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In-vivo mutagenesis of the rock inhabitant Knufia petricola by a customized Ac/Ds transposon system
(2023)
Microcolonial black fungi ubiquitously inhabit sun-exposed natural and man-made surfaces of our planet. To promote genetic studies, CRISPR/Cas9-based genome editing was implemented in the rock-inhabiting fungus Knufia petricola (Eurotiomycetes/ Chaetothyriales). Now efficient targeted mutagenesis of K. petricola - as a representative of the polyphyletic group of black fungi - enables the elucidation of extremotolerance, oligotrophism, unusual types of cell division, mineral weathering and symbiotic interactions. Even more progress on assigning functions to yet unknown genes can be achieved by a forward genetics approach. We chose the two-component Activator/Dissociation (Ac/Ds) transposon system from maize for generating K. petricola insertional mutants by in-vivo mutagenesis. For the optimal use of this genetic tool, an inducible promoter i.e, from the metabolism-independent Tet-on system, was combined with the AcTPase-coding sequence enabling the regulatable transposition of the resistance cassette-containing Ds transposon. In total, six auxotrophic Ac/Ds starter strains containing the Ds transposon at different position of ade2, ura3 or ppt1 were generated. The cultivation of these strains with doxycycline for induction of TET::Ac and subsequent selection of cells on ADE/URA/LYS-lacking media resulted in prototrophic colonies (revertants) for most Ac/Ds strains. Amplicon sequencing of excision sites revealed characteristic footprint patterns, proving that the transposon jumped. For identifying unknown Ds re-insertions sites, the thermal asymmetric interlaced (TAIL)-PCR was successfully implemented. First identified Ds re-insertion sites suggest that the distribution pattern may depend on the excision site. Currently, transposition frequencies and genome-wide distribution of re-insertion sites are studied in different Ac/Ds starter strains to identify the best candidate for generating saturated mutant libraries. This transposon mutagenesis strategy is also interesting for studying other black fungi, because once the Ac and Ds components are integrated in the genome, the fungus ‘only’ needs to be cultivated for generating insertional mutants.
Microcolonial black fungi ubiquitously inhabit sun-exposed natural and man-made surfaces of our planet. To promote genetic studies, which are hindered by slow growth, lack of sexual cycles and transformation difficulties, CRISPR/Cas9-based genetic tools were implemented (Erdmann et al. 2022, Front Fungal Biol). Now efficient targeted mutagenesis of the rock inhabitant Knufia petricola (Eurotiomycetes/Chaetothyriales) - as a representative of the polyphyletic group of black fungi - enables the elucidation of extremotolerance, oligotrophism, unusual types of cell division, mineral weathering and symbiotic interactions. Still more progress on assigning functions to yet unknown genes can be expected if a forward genetics approach is available. We chose the two-component Activator/ Dissociation (Ac/Ds) transposon system from maize for generating a collection of insertional mutants by in-vivo mutagenesis of K. petricola. For the optimal use of this genetic tool, an inducible promoter for the expression of the Ac transposase (AcTPase) and by this the regulatable transposition of the resistance cassette-containing Ds transposon is desired. However, endogenous promoters for nitrate assimilation and galactose catabolism - often used in fungi for regulatable gene expression - are not inducible by their substrates in K. petricola suggesting that the regulatory networks for nutrient acquisition differ significantly in oligotrophic fungi. Therefore, the metabolism-independent Tet-on system was combined with the AcTPase coding sequence and subsequently transformed into Ds-carrying K. petricola strains. In total, four auxotrophic Ac|Ds starter strains containing the Ds transposon at different position of ade2 or ura3 were generated. The cultivation of these strains with doxycycline for induction of TET::Ac and subsequent selection of cells on ADE/URA-lacking media resulted in prototrophic colonies (revertants) for some but not all Ac|Ds strains. Currently, the transposition events in the obtained revertants are studied to validate the procedure. First amplicon sequencing of excision sites revealed footprint patterns, proving the transposon jumped.