FIG 1.

Generation of A. fumigatus mtfA deletion and complementation strains. Confirmation of mtfA deletion (ΔmtfA) and complementation (com) strains by Southern blotting, PCR, and qRT-PCR. (A) Diagram showing the replacement of mtfA with the marker gene pyrG by a double-crossover event. EcoRI restrictions sites (E) and probe template are shown. (B) X-ray image showing the Southern blot results confirming the deletion of mtfA. Genomic DNA samples were digested with EcoRI. The expected band sizes were 1.8 kb (wild type [WT]) and 6.1 kb (ΔmtfA). (C) Linear representation of plasmid pTDS10 used for complementation. (D) PCR confirmation of the mtfA wild-type allele integration in the ΔmtfA genome. The wild-type strain was used as a control. The PCR yielded the predicted 1-kb PCR product. (E) qRT-PCR expression analysis of mtfA with primers AfumRM7qrtPCRF and AfumRM7qrtPCRR (Table 2). mtfA expression is recovered in the complementation strain. The relative expression was calculated using the 2^−ΔΔCT method, as described by Livak and Schmittgen (70). Expression of 18S rRNA was used as an internal reference gene. Values were normalized to the expression levels in the wild type, considered 1. The error bars represent standard errors.