered with independent data and different tree reconstruction methods are likely to reflect true evolutionary relationships. Two complementary phylogenomic data sets based on 99 fungal genomes and 109 fungal expressed sequence tag (EST) sets analyzed with four different tree reconstruction methods shed light from different angles on the fungal tree of life. Eleven additional data sets address specifically the phylogenetic position of Blastocladiomycota, Ustilaginomycotina, and Dothideomycetes, respectively. The combined evidence from the resulting trees supports the deep-level stability of the fungal groups toward a comprehensive natural system of the fungi. In addition, our analysis reveals methodologically interesting aspects. Enrichment for EST encoded data—a common practice in phylogenomic analyses—introduces a strong bias toward slowly evolving and functionally correlated genes. Consequently, the generalization of phylogenomic data sets as collections of randomly selected genes cannot be taken for granted. A thorough characterization of the data to assess possible influences on the tree reconstruction should therefore become a standard in phylogenomic analyses.

Estimating the Phanerozoic history of the Ascomycota lineages: Combining fossil and molecular data

The phylum Ascomycota is by far the largest group in the fungal kingdom. Ecologically important mutualistic associations such as mycorrhizae and lichens have evolved in this group, which are regarded as key innovations that supported the evolution of land plants. Only a few attempts have been made to date the origin of Ascomycota lineages by using molecular clock methods, which is primarily due to the lack of satisfactory fossil calibration data. For this reason we have evaluated all of the oldest available ascomycete fossils from amber (Albian to Miocene) and chert (Devonian and Maastrichtian). The fossils represent five major ascomycete classes (Coniocybomycetes, Dothideomycetes, Eurotiomycetes, Laboulbeniomycetes, and Lecanoromycetes). We have assembled a multi-gene data set (18SrDNA, 28SrDNA, RPB1 and RPB2) from a total of 145 taxa representing most groups of the Ascomycota and utilized fossil calibration points solely from within the ascomycetes to estimate divergence times of Ascomycota lineages with a Bayesian approach. Our results suggest an initial diversification of the Pezizomycotina in the Ordovician, followed by repeated splits of lineages throughout the Phanerozoic, and indicate that this continuous diversification was unaffected by mass extinctions. We suggest that the ecological diversity within each lineage ensured that at least some taxa of each group were able to survive global crises and rapidly recovered.

In silico analysis of nickel containing superoxide dismutase evolution and regulation

Superoxide dismutases are essential enzymes involved in detoxification of reactive oxygen by dismutation of the superoxide radical anion. A class of nickel containing superoxide dismutases has been described for streptomycetes and cyanobacteria. In silico analysis was used to study the distribution of genes coding for NiSOD in other taxa and to elucidate signals linked to nickel incorporation and maturation of NiSOD. Data mining revealed homologous proteins from actinobacteria, proteobacteria, chlamydiae, and eukarya (green algae) thus allowing a comparison of protein structural elements. Nickel ligands and maturation signals for N‐terminal proteolysis were highly conserved. Genomic sequences surrounding genes encoding NiSOD homologs were compared in order to detect putative accessory enzymes involved in maturation. An endopeptidase gene linked to sodN coding for NiSOD was found in actinobacteria and cyanobacteria, but not in other taxa. The distribution of NiSOD encoding sequences showed four clusters which are not consistent with the phylogeny of the species. In addition, the different genomic context argues for heterologous gene transfer, most likely from actinobacteria to other taxa. In order to address regulation by nickel availability and incorporation into the mature protein, we present first evidence for putative regulatory nucleotide sequences which will be useful in future studies on nickel uptake and incorporation. (© 2009 WILEY‐VCH Verlag GmbH & Co. KGaA, Weinheim)

Polyphasic study of plant- and clinic-associated Pantoea agglomerans strains reveals indistinguishable virulence potential.

Pantoea species are ubiquitous in nature and occasionally associated with infections caused by contaminated clinical material. Hence, Pantoea agglomerans is considered as an opportunistic pathogen of humans. Since species of the genus Pantoea and closely related species of other Enterobacteriaceae genera are phenotypically very similar, many clinical isolates are misassigned into P. agglomerans based on the use of quick commercial-offered biochemical tests. Our objective was to find markers enabling discrimination between clinical and plant isolates and to assess their virulence potential. We characterized 27 Pantoea strains, including 8 P. agglomerans isolates of clinical, and 11 of plant origin by biochemical tests and genotyping, including analysis of 16S rDNA and gapA gene sequences, and pattern polymorphisms of ITS- and ERIC/REP-DNA. All data showed that no discrete evolution occurred between plant-associated and clinical P. agglomerans isolates. Based on the typing results, five clinical- and five plant-associated P. agglomerans strains representing the majority of clades were tested on a model plant and in embryonated eggs. On soybean plants P. agglomerans strains independent of their origin could develop stable epiphytic populations. Surprisingly, in the embryonated egg model there was no difference of virulence between clinical and vegetable P. agglomerans isolates. However, these strains were significantly less virulent than a phytopathogenic P. ananatis isolate. We suggest that, independent of their origin, all P. agglomerans strains might possess indistinguishable virulence potential.

Bioremediation and Heavy Metal Uptake: Microbial Approaches at Field Scale

The remediation of metal-contaminated soil is generally achieved by technical solutions. Bioremediation approaches include phytoremediation with plants taking up metals from the soil with the water phase and enriching these in above-ground biomass, which then can be further processed. On the other hand, phytostabilization is achieved when plants exclude the metals from uptake into above-ground tissue, thus allowing for the use of biomass for downstream applications such as energy production. The performance of plants is dependent on metal mobility in the soil, which is greatly influenced by the soil microbial population. Thus, phytoremediation strategies are evaluated here with regard to microbial impact. Emphasis is laid on field-scale experiments, which are performed to allow for assessing the function of soil bacteria and fungi for enhancement of plant performance in either phytoextraction or phytostabilization. Another bioremediation strategy is the use of fungal fruiting bodies for the accumulation of metals from the soil environment. This mycoremediation depends on the performance of fungi for metal uptake, which generally exceeds plant rates for metal uptake by far. Microbially enhanced phytoremediation is tested for its performance at the former uranium mining site near Ronneburg, Germany, where a test field site has been established. Furthermore, naturally occurring fungal fruiting bodies are collected and analyzed for use in mycoremediation.

The anamorphic genus Monotosporella (Ascomycota) from Eocene amber and from modern Agathis resin.

The anamorphic fungal genus Monotosporella (Ascomycota, Sordariomycetes) has been reco-vered from a piece of Early Eocene Indian amber, as well as from the surface of extant resin flows in New Caledonia. The fossil fungus was obtained from the Tarkeshwar Lignite Mine of Gujarat State, western India, and was part of the biota of an early tropical angiosperm rainforest. The amber inclusion represents the second fossil record of Sordariomycetes, as well as the first fossil of its particular order (either Savoryellales or Chaetosphaeriales). The fossil fungus is distinguished from extant representatives by possessing both short conidiophores and small two-septate pyriform conidia, and is described as Monotosporella doerfeltii sp. nov. Inside the amber, the anamorph is attached to its substrate, which is likely the degraded thallus of a cladoniform lichen. The extant New Caledonian species is assigned to Monotosporella setosa. It was found growing on semi-solidified resin flows of Agathis ovata (Araucariaceae), and is the first record of Monotosporella from modern resin substrates.

Hydrophobins in the Life Cycle of the Ectomycorrhizal Basidiomycete Tricholoma vaccinum.

Hydrophobins—secreted small cysteine-rich, amphipathic proteins—foster interactions of fungal hyphae with hydrophobic surfaces, and are involved in the formation of aerial hyphae. Phylogenetic analyses of Tricholoma vaccinum hydrophobins showed a grouping with hydrophobins of other ectomycorrhizal fungi, which might be a result of co-evolution. Further analyses indicate angiosperms as likely host trees for the last common ancestor of the genus Tricholoma. The nine hydrophobin genes in the T. vaccinum genome were investigated to infer their individual roles in different stages of the life cycle, host interaction, asexual and sexual development, and with respect to different stresses. In aerial mycelium, hyd8 was up-regulated. In silico analysis predicted three packing arrangements, i.e., ring-like, plus-like and sheet-like structure for Hyd8; the first two may assemble to rodlets of hydrophobin covering aerial hyphae, whereas the third is expected to be involved in forming a two-dimensional network of hydrophobins. Metal stress induced hydrophobin gene hyd5. In early steps of mycorrhization, induction of hyd4 and hyd5 by plant root exudates and root volatiles could be shown, followed by hyd5 up-regulation during formation of mantle, Hartig’ net, and rhizomorphs with concomitant repression of hyd8 and hyd9. During fruiting body formation, mainly hyd3, but also hyd8 were induced. Host preference between the compatible host Picea abies and the low compatibility host Pinus sylvestris could be linked to a stronger induction of hyd4 and hyd5 by the preferred host and a stronger repression of hyd8, whereas the repression of hyd9 was comparable between the two hosts.

4 Fungal Molecular Response to Heavy Metal Stress

Heavy metal contamination of soil is a serious threat to public health and the environment and is becoming more and more relevant. Fungi are able to tolerate and even enrich high metal loads, which allows for exploration and colonization of metal contaminated habitats. This is facilitated by the numerous ways of metal resistance which have evolved in both yeasts and filamentous species. These include extracellular chelation to excreted compounds, sorption to cell wall components, regulation of metal ion import and efflux, intracellular chelation and compartmentalization, and alleviation of the toxic effects metal ions may cause. All these have to be coordinated to maintain metal homoeostasis. A deeper knowledge of the molecular mechanisms responsible for fungal heavy metal uptake and resistance is critical to the understanding of the fungal role in the environment.

Tricholoma vaccinum host communication during ectomycorrhiza formation

The genome sequence of Tricholoma vaccinum was obtained to predict its secretome in order to elucidate communication of T. vaccinum with its host tree spruce (Picea abies) in interkingdom signaling. The most prominent protein domains within the 206 predicted secreted proteins belong to energy and nutrition (52%), cell wall degradation (19%) and mycorrhiza establishment (9%). Additionally, we found small secreted proteins that show typical features of effectors potentially involved in host communication. From the secretome, 22 proteins could be identified, two of which showed higher protein abundances after spruce root exudate exposure, while five were downregulated in this treatment. The changes in T. vaccinum protein excretion with first recognition of the partner were used to identify small secreted proteins with the potential to act as effectors in the mutually beneficial symbiosis. Our observations support the hypothesis of a complex communication network including a cocktail of communication molecules induced long before physical contact of the partners.

Dynein Heavy Chain, Encoded by Two Genes in Agaricomycetes, Is Required for Nuclear Migration in Schizophyllum commune.

The white-rot fungus Schizophyllum commune (Agaricomycetes) was used to study the cell biology of microtubular trafficking during mating interactions, when the two partners exchange nuclei, which are transported along microtubule tracks. For this transport activity, the motor protein dynein is required. In S. commune, the dynein heavy chain is encoded in two parts by two separate genes, dhc1 and dhc2. The N-terminal protein Dhc1 supplies the dimerization domain, while Dhc2 encodes the motor machinery and the microtubule binding domain. This split motor protein is unique to Basidiomycota, where three different sequence patterns suggest independent split events during evolution. To investigate the function of the dynein heavy chain, the gene dhc1 and the motor domain in dhc2 were deleted. Both resulting mutants were viable, but revealed phenotypes in hyphal growth morphology and mating behavior as well as in sexual development. Viability of strain Δdhc2 is due to the higher expression of kinesin-2 and kinesin-14, which was proven via RNA sequencing.