Generation of <i>mac1</i> knockout and complemented strains.

<p><b>(A)</b> Physical map of the <i>F</i>. <i>oxysporum mac1</i> locus in the wt and the <i>mac1</i>Δ strains. Relative positions of restriction sites, PCR primers and the probe used in the Southern blot analysis are indicated. <i>Hyg</i><sup>R</sup>, hygromycin resistance gene. <b>(B)</b> Southern blot analysis of putative <i>mac1</i>Δ deletion mutants. Genomic DNA of the wt strain and twelve independent hygromycin resistant transformants was treated with <i>Bam</i>HI, separated on a 0.7% agarose gel, transferred to a nylon membrane, and hybridized with the DNA probe corresponding to the 5’ flanking region of <i>mac1</i> indicated in <b>(A)</b>. Molecular sizes of the hybridizing bands are indicated on the left. Transformants #1, #7, #10 and #11 show hybridizing bands consistent with homologous replacement of the <i>mac1</i> gene with the hygromycin resistance cassette. <b>(C)</b> Physical map of the <i>F</i>. <i>oxysporum mac1</i> locus in the <i>mac1</i>Δ mutant and the complemented <i>mac1</i><sup>Stag</sup> strain. Relative positions of PCR primers are indicated. <b>(D)</b> Agarose gel electrophoresis of PCR products obtained using the primer pair Mac1-5’-F and Mac1-Stag-R with genomic DNA extracted from the indicated strains. M, molecular size markers.The presence of the 2916 bp amplification band is consistent with insertion of the <i>mac1</i><sup>Stag</sup> allele at the native <i>mac1</i> locus.</p> <p>(PDF)</p>