TY - CONF A1 - Nitsche, Sarah A1 - Gerrits, Ruben A1 - Gorbushina, Anna A1 - Schumacher, Julia T1 - The role of the stress-activated MAP kinase SAK1 in Knufia petricola N2 - Mitogen-activated protein (MAP) kinases are highly conserved in animals, plants, and fungi and represent fundamental parts of signaling networks in eukaryotic cells. Black DHN (1,8-dihydroxynaphthalene) melanin and orange carotenoids are produced by many fungi in specific cell types/under certain conditions for protecting cells from abiotic and/or biotic stresses. Microcolonial black fungi constitutively produce DHN melanin, contain the conserved carotenogenic gene cluster, exhibit slow yeast-like growth and survive in extreme environments. Thus, the question arises to which extent pigment formation and responses mediated by the stress-activated MAP kinase module contribute to the observed extremotolerance. We address this question in the rock inhabitant Knufia petricola, the only representative of the extremotolerant black fungi that is genetically amendable. Here, the mutations of pks1, phs1 and both genes result in melanin-free (pink), carotenoid-free (black) and pigment-free (white) strains, respectively (Voigt Knabe et al. 2020, Sci Rep). The gene encoding the stress-activated MAP kinase was deleted in the wild-type and different pigment-deficient backgrounds. In addition, strains were generated that express a GFP-SAK1 fusion protein from the sak1 locus to follow the cytosolic/nuclear shuttling of SAK1 upon stress. Growth of the obtained single, double and triple deletion mutants was tested by dropping cell suspensions on solid media supplemented with different stress-inducing agents. The Δsak1 mutants show slightly reduced growth rates even under non-stress conditions and are hypersensitive to different stress conditions: reduced growth is observed on media inducing, for instance, osmotic, oxidative, membrane, and pH stress, and upon incubation at 30 °C (heat stress). Melanin-free Δsak1 mutants are more sensitive than black Δsak1 mutants to some but not all stress conditions, suggesting that melanin and the SAK1 pathway have complementary roles in protecting K. petricola from stress. T2 - VAAM Fachgruppentragung "Molecular Biology of Fungi" CY - Kaiserslautern, Germany DA - 07.09.2022 KW - Fungus KW - Extremotolerance KW - Signal transduction PY - 2022 AN - OPUS4-55677 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Erdmann, Eileen A. A1 - Nitsche, Sarah A1 - Gerrits, Ruben A1 - Heeger, Felix A1 - Gorbushina, Anna T1 - Genetic engineering of black fungi: lessons learned from Knufia petricola N2 - The exponential rise in the number of fungal genomes sequenced by next-generation sequencing techniques makes it necessary to increase efforts to correctly annotate and assign gene functions. There are two possibilities to explore a genome and its gene functions. The hypothesis-based method proves the function of already existing gene/allele candidates by targeted mutagenesis - so called reverse genetics. The basis of forward genetics approaches is the random mutagenesis of the genome, followed by screening of obtained mutants for the phenotype of interest, and identification of the mutated genes in the respective mutants. This strategy is hypothesis-generating, means it is necessary to verify the relationship between the detected mutations and the observed phenotype by targeted mutagenesis of the identified gene. We developed a toolbox for editing the genome of the rock inhabitant Knufia petricola [Eurotiomycetes, Chaetothyriales] that allows the study of the phenotypic characteristics of black fungi such as the regulation of pigment synthesis, general stress responses, oligotrophy, and the unusual modes of cell division by advanced reverse and forward genetics approaches. The toolbox includes the annotated genome sequence of strain A95, efficient strategies for CRISPR/Cas9-based genome editing and live-cell imaging using genetically encoded fluorescent proteins, as well as protocols for -omics approaches and for simulation of mineral weathering. A forward genetics approach using transposon mutagenesis is currently developed for identifying essential genes. The established protocols and knowledge gained from K. petricola form a starting point for making other fungi from extreme environments accessible to genetic manipulation. T2 - IUBMB Focused Meeting on Extremophilic Fungi (FUN-EX) CY - Ljubljana, Slovenia DA - 19.09.2023 KW - Fungus KW - Extremotolerance KW - Genetics KW - Model organism PY - 2023 AN - OPUS4-58424 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Brandhorst, Antonia A1 - Nitsche, Sarah A1 - Erdmann, Eileen A1 - Gerrits, Ruben A1 - Gorbushina, Anna T1 - The roles of DHN melanin and the stress-activated MAP kinase in the rock inhabitant Knufia petricola N2 - Black fungi/yeasts exhibit high stress tolerance, yeast‑like or meristematic growth, and constitutive 1,8-dihydroxynaphthalene (DHN) melanin formation. Due to their slow growth, robust cell walls and the lack of sexual cycles and genetic tools, the underlying mechanisms of their phenotypic traits have remained largely unexplored. Using recently developed genetic tools, it is now possible to manipulate the genome of the rock-inhabiting model fungus Knufia petricola. Thus, gene functions and the cell biology of black fungi can be studied using CRISPR/Cas9-based genome editing and live-cell imaging with genetically encoded fluorescent proteins. Here, we are addressing the question to which extent constitutive pigment formation (melanin and carotenoids) and responses mediated by the stress-activated mitogen-activated protein (MAP) kinase contribute to the observed extremotolerance of K. petricola. The mutations of pks1, phs1 and both genes result in melanin-free (pink), carotenoid-free (black) and pigment-free (white) strains, respectively. The other putative melanogenic genes were identified in the genome, deleted to confirm their involvement in DHN melanogenesis and co-expressed in Saccharomyces cerevisiae for reconstruction of the synthesis pathway. Sak1 encoding the stress-activated MAP kinase was deleted in the wild-type and different pigment-deficient backgrounds. Growth of the obtained single, double and triple deletion mutants was tested by droplet tests on media supplemented with different stress-inducing agents. The Δsak1 mutants show slightly reduced growth rates even without environmental pressure and are hypersensitive to different stresses: e.g. osmotic, oxidative, membrane, pH and heat stress. Melanin-free Δsak1 mutants are more sensitive than black Δsak1 mutants to some but not all stress conditions, suggesting that melanin and the SAK1 pathway have complementary roles in protecting K. petricola from stress. T2 - 16th European Conference on Fungal Genetics CY - Innsbruck, Austria DA - 05.03.2023 KW - Black fungus KW - Extremotolerance KW - Pigments PY - 2023 AN - OPUS4-57147 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -