Abstract

Can we live without fungi? Microscopic entities or giants, friends or killers, degraders or producers, they all play crucial roles for life on our planet. Their presence has accompanied the history of humanity, but for a long time, due their largely hidden and unseen actions, their importance was not fully acknowledged and their phylogenetic relationships with animals and plants were erroneously described. Nowadays we are aware that fungi are powerful organisms, which can offer us new pharmaceuticals, help cleaning up waters and soils from contaminants, and provide crucial support to the green inhabitants of the planet. The aim of this presentation is to illustrate different strategies developed by fungi in order to beneficially interact with land plants. Fossil data reveal that fungi resembling modern Glomeromycotina were already associated with first land plants around 450 MYA. However, only today, thanks to the use of -omics technology, we can decipher their enigmatic genomes, reconstruct their metabolic pathways, describe their impact on plants, and identify the molecules involved in the dialogue occurring with their hosts. Thanks to this wealth of data, we can finally make hypotheses on the evolution and molecular mechanisms that make fungi so successful in time and space. Mycorrhizal fungi create networks not only with plants, but also with other soil inhabitants like animals, other fungi and bacteria. The dialogue with bacteria is particularly fascinating. Bacteria can live on the surface of mycelia and spores or, in a more intimate way, as endocellular symbionts inside fungal cells. In the past, the interactions between bacteria and fungi were mostly described as antagonistic in nature, however, most recent data describe cooperative activities between fungi and bacteria. Increasing attention is currently given to the concepts of microbiota and holobiont. In this context, on one hand mycorrhizal fungi are part of the plant microbiota, and represent a key component of the plant holobiont; on the other hand, they also possess their own microbiota. Thus, analogous to animals and plants, a mycorrhizal fungus may be seen at the center of a complex network of inter-kingdom interactions. An example of this tripartite symbiosis is Gigaspora margarita, an arbuscular mycorrhizal fungus that associates both with many plants and diverse endobacterial populations. Deciphering these multiple interactions will be a future goal, which may provide interesting insights into the capacity of mycorrhizal fungi to modulate their responses depending on the organism with whom they interact.

Abstract

Abstract

Water is crucial for life as we know it. High salinity, drought and freezing all lead to decreased water activity and thus disturb the functioning of biological systems. In addition to this, ions of inorganic salts are directly toxic to the cells. Halophilic/halotolerant and psychrophilic/psychrotolerant fungi have evolved specialized molecular mechanisms for avoiding and managing these detrimental effects. Due to their excellent adaptability many of these fungi have great biotechnological potential, due to two reasons in particular: 1) Hypersaline, arid and polar environments are very specific and promote competition for the scarce resources, and are thus promising sources of novel and unique antibacterial, antifungal and/or antialgal compounds. 2) Enzymes from psychrophiles and halophiles are functional at low temperatures and high salinity and therefore interesting for sustainable cleantech biotechnological applications, such as degradation of macro-algae in cold Arctic, marine, hypersaline waters and snow and ice algae covering the surface of glaciers and ice sheets.

Different life strategies of extremophilic fungi will be exemplified by five representative species: Aureobasidium pullulans, Hortaea werneckii, A. subglaciale, Penicillium sp. nov. and Articulospora sp. nov., which inhabit hypersaline waters of salterns around the world, Arctic glaciers and black Greenland ice sheet, respectively. Environmental data and information on their molecular mechanisms of adaptations combined with the knowledge produced by their genome sequencing will be presented for Aureobasidium pullulans, A. subglaciale and Hortaea werneckii, while Penicillium sp. nov. and Articulospora sp. nov. will be presented in the context of environmental data related to their recent discovery in the Greenland ice in association with the non-cultivable black ice algae. In the analysis of the genomes and transcriptomes we focussed on (i) the presence and characteristics of genes involved in stress tolerance, (ii) the presence of biotechnologically important genes, in particular enzymes relevant for decomposition of abundant algal biomass.Introduction

Abstract

Abstract

Wood-degrading white-rot basidiomycetes are exclusively found on wood in nature, where they play a significant role in the degradation of all polymeric components of wood cell walls, including both polysaccharides and the extremely recalcitrant aromatic polymer lignin. The increasing number of fungal genome sequences has revealed that white-rot basidiomycete genomes typically possess a wider repertoire of genes predicted to encode diverse plant cell wall modifying enzymes compared to ascomycete fungi. Therefore, wood-degrading white-rot fungi have a high potential as a source of industrially interesting enzymes or enzyme sets. The white-rot fungus Dichomitus squalens is an efficient wood degrader, which is commonly found in the northern regions of Europe, Asia and North America. When grown on different wood and non-woody plant biomasses, D. squalens upregulates specific sets of genes and secretes the corresponding enzymes matching the composition of the different substrates. The ability of D. squalens to respond to the various plant biomass types, including those that do not exist in its natural habitat, makes it an ideal species to study modification and degradation of plant biomass. Four genome sequenced D. squalens strains providing the best coverage of a filamentous basidiomycete species to date together with recently established genetic transformation system further facilitate the use of this species to understand basidiomycete gene function and development of improved strains for biotechnological applications. We have also detected differences between mono- and dikaryotic strains of D. squalens to grow on and degrade plant biomass. To study this in more detail, we grew four dikaryotic and three monokaryotic strains of D. squalens on spruce wood sticks and analysed the cultures after two and four weeks for their transcriptome, proteome and metabolome. Highlights from this study will also be presented.

Abstract

For many fungi, plant biomass is the predominant carbon source, but also a highly challenging substrate due to its complex and variable composition. It consists mainly of polymers, of which the polysaccharides are the major carbon sources used by fungi. Secreted enzymes degrade these polymeric compounds to mono- and small oligomers that are taken up by the fungal cell.

Filamentous fungi typically contain between 120 and 350 genes in their genome that encode plant biomass degrading enzymes. Therefore it is important that the genes expressed by a fungus encode those enzymes that match the composition of the prevailing substrate. For this fungi have evolved an intricate regulatory system that responds to the various mono- and disaccharides that are released from plant biomass. This system does not consist of a set of independent regulators, but rather of a network in which links between the individual regulators exist not only with respect to their target genes, but also by influencing each other’s expression level.

In this presentation the current knowledge on regulation of plant biomass degradation from several well-studied ascomycete fungi will be compared and linked, to provide an overall view of this highly complex process. The recent identification of the L-arabinose responsive regulatory systems in eurotiomycetes and sordariomycetes, which are a clear example of parallel evolution, will also be discussed, as well as the regulatory differences between species.

Abstract

The reduction of unwanted endogenous proteases has already been an important target for strain improvement of fungal host strains used in protein production for many years. Targeted deletion of specific proteases has been used extensively for this purpose. Surprisingly, only very little is known about regulatory circuits that specifically control fungal protease production. The only protease-specific regulator gene discovered to date is the prtT gene from Aspergillus niger, which encodes a canonical Zn2-Cys6 activator protein (Punt et al., 2008) involved in the expression of a wide range of protease genes. Interestingly, homologues of prtT are only found in Aspergillus species, whereas no prtT homologue is present in Trichoderma reesei. We aimed to discover a similar protease master switch in our research. T. reesei mutants with strongly reduced overall protease levels and strongly reduced expression of a number of protease genes were obtained using a biological screen for the selection of protease-deficient mutants (Braaksma et al., 2008). Genome sequencing of a number of these strains followed by SNP analysis revealed that several of these mutants carried mutant alleles from a single gene, which we termed pea1 for protease-expression-affected. Disruption of this gene in both T. reesei and Fusarium sp. confirmed the role of pea1 in protease gene expression. Intriguingly, the encoded protein does not show any similarity to known regulatory proteins, indicating that a completely new regulatory circuit may be governing protease gene expression in T. reesei, which opens the way to further research in this area.

Abstract

Active transport and local translation of mRNAs ensure the appropriate spatial organization of proteins within cells. Recent work has shown that this process is intricately connected to membrane trafficking. We study the model organism Ustilago maydis. In highly polarized cells of this fungus microtubule-dependent co-transport of mRNAs and endosomes is essential for efficient polar growth. We discuss a novel concept of endosome-coupled translation that loads shuttling endosomes with septin cargo, a process important for correct septin filamentation. Key players are RNA-binding proteins containing RNA recognition motifs for mRNA binding as well as Mademoiselle domains for protein/protein interaction. Here, new insights on protein RNA as well as protein-protein interactions will be presented. Interestingly, evidence is accumulating that RNA and membrane trafficking are also tightly interwoven in higher eukaryotes suggesting that this phenomenon is a common theme and not an exception restricted to fungi.

Abstract

In fungal cells, specialized proteins gather in specific places to break cell symmetry and produce hyphae. This organization includes the orchestration of two distinct vesicle processes, endocytosis, and exocytosis that take place in tandem in different areas of the apical compartment in growing hyphae. Part of the signals for endocytosis and endocytosis include the asymmetry of the plasma membrane phospholipid bilayer. We studied the flippases, DNF-1, DRS-2 and DNF-4 that seems to be responsible for this membrane asymmetry in Golgi, vesicles and the plasma membrane. The mutation of dnf-1 and drs-2 genes produced alterations in the maintenance and stability of the Spitzenkörper and affected the actin cytoskeleton organization in the apical compartment. Surprisingly, neither of the flippases DNF-1 and DRS-2 was present in the plasma membrane; both were localized in different layers of the Spitzenkörper, associated to different secretory vesicles. DRS-2 was associated with vesicles transporting chitin synthases. DNF-4 seemed to be present in the Golgi equivalent. Each flippase is in charge of the localization of different phospholipids, their presence in different compartments can predict which phospholipid is more abundant. These results indicate that phospholipid flippases (P4 ATPases) may be important for the polarization of secretory vesicles, Spitzenkörper integrity and thus for the localization of many tip growing proteins.

Abstract

The endomembrane network is a system of cytoplasmic membranes that partition the cell into functional and structural organelles and compartments, which together are involved in biomolecular synthesis, break down, and transport. The endomembrane network of Neurospora crassa hyphae was examined using transmission electron microscopy. All hyphae were prepared by cryofixation and freeze substitution protocols. Neurospora crassa hyphal tip cells contained three cytoplasmic regions based on content and organization. Region I corresponded to the hyphal apex and contained a well-defined Spitzenkörper composed of macro and microvesicles, plus cytoskeletal elements. Golgi equivalents (GE) and aggregations of cisternae with electron-dense lumen were present in this region. Region II extended behind region I approximately 10 to 20 µm and contained abundant vesicles, GE, and rough endoplasmic reticulum (rER). Smooth, flattened cisternae with electron-transparent contents were also present in region II. Endocytotic profiles along the plasma membrane were not common in regions I and II. The transition into region III was marked by abundant nuclei, multivesicular bodies (MVBs), vacuoles containing granular-like material, rER, GE, and flattened cisternae. Cytosolic surfaces of flattened cisternae were coated with a fibrous, electron-dense material. These coated surfaces were restricted to the edges of the flattened cisternae. Microtubules were in close proximity to GE, MVBs, and flattened cisternae. MVBs were diverse in size and shape. Observations of serial sections revealed that vacuoles and flattened cisternae were continuous

Abstract

In Neurospora crassa hyphae the localization of all seven chitin synthases (CHSs) at the Spitzenkörper (Spk) and at developing septa has been well analyzed. Hitherto, the mechanisms of CHSs traffic and sorting from synthesis to delivery sites remain largely unexplored. In Saccharomyces cerevisiae exit of Chs3p from the endoplasmic reticulum (ER) requires chaperone Chs7p. Here, we analyzed the role of CSE-7, N. crassa Chs7p orthologue in the biogenesis of CHS-4 (orthologue of Chs3p). In a N. crassa Δcse-7 mutant, CHS-4-GFP no longer accumulated at the Spk and septa. Instead, fluorescence was retained in hyphal subapical regions in an extensive network of elongated cisternae (NEC) referred to previously as tubular vacuoles. In a complemented strain expressing a copy of cse-7 the localization of CHS-4-GFP at the Spk and septa was restored, providing evidence that CSE-7 is necessary for CHS-4 to exit the NEC and for its localization at hyphal tips and septa. CSE-7 was revealed at delimited regions of the ER at the immediacies of nuclei, at the NEC, and remarkably also at septa and the Spk. The organization of the NEC was dependent on the microtubule cytoskeleton. SEC-63, an extensively used ER marker, and NCA-1, a SERCA-type ATPase previously localized at the nuclear envelope, were used as markers to discern the nature of the membranes containing CSE-7. Both SEC-63 and NCA-1 were found at the nuclear envelope, but also at regions of the NEC. However, at the NEC only NCA-1 co-localized extensively with CSE-7. Observations by transmission electron microscopy revealed abundant rough ER sheets and distinct electron translucent smooth flattened cisternae, which could correspond collectively to the NEC, thorough the subapical cytoplasm. This study identifies CSE-7 as the putative ER receptor for its cognate cargo, the polytopic membrane protein CHS-4, and elucidates the complexity of the ER system in filamentous fungi.

Abstract

Schizophyllum commune belongs to the white rot basidiomycetes and is relevant for wood degradation worldwide. As early colonizer after forest fire and of tree wounds, the fungus has also phytopathogenic importance. Its high competitive ability is based on the recognition of other bacteria and fungi, the production of specific extracellular metabolites and a strategy of fast growth.The fungal cytoskeleton, composed of a complex network of microtubules and actin structures, has a major impact on transport of vesicles as well as endo- and exocytosis processes. Visualization of the actin cytoskeleton in actively growing hyphae was performed with Lifeact-GFP. Thereby cortical actin patches were visualized at cell tips and clamps and as well as in subapical cells, preceded septation. The actin cytoskeleton in living hyphae during septum development shows close association with nuclear division. Clamp cell formation, typical of many model basidiomycetes including S. commune, indicated an aggregation of actin filaments to ring structures at the future site of nuclear division. Additionally, GFP-labeling of histone H2B enables visualization of nuclear movement and mitosis events in monokaryotic and dikaryotic cells. After mating events, fast nuclear exchange in anastomoses and hyphal cells were observed.

Abstract

During growth, filamentous fungi uniquely produce polarized cells called hyphae. It is generally presumed that polarization of hyphae is dependent upon a mechanism called apical recycling, which maintains a balance between the tightly coupled processes of endocytosis and exocytosis. Endocytosis predominates in an annular domain deemed the sub-apical endocytic collar, which is located in the region of plasma membrane 1-5μm distal to the Spitzenkörper (SPK). Here, a bioinformatics approach was utilized to methodically identify 42 Aspergillus nidulans proteins that are predicted to be cargo of endocytosis based on the presence of an NPFxD (or similar DPFxD) peptide motif. This motif is a necessary endocytic signal sequence first established in the model yeast Saccharomyces cerevisiae, where it marks proteins for endocytosis. The focus of this project is to examine the predicted endocytic association and function of these motif-containing proteins during hyphal growth using fluorescent markers and live-cell imaging. Many of these proteins have orthologs in budding and fission yeasts that have previously been shown to play a role in cell development and regulation of polar cellular morphology. Based on this data, we hypothesize that NPFxD or DPFxD motif-containing proteins in A. nidulans that are cargo for endocytosis will localize to at least one of three regions where cargo are characteristically observed. These predicted regions include the sub-apical collar, where cargo is actively endocytosed and internalized into the hypha, as well as the apical crescent, which lines the membrane at the apex of the hypha and terminates roughly where the sub-apical collar is predicted to begin. The third and final anticipated area of localization is the SPK, which contains two subsets of differently sized vesicles, and is considered to be the key determinant of hyphal growth and directionality. Proteins that are cargo for endocytosis can be observed in each of these areas based on the step(s) of the recycling process they are involved with during membrane turnover. At this time, we have observed localization to the predicted regions associated with hyphal growth for 9 of the 42 motif-containing proteins in A. nidulans. Mutants in these genes have varied in their ability to establish or maintain polarity and also display atypical development in various cell types, which suggests that the genes in question are involved with membrane turnover.