1. SUPPLEMENTAL FIGURE S1 Cloning strategy of the Tet-On expression system Construction of pVG1-47 (PgpdA::rtTA::TcgrA – TetO7::Pmin::TtrpC) A 520 bp TetO7-Pmin fragment and a 436 bp TtrpC fragment were PCR amplified from plasmid p500 (Vogt et al., BMC Microbiol 5:1., 2005) by introducing the indicated restriction site overhangs. The PCR product obtained were cloned into pJET (Fermentas) giving plasmids pSA2-5 and pSA3-6, respectively. Both the TetO7-Pmin fragment and the TtrpC fragment were cut out from pSA2-5 and pSA3-6 via a NotI x PmeI double restriction and cloned into the NotI site of p474 (Vogt et al., BMC Microbiol 5:1., 2005) via a three-way-ligation. Construction of pVG2.2 (PgpdA::rtTA::TcgrA– TetO7:: Pmin::TtrpC– pyrG*) The 2.2 kb pyrG* fragment was PCR amplified from pAB94 (van Gorcom and van den Hondel, Nucleic Acids Res 16:9052, 1988) by introducing AscI overhangs and cloned into the AscI site of pVG1-47. Construction of pVG3 (PgpdA::rtTA::TcgrA – TetO7::Pmin::mluc::TtrpC) The 1.6 kb mluc open reading fragment was PCR amplified from pLUC6 (Morgan et al., Fungal Genet Biol 38:327-32, 2003) by introducing PmeI overhangs and cloned into pJET vector (Fermentas) yielding in plasmid pSA1-2. The mluc fragment was cut from pSA1-2 via PmeI restriction and cloned into the PmeI site of pVG1. Construction of pVG4.1 (PgpdA::rtTA::TcgrA – TetO7::Pmin::mluc::TtrpC – pyrG*) The pyrG* AscI-fragment (see above) was ligated into the AscI site of pVG3, creating pVG4.1. Color code: Green (A) = PgpdA::rtTA::TcgrA, purple (B) = tetO7::Pmin::TtrpC, red (C) = pyrG* and orange = mluc. The sizes of the fragments and the plasmids are indicated.

2. SUPPLEMENTAL FIGURE S2 Southern analysis of A. niger strains gained after transformation with pVG2.2 or pVG4.1 A B A. To confirm homologous integration of the constructs at the A. niger pyrG locus, genomic DNAs of selected transformants were restricted with NcoI and subjected to Southern hybridization using pyrG as a probe. Strain AB4.1 served as a wild type control. NcoI does not cut within pVG2.2 but once within the mluc fragment of pVG4.1. The expected fragment size for the wild type pyrG is 3.2 kb. For a single-copy integration of construct pVG4.1 at pyrG, two signals are expected (5 kb, 7.7 Kb). For a single- copy integration of construct pVG2.2 at pyrG, one signal at 11.8 Kb is expected. For a tandem-copy integration of construct pVG4.1 at pyrG, three signals are expected (5 kb, 7.7 kb, 9.4 kb). B. Lanes 1 and 15: control strain AB4.1 showing a single band of 3.2 kb Lane 3: strain VG5.1, single-copy integration of pVG2.2 at pyrG (one signal at 11.8 Kb) Lane 6: heterokaryon, single-copy integration of pVG4.1 at pyrG (two signals at 5 and 7.7 kb plus a wild type signal at 3.2 kb) Lane 7: strain VG6.1, single-copy integration of pVG4.1 at pyrG (two signals at 5 and 7.7 kb) Lane 10: strain VG8.1, integration of two copies of pVG4.1 at pyrG resulting in three signals (5, 7.7, 9.44 kb) Lane 11: strain VG8.2, integration of two copies of pVG4.1 at pyrG resulting in three signals (5, 7.7, 9.44 kb) & heterologous integration of one pVG4.1 copy (signal > 11 kb) Lanes 2, 4, 5, 8, 9, 12-14 show wild type signal, indicating restoration of a functional pyrG allele without plasmid integration.