Annual Conference 2025 of the Association for General and Applied Microbiology - Jahrestagung 2025 der Vereinigung für Allgemeine und Angewandte Mikrobiologie

Prof. Dr.

The tRNA splicing endonuclease (SEN) complex localization in the model fungus Ustilago maydis

Biology of yeast and fungi

Transfer RNAs (tRNAs) are synthesized by RNA-polymerase III in the nucleus and modified by multiple enzymes before entering the protein translation process. In recent years intron-containing tRNAs have been detected in all three kingdoms of life. Splicing of intron-containing tRNAs is accomplished by the well-conserved splicing endonuclease (SEN) complex. Although splicing of intron-containing tRNAs is an essential process, it has been discovered that the localization of the SEN complex differs significantly between species.In eukaryotes, the SEN complex comprises four essential subunits, with Sen2 and Sen34 forming the catalytically active site and, Sen15 and Sen54 forming structural components of the complex. Surprisingly, in humans and vertebrates, it was discovered that the SEN complex is located in the nucleus whereas tRNA splicing in the budding and fission yeast ascomycetes takes place on the cytosolic surface of mitochondria. Despite its high grade of conservation, this finding indicates that species-specific mechanisms evolved to ensure subcellular trafficking of intron-containing tRNAs and the SEN complex.The basidiomycete fungus Ustilago maydis has emerged as a new model system for cell biology. The genome-wide analysis demonstrated that U. maydis is more closely related to humans than to budding yeast and therefore offers a unique system to study mammalian processes. Interestingly, almost all tRNAs in U. maydis contain introns and sequence identities of SEN complex subunits with a human range between 28% to 50%, indicating a very high degree of conservation between the two organisms. Using state-of-the-art life-cell imaging coupled with genetic engineering we unravel the subcellular localization of the SEN complex in U. maydis, which might help to understand tRNA biology and its associated human diseases in the future.

Poster

Unraveling the complete toolbox U. maydis utilizes for sucrose metabolization

Sucrose is a common carbon source in nature, which is utilized in plants as a transport sugar and metabolized by microbes. One microbe which thrives on sucrose during infection is the corn smut fungus Ustilago maydis. U. maydis has a dimorphic lifestyle. It grows saprotrophically in a haploid yeast form, while together with a compatible mate it forms a dikaryotic hyphae, which infects the plant. It infects Zea mays in a biotrophic manner, resulting in the formation of tumors that spread spores. Till now, the sucrose metabolism of U. maydis during the different stages of the life cycle is not described in detail. So far it was assumed that U. maydis uses a high-affinity sucrose transporter (Srt1) for import, a dual localized invertase (Suc2), which can act both intra- and extracellularly and an additional intracellular invertase that we termed Suc1. The overall aim of this study was to understand the sucrose metabolism in the biotrophic plant pathogen Ustilago maydis.We identified relevant enzymes bioinformatically and confirmed their activities by yeast complementation assays. Deletion mutants were generated to verify the observations. Axenic growth experiments on media supplemented with sucrose were conducted with U. maydis deletion mutants growing in the yeast form to determine the need of transporters and invertases. Maize infections with deletion mutants were conducted to study their relevance during infection.A combination of bioinformatics and yeast complementation assays revealed that U. maydis employs additional two intracellular invertases. This results in a total of four invertases of which three are only intracellular and one can be secreted but also has an intracellular form. Additionally, to the known transporter Srt1, U. maydis utilizes Agt1 for sucrose import, which was described in S. cerevisiae as a maltose transporter. Growth experiments with deletion mutants uncovered, that Ima1 and Agt1 are the main responsible invertase and transporter, respectively, during axenic growth on sucrose.We display that U. maydis employs a complex toolbox of invertases and transporters, which may result from its different life stages and reflect the competition between the plant immune system and the infection strategy of the fungus.

Morphological traits of ectomycorrhizal fungi in symbiosis

Mycorrhiza are a mutualistic interaction between numerous fungi and approximately 90 % of land plants, making them important for diverse ecosystems. Boreal and temperate forest trees interact with ectomycorrhizal fungi (EMF) that form three characteristic morphological traits: a hyphal mantle around the fine root tips, a network of hyphae between the root cortex cells (the Hartig net) and hyphae reaching into the soil for certain distances (extraradical mycelium). In ectomycorrhizal interactions, fungi gain carbohydrates from the trees, while the trees gain access to mineral nutrients and water. Additionally, current studies underline the role of EMF in protection of trees from biotic and abiotic stressors, such as drought and herbivore attack. Still, the role of mantle, Hartig net and extraradical mycelium in these different aspects of EMF function has not been elucidated.Here, we study the symbiotic interaction between the basidiomycete EMF Paxillus involutus and the poplar tree Paxillus x canescens in a laboratory system. We employ different P. involutus strains that differ in their ability to form a Hartig net in poplar roots. Using Petri dish mycorrhization experiments, followed by microscopic analysis of root cross sections, we examine the extent to which different strains penetrate the root cortex. These experiments are carried out in different physiological conditions regarding the nutrition status of the fungus and / or the plant partner. We aim to test the impact of Hartig net formation on nutrient exchange and protection from drought stress in potted poplar plants. Further, we perform phosphorus uptake experiments with P. involutus to understand the dependency of nutrient transfer from the substrate to the EMF and further into the plant on Hartig net formation and on environmental conditions. Our results will further the understanding of EMF functions and point out means to conquer the effects of climate change on forest ecosystems.

Establishing transformation systems for ectomycorrhizal fungi

Mycorrhizal symbiosis between fungal microorganism and plants is an important feature in many ecosystems. Ectomycorrhizal fungi that remain outside of plants cells are important as partners for trees of temperate and boreal forests. The fungal partners are mostly ascomycetes or basidiomycetes that can be generalists or specialists. During symbiosis, the fungus transfers water and minerals to the plant, while the plant transfers carbohydrates to the fungus. Although many aspects of ectomycorrhizal fungi have been studied in detail, molecular genetic work and functional characterization of genes remains scarce.Here, we aim to establish transformation methods for two ectomycorrhizal fungi, the ascomycete Cenococcum geophilum and the basidiomycete Paxillus involutus, which both undergo symbiosis with different tree species, including the genetically tractable poplar. We test selection markers for transformation, including different antibiotics and carboxin, and show strategies for cloning of the corresponding genes into suitable vectors. An important aspect in genetic transformation of basidiomycetes is the promoter; thus, we further show data on Golden Gate vectors for easy promoter exchange. Concerning transformation methods, we work on both, protoplast-mediated and Agrobacterium-mediated transformation. Since P. involutus is dikaryotic, we aim to generate monokaryons, which are easier to transform in some basidiomycete species due to their single nucleus and the formation of vegetative spores. With a robust transformation system for ectomycorrhizal fungi, we will be able to study gene functions and to learn more about the molecular mechanisms underlying ectomycorrhizal symbiosis, which is of major importance for temperate and boreal forests.

Biology of yeast and fungi 1

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