FIG 4.

Disruption of the MDR3 (TinMDR3) gene of T. indotineae TIMM20118 and TIMM20119 by gene replacement strategy. (A) Schematic representation of a part of the TinMDR3-targeting vector pAg1-TinMDR3/T. The hph cassette is composed of Aspergillus nidulans trpC promoter (PtrpC), E. coli hygromycin B phosphotransferase gene (hph), and A. fumigatus cgrA terminator (TcgrA). Border sequences are the specific regions that delineate the DNA to be transferred during Agrobacterium tumefaciens-mediated transformation. Restriction enzyme site: A, ApaI; Ba, BamHI; E, EcoRI; K, KpnI; P, PstI; S, SpeI. (B) Schematic representation of the TinMDR3 locus before and after homologous recombination. (C) Southern blotting. Aliquots of approximately 10 μg of genomic DNA from each strain were digested with EcoRI and separated by electrophoresis on 0.8% (wt/vol) agarose gels. Lanes 1 to 6, TIMM20118 (parent strain), ΔTinMDR3-45MM-9, ΔTinMDR3-45MM-15, TIMM20119 (parent strain), ΔTinMDR3-122MM-1, and ΔTinMDR3-122MM-17, respectively. A fragment of about 530 bp of the TinMDR3 locus in TIMM20119 was amplified by PCR with a pair of the primers P47 and P48 (Table S1) and used as a hybridization probe. DNA standard fragment sizes are shown on the left. (D) Evaluation of ITC susceptibility in the TinMDR3 disruptants produced from TIMM20118 and TIMM20119, by Etest assays. The plates were incubated at 28°C for 4 days.