TY - CONF A1 - Gorbushina, Anna A1 - Knabe, Nicole A1 - Voigt, Oliver A1 - Heeger, Felix A1 - Schumacher, Julia T1 - A Genetic Toolbox for Exploring the Life Style of the Rock-inhabiting Black Fungus Knufia petricola N2 - After their discovery on rock surfaces in cold and hot deserts, a polyphyletic group of ascomycetous black fungi was found to dominate a range of hostile environments – natural and man-made, from salterns to dishwashers, roofs and solar panels. Together with bacteria and algae they may establish sub-aerial biofilms and cause weathering of the surfaces they grow on. Their impressive survival abilities as well as their constitutive protective pigmentation and cluster-like microcolony organisation are similar in environmental isolates as well as in heat-tolerant opportunistic pathogens of animals and humans. The exact genetic properties that ensure their survival in extreme environments can be studied if some black fungi were amenable to genetic manipulations. We selected the rock-inhabiting fungus Knufia petricola (class Eurotiomycetes, order Chaetothyriales) that grows moderately in axenic culture and exhibits all the characteristics of black yeasts such as yeast-like cell growth, absence of reproductive structures and constitutive dihydroxynaphthalene (DHN) melanogenesis. For this environmental strain we developed protocols to efficiently generate and transform protoplasts resulting in stable homokaryotic transformants by targeting genes involved in pigment synthesis and expressing fluorescent reporter genes. Hence, endogenous and foreign genes can be expressed from episomal AMA1-containing plasmids and genome-integrated DNA constructs. Moderate rates of homologous recombination allow for both ectopic and targeted integrations. CRISPR-Cas9 was further validated as a strategy for obtaining selection marker-free mutants and silencing via RNA interference as an approach to study essential genes. Availability of this genetic toolbox and an annotated genome sequence is paving the way for studying interactions of K. petricola and other black yeasts with environmental stressors, material surfaces, soil matrices and phototrophic symbionts. T2 - VAAM symposium 'Molecular Biology of Fungi' CY - Göttingen, Germany DA - 19.09.2019 KW - Knufia petricola KW - Rock-inhabiting fungus KW - Genetics KW - Crispr-Cas9 PY - 2019 AN - OPUS4-49634 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Abdallah, Khaled A1 - Knabe, Nicole A1 - Breitenbach, Romy A1 - Dementyeva, Polina A1 - Voigt, Oliver A1 - Gerrits, Ruben A1 - Schumacher, Julia A1 - Gorbushina, Anna T1 - Developing a genetic toolbox for Knufia petricola A95: a model for stress-tolerant and symbiose-competent rock-inhabiting fungi N2 - Black microcolonial fungi (MCF) are persistent inhabitants of rock surfaces in hostile desert environments. In these niches, MCF have evolved mineral-weathering and symbiotic capabilities as well as mechanisms to cope with multiple stresses such as solar irradiation, temperature extremes and low water activity. Due to their stress tolerance these ascomycetes are prominent in modern terrestrial ecosystems – like man-made material surfaces from roof to solar panels. MCF interactive capabilities support their facultative symbiotic relationships with cyanobacteria and ensure their rock-weathering geochemical activity. Using the rock-inhabiting fungus K. petricola A95 (Chaetothyriales), we developed transformation protocols and deleted genes responsible for production of the protective pigments melanins and carotenoids. To confirm that the mutant phenotypes were not due to hidden mutations, melanin synthesis was restored by complementing the mutants with the respective wild type genes. Strains of K. petricola carrying gene variants for fluorescent proteins EGFP and DsRed are available. We successfully labelled the cytoplasm, nuclei, peroxisomes and mitochondria. Targeted and ectopic integrations result in stable transformants suitable for further phenotypical characterization. As K. petricola is a non-pathogenic fungus with all characteristic features of MCF, including meristematic growth, melanized cell-walls, extracellular polymeric substances and extensive pigment production, our results will shed light on protective role of pigments during cell wall maturation and oxidative stress defence in rock-inhabiting MCF. Genes involved in environmental sensing or substrate and phototroph interactions are currently targeted. With the help of a mutant collection and fluorescently labelled K. petricola we will be able to investigate interactions of MCF with environmental stressors, mineral substrates, soil matrices and phototrophic symbionts. T2 - Fungal Genetics Conference 2019 CY - Asilomar, CA, USA DA - 12.03.2019 KW - Knufia petricola KW - Genetics KW - Melanin PY - 2019 AN - OPUS4-49635 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER - TY - CONF A1 - Schumacher, Julia A1 - Heeger, Felix A1 - Whitfield, Daniel A1 - Knabe, Nicole A1 - Nai, Corrado A1 - Schumacher, Julia A1 - Broughton, William A1 - Cuomo, C. A1 - de Souza, E. A1 - Lespinet, O. A1 - Mazzoni, C. A1 - Monaghan, M. A1 - Gorbushina, Anna T1 - Exploring the genome of the black yeast Knufia petricola N2 - Black yeasts form a polyphyletic group of Ascomycota that colonize bare surfaces like rocks, facades of buildings, and solar panels. Their protective adaptations enable an adequate response to fluctuating and diverse temperature, water and UV radiation stresses. Together with bacteria and algae they form sub-aerial biofilms (SAB) this way discoloring and weathering the surfaces they grow on. Strain A95 of Knufia petricola (Eurotiomycetes, Chaetothyriales) displays both typical yeast-like cell growth and constitutive dihydroxynaphthalene (DHN) melanogenesis. Along with the cyanobacterium Nostoc punctiforme as photobiont, it is already used in a model system for studying SAB formation and bio-weathering. Applying the recently developed tools for the generation of deletion mutants will allow to define gene functions and to identify genes critical for abiotic and biotic interactions. We present a chromosome-level genome assembly and annotation for K. petricola A95. The genome was assembled with MaSuRCA using a hybrid assembly approach of Illumina MiSeq and PacBio SMRT sequencing data. The resulting assembly consists of 17 contigs including the complete mitochondrial genome and five complete chromosomes. It shows indication of repeat-induced point mutations (RIP). Supported by RNA sequencing data from eight different growth conditions, 10,994 genes were predicted with the BRAKER2 pipeline. Functional annotation of genes was obtained from general functional annotation databases and the fungal specific database FungiPath. Comparative analyses are in progress to identify genes specific to black yeasts, that may facilitate the survival on exposed surfaces. In sum, the genome sequence of K. petricola is a valuable resource to gain insight into the protein inventory and functional pathways of extremotolerant and symbiosis-capable fungi. T2 - Fungal Genetics Conference 2019 CY - Asilomar, CA, USA DA - 12.03.2019 KW - Knufia petricola KW - Black fungus KW - Genome sequence PY - 2019 AN - OPUS4-49636 LA - eng AD - Bundesanstalt fuer Materialforschung und -pruefung (BAM), Berlin, Germany ER -