TY - JOUR A1 - Voigt, Oliver A1 - Knabe, Nicole A1 - Nitsche, Sarah A1 - Erdmann, Eileen A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - An advanced genetic toolkit for exploring the biology of the rock‑inhabiting black fungus Knufia petricola N2 - Microcolonial black fungi are a group of ascomycetes that exhibit high stress tolerance, yeast-like growth and constitutive melanin formation. They dominate a range of hostile natural and man-made environments, from desert rocks and salterns to dishwashers, roofs and solar panels. Due to their slow growth and a lack of genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We chose to address this gap by genetically engineering the rock-inhabiting fungus Knufia petricola (Eurotiomycetes, Chaetothyriales), a species that exhibits all characteristics of black fungi. A cell biological approach was taken by generating K. petricola strains expressing green or red fluorescent protein variants. By applying: (1) traditional gene replacement; (2) gene editing and replacement via plasmid-based or ribonucleoprotein (RNP)-based CRISPR/Cas9, and (3) silencing by RNA interference (RNAi), we constructed mutants in the pathways leading to melanin, carotenoids, uracil and adenine. Stable single and double mutants were generated with homologous recombination (HR) rates up to 100%. Efficient, partially cloning-free strategies to mutate multiple genes with or without resistance cassettes were developed. This state-of-the-art genetic toolkit, together with the annotated genome sequence of strain A95, firmly established K. petricola as a model for exploring microcolonial black fungi. KW - Subaerial biofilms KW - Biodeterioration KW - Fluorescent microscopy KW - CRISPR/Cas9 KW - RNA interference PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-518601 DO - https://doi.org/10.1038/s41598-020-79120-5 VL - 10 IS - 1 SP - 22021 PB - Springer Nature AN - OPUS4-51860 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Selbmann, L. A1 - Benkő, Z. A1 - Coleine, C. A1 - de Hoog, S. A1 - Donati, C. A1 - Druzhinina, I. A1 - Emri, T. A1 - Ettinger, C. L. A1 - Gladfelter, A. S. A1 - Gorbushina, Anna A1 - Grigoriev, I. V. A1 - Grube, M. A1 - Gunde-Cimerman, N. A1 - Karányi, Z. A. A1 - Kocsis, B. A1 - Kubressoian, T. A1 - Miklós, I. A1 - Miskei, M. A1 - Muggia, L. A1 - Northen, T. A1 - Novak-Babič, M. A1 - Pennacchio, C. A1 - Pfliegler, W. P. A1 - Pòcsi, I. A1 - Prigione, V. A1 - Riquelme, M. A1 - Segata, N. A1 - Schumacher, Julia A1 - Shelest, E. A1 - Sterflinger, K. A1 - Tesei, D. A1 - U’Ren, J. M. A1 - Varese, G. C. A1 - Vázquez-Campos, X. A1 - Vicente, V. A. A1 - Souza, E. M. A1 - Zalar, P. A1 - Walker, A. K. A1 - Stajich, J. E. T1 - Shed Light in the DaRk LineagES of the Fungal Tree of Life—STRES N2 - The polyphyletic group of black fungi within the Ascomycota (Arthoniomycetes, Dothideomycetes, and Eurotiomycetes) is ubiquitous in natural and anthropogenic habitats. Partly because of their dark, melanin-based pigmentation, black fungi are resistant to stresses including UV- and ionizing-radiation, heat and desiccation, toxic metals, and organic pollutants. Consequently, they are amongst the most stunning extremophiles and poly-extreme-tolerant organisms on Earth. Even though ca. 60 black fungal genomes have been sequenced to date, [mostly in the family Herpotrichiellaceae (Eurotiomycetes)], the class Dothideomycetes that hosts the largest majority of extremophiles has only been sparsely sampled. By sequencing up to 92 species that will become reference genomes, the “Shed light in The daRk lineagES of the fungal tree of life” (STRES) project will cover a broad collection of black fungal diversity spread throughout the Fungal Tree of Life. Interestingly, the STRES project will focus on mostly unsampled genera that display different ecologies and life-styles (e.g., ant- and lichen-associated fungi, rock-inhabiting fungi, etc.). With a resequencing strategy of 10- to 15-fold depth coverage of up to ~550 strains, numerous new reference genomes will be established. To identify metabolites and functional processes, these new genomic resources will be enriched with metabolomics analyses coupled with transcriptomics experiments on selected species under various stress conditions (salinity, dryness, UV radiation, oligotrophy). The data acquired will serve as a reference and foundation for establishing an encyclopedic database for fungal metagenomics as well as the biology, evolution, and ecology of the fungi in extreme environments. KW - Adaptation KW - Black fungi KW - Dothideomycetes KW - Eurotiomycetes KW - Extremophiles KW - Genomics KW - Metabolomics KW - Secondary metabolites KW - Stress conditions KW - Transcriptomics PY - 2020 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-519328 DO - https://doi.org/10.3390/life10120362 VL - 10 IS - 12 SP - 362 PB - MDPI CY - Basel AN - OPUS4-51932 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - Schwarze Pilze – Wüstenbesiedler finden neue Lebensräume N2 - Schwarze mikrokoloniale Pilze besiedeln zunehmend von Menschen geschaffene Habitate, wie schadstoffbelastete Böden, Statuen, Gebäudefassaden und Dächer. Sie verfärben und zersetzen die Oberflächen von anfälligen Materialien oder reduzieren die Lichtausbeute von Solaranlagen. Die Biologie dieser Pilze und ihre Relevanz für die Materialforschung stehen im Fokus unserer Studien an der Bundesanstalt für Materialforschung und -prüfung (BAM). Mit einer Kollektion schwarzer Pilze, die von Solaranlagen isoliert wurden, bringen wir klima- und materialrelevante Biodiversität in den Stammbaum des Lebens. Die Überlebensstrategien dieser Organismen versuchen wir mit molekularbiologischen und genetischen Untersuchungsansätzen zu entschlüsseln. KW - Pilze KW - Genetik KW - Diversität PY - 2021 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-541037 DO - https://doi.org/10.1007/s12268-021-1646-9 VL - 27 IS - 6 SP - 665 EP - 666 PB - Springer AN - OPUS4-54103 LA - deu AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - Light sensing in plant- and rock-associated black fungi N2 - Fungi that share light-flooded habitats with phototrophs may profit from their excess photosynthetic products. But to cope with sunlight-associated stresses [e.g. high temperatures, UV radiation with associated DNA damage, accumulation of reactive oxygen species (ROS), desiccation and osmotic stresses] it is important for fungi to accurately sense and respond to changes in light. To test the hypothesis that light is an environmental cue that Ascomycota use to coordinate growth, stress responses as well as to establish pathogenic or symbiotic relationships, the photoreceptor (PR) distribution in species from different ecological niches was analysed. The genomes of black [dihydroxynaphthalene (DHN) melanin-containing] fungi from phyllosphere and exposed solid surfaces contain multiple photoreceptors (PRs). The plant pathogen Botrytis cinerea (Leotiomycetes) has a highly sophisticated photosensory and signalling system that helps to avoid light and to locate susceptible hosts. Rock-inhabiting Dothideomycetes and Eurotiomycetes including Knufia petricola possess equal numbers ofPRs along with the same set of protective pigments. This similarity between black fungi from plant and rock surfaces suggests that photoperception and -regulation are important for fungi that receive nutrients through cooperation with phototrophs. Genetic tools for manipulating K. petricola exist and will be used to test this idea. KW - Botrytis cinerea KW - DHN melanin KW - Knufia petricola KW - Phyllosphere KW - Rock biofilm PY - 2020 DO - https://doi.org/10.1016/j.funbio.2020.01.004 VL - 124 IS - 5 SP - 407 EP - 417 AN - OPUS4-50786 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schumacher, Julia T1 - CRISPR-Cas9 in der Materialforschung - Den gesteinsbesiedelnden Pilzen auf der Spur T1 - CRISPR-Cas9 in materials research - On the trail of rock-inhabiting fungi N2 - Das mikroskopische Leben auf exponierten Oberflächen ist genügsam und kooperativ. Gesteinsbesiedelnde schwarze Pilze, Grünalgen und Cyanobakterien unterstützen einander in der Eroberung von Felsen, Mauern, Denkmälern, Dächern, Fassaden und Sonnenkollektoren. Bedeutend sind die schwarzen Pilze als Gesteinszerstörer und Biofilmbildner. Ihre massiven Zellwände und ihr langsames Wachstum machen sie stresstolerant und fordern zugleich die experimentelle Forschung heraus. In der Materialforschung können Biofilme erwünscht oder unerwünscht sein. Biofilme auf Fassaden können das Innenstadtklima positiv beeinflussen, während sie auf einem Marmordenkmal unwillkommen sind. Ohne tieferes Verständnis der angepassten Mikroben ist weder ihre Bekämpfung noch ihre gezielte Förderung auf Materialien möglich. Hier treffen sich Genetik und Materialforschung: Die CRISPR-Cas9-Technologie ermöglicht es, die Genome der Pilze für funktionale Analysen zu editieren, um die Mechanismen der Materialbesiedlung und Materialschädigung zu entschlüsseln. N2 - Rock-inhabiting black fungi are adapted to the harsh life on rocks in deserts and release minerals from the rocks. The same adaptations enable these fungi to colonize man-made surfaces such as monuments, building facades and solar systems. Black fungi are often associated with phototrophic microorganisms. The slow growth and the melanized cell walls, which protect the fungi from extreme environmental infuences, render molecular biological and genetic engineering methods diffcult, which is why little is known about the biology of these fungi. Knufa petricola was selected to understand the processes of material colonization and damage with the help of adapted methods such as CRISPR-Cas9-mediated genome editing. KW - Knufia petricola KW - Schwarze Pilze KW - Bioflme KW - Genomeditierung KW - Pigmente KW - Multiplexing KW - Resistenzkassette KW - Transformanten KW - Knufia petricola KW - Black fungi KW - Biofilms KW - Pigments KW - Genome editing KW - Multiplexing KW - Resistance cassette KW - Transformants PY - 2024 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-620500 DO - https://doi.org/10.11576/biuz-7595 SN - 0045-205X SN - 1521-415X N1 - Volltext (PDF) in deutsch und englisch - Full text (PDF) in German and English VL - 54 SP - 41 EP - 50 PB - Verband Biologie, Biowissenschaften und Biomedizin in Deutschland (VBiO) CY - München AN - OPUS4-62050 LA - mul AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - JOUR A1 - Schumacher, Julia T1 - Schwarze Pilze: Robuste Multitalente für die Biotechnologie T1 - Black fungi: Robust all-rounders for biotechnology N2 - Knufia petricola ist ein gesteinsbesiedelnder Pilz aus der polyphyletischen Gruppe der schwarzen Pilze. Diese Pilze entwickelten außergewöhnliche – und bisher kaum erforschte – Fähigkeiten, in verschiedenen extremen Umgebungen zu gedeihen. Die Nutzung der genetischen Vielfalt des Genoms von schwarzen Pilzen und die Nutzung von K. petricola als alternativer Wirt für die Produktion von Enzymen und Sekundärmetaboliten eröffnen Perspektiven für die Nutzung dieser bislang wenig bekannten Pilze für biotechnologische Anwendungen. N2 - Knufia petricola is a rock-inhabiting fungus belonging to the polyphyletic group of black fungi. These fungi developed extraordinary – and so far hardly studied – capabilities to thrive in different extreme environments. Accessing the genetic diversity of black fungal genomes and using K. petricola as an alternative host for producing enzymes and secondary metabolites opens perspectives for utilizing these so far little recognized fungi for biotechnological applications. KW - Knufia petricola KW - Fungal pigments KW - Genetic engineering KW - Knufia petricola KW - Pilzpigmente KW - Gentechnik PY - 2025 UR - https://nbn-resolving.org/urn:nbn:de:kobv:b43-628150 DO - https://doi.org/10.1007/s12268-025-2442-8 SN - 0947-0867 SN - 1868-6249 VL - 31 IS - 2 SP - 159 EP - 162 PB - Springer CY - Heidelberg AN - OPUS4-62815 LA - mul AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia 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 - TY - CHAP A1 - Schumacher, Julia ED - Scott, B. ED - Mesarich, C. T1 - Role of light in the life cycle of Botrytis cinerea N2 - The fungus Botrytis cinerea (Botryotinia fuckeliana) infects more than 500 plant species and causes a wide range of symptoms: soft rots, accompanied by collapse and water-soaking of tissues followed by the appearance of gray masses of conidia on leaves and soft fruits (gray mold), and spots that may turn brown to full-scale soft rotting on flower petals (Botrytis blight). In general, B. cinerea is responsible for severe economic losses that are either due to the damage of growing plants in the field or the rot of harvested fruits, flowers, and vegetables during storage under cold and humid conditions. B. cinerea has adapted to the plant host and its environment by evolving strategies to use plant tissues for proliferation in terms of a necrotrophic lifestyle, and to survive biotic stresses (host responses) as well as abiotic factors of the host’s environment such as sunlight and concomitant stresses. B. cinerea maintains a complex regulatory network of light-sensitive proteins and signal transduction pathways to use light for coordinating stress responses, virulence, and reproduction. Different light-controlled reproduction cycles enable B. cinerea to live in moderate climate zones by infecting and propagating in summer and resting in winter when green host tissues are unavailable. KW - Gray mold fungus KW - Plant pathogen KW - Light KW - Photoreceptors KW - Development PY - 2023 DO - https://doi.org/10.1007/978-3-031-16503-0_14 VL - 5 SP - 329 EP - 346 PB - Springer, Cham AN - OPUS4-56724 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Knufia petricola – a model for exploring the biology of black rock-inhabiting fungi N2 - Black fungi also called black yeasts, rock-inhabiting fungi or microcolonial fungi are a group of Ascomycetes [Eurotiomycetes, Arthoniomycetes and Dothideomycetes] that exhibit high stress tolerance, yeast‑like or meristematic growth, and constitutive 1,8-dihydroxynaphthalene (DHN) melanin formation. They dominate a range of hostile natural and man‑made environments – from desert rocks and salterns to dishwashers, roofs, and solar panels. Due to their slow growth and the lack of sexual cycles and genetic tools, the underlying mechanisms of black fungi’s phenotypic traits have remained largely unexplored. We consider the rock inhabitant K. petricola [Eurotiomycetes, Chaetothyriales] a suitable model for studying the phenotypic characteristics of black fungi. With K. petricola the regulation of pigment synthesis, general stress responses and the unusual modes of cell division can be dissected by advanced reverse and forward genetics approaches. The genome of K. petricola strain A95 was sequenced using a combination of short high quality Illumina reads and long PacBio reads. The final assembly consists of twelve contigs: five complete chromosomes and six contigs with one telomer each. Gene annotation supported by transcriptomics and proteomics data was manually curated. Recently, we developed a set of genetic tools to manipulate the genome for analyzing gene functions and studying the cell biology. This set includes 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 in the laboratory. Mutants defective in DHN melanogenesis, carotenogenesis or both processes are currently studied to elucidate the role of these protective pigments in tolerance of natural and man-made stresses, weathering of olivine, penetration of marble, and adhesion to surfaces. Further, the established protocols and knowledge gained from K. petricola form a starting point for making other extremotolerant black fungi accessible to genetic manipulation. T2 - 31st Fungal Genetics Conference CY - USA, CA, Pacific Grove DA - 15.03.2022 KW - Black fungi KW - Melanin KW - Genome editing PY - 2022 AN - OPUS4-54585 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia T1 - Many-sided DHN melanin – spotlight on its function in microcolonial black fungi N2 - Dihydroxynaphthalene (DHN) melanin is produced by different Ascomycetes via slightly differing biosynthetic routes. The polyketide synthases (PKS) release the heptaketide YWA1, the hexaketide AT4HN or the pentaketide T4HN. The first two products are deacetylated by ‘yellowish-green’ hydrolases to T4HN, and T4HN is further converted by a core set of enzymes to DHN. Final polymerization steps are accomplished by multicopper oxidases. DHN melanogenesis is often regulated in a spatial and temporal fashion resulting e.g. in melanized reproduction, survival and/or infection structures. Remarkable is the regulation of the DHN melanogenesis in the foliar plant pathogen Botrytis cinerea: it involves two differently expressed PKSs providing the precursor in conidia and sclerotia, respectively (Schumacher 2016, Mol Microbiol). In contrast, a polyphyletic group of Ascomycetes (microcolonial fungi/ black yeast) dwelling in hostile habitats such as bare rock surfaces in hot and cold deserts, exhibits constitutive melanogenesis. Here, DHN melanin builds a protective layer around all vegetative cells thus contributing to the survival of diverse environmental stresses even without specialized reproduction structures. As part of our continuing research on microcolonial rock-inhabiting fungi, we chose the genetically amenable Knufia petricola strain A95 (Nai et al. 2013, Fungal Genet Biol; Noack-Schönmann et al. 2014, AMB Express) for detailed studies. DHN-deficient mutants generated by targeted mutation of biosynthetic genes were studied with regard to the architecture of the cell wall and the EPS (extracellular polymeric substances) matrix, attachment to and weathering of olivine, as well as the tolerance to abiotic and biotic stresses. We will discuss the critical role of the outer cell surface (DHN melanin and EPS) in adhesion to the substrate and subsequent damage of the colonized surface. T2 - European Conference on Fungal Genetics (ECFG15) CY - Rome, Italy DA - 17.02.2020 KW - fungus KW - melanin KW - pigmentation PY - 2020 AN - OPUS4-50591 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -