Genetic Engineering of the Rock Inhabitant Knufia petricola Provides Insight Into the Biology of Extremotolerant Black Fungi

Black microcolonial fungi (Ascomycetes from Arthonio-, Dothideo-, and Eurotiomycetes) are stress-tolerant persistent dwellers of natural anthropogenic extreme habitats. They exhibit slow yeast-like or meristematic growth, do not form specialized reproduction structures accumulate the black pigment 1,8-dihydroxynaphthalene (DHN) melanin in multilayered cell walls. To understand how live, survive, colonize mineral substrates, interact with phototrophs genetic methods needed to test these functions interactions. We chose rock inhabitant Knufia petricola Chaetothyriales as a model for developing manipulation. Here, we report on expansion toolkit by more efficient multiplex CRISPR/Cas9 using plasmid-based system expression Cas9 multiple sgRNAs implementation three resistance selection markers genR (geneticin/ nptII ), baR (glufosinate/ bar suR (chlorimuron ethyl/ sur ). The targeted integration constructs replacement essential genes synthesis allows an additional color screening transformants. black-pink due elimination pks1 (melanin) was applied promoter studies GFP fluorescence reporter. black-white concurrent phs1 (carotenoids) identify transformants that contain two co-localization bimolecular complementation (BiFC) studies. interaction K. White Collar orthologs were demonstrated. Two intergenic regions ( igr1, igr2 ) identified which can be inserted without causing obvious phenotypes. Plasmids pNXR-XXX series new compatible entry plasmids used fast easy generation suitable broad other fungi. This variety tools is opening completely perspective mechanistic very detailed study expression, functioning regulation genes/proteins encoded genomes