Southern blot analysis

15 µg of chromosomal DNA was digested with 30 U of the given restriction enzymes (NEB). The resulting DNA fragments were separated by electrophoresis on an 0.8% agarose gel, then denatured in 0.4-M NaOH, and transferred by capillary forces onto a Biodyne B 0.45-µm nylon membrane (Pall Corporation, Port Washington, NY, USA) using 10 × SSC. 1.5 µg of biotinylated DNA probe was used for hybridization at 65 °C overnight. Probes were generated by PCR using the primers 5Xyr1_fwd and Xyr1_1760rev-NotI (Fig. 1a, c) or 5pyr4_fwd(BglII) and 5pyr4_rev-BspEI (Fig. 3a, c) using chromosomal DNA of T. reesei Δtmus53 as template. Labeling of the probe was performed using a Klenow Fragment (exo-) (Thermo Scientific), random hexamer primers, and biotin-11-dUTP (Jena Bioscience, Jena, Germany). Signals were visualized using Poly-HRP conjugated to streptavidin and ECL Plus Western Blotting substrate (both Thermo Scientific) on a ChemiDoc MP (Bio-Rad Laboratories, Hercules, USA).