Targeted deletion of the high-affinity copper transporters Ctr3 and Ctr1a in <i>F</i>. <i>oxysporum</i>.

<p><b>(A, C)</b> Physical maps of the <i>F</i>. <i>oxysporum ctr3</i> and <i>ctr1a</i> loci in the wt and in the <i>ctr3</i>Δ <b>(A)</b> and <i>ctr1a</i>Δ <b>(C)</b> strains, respectively. Relative positions of restriction sites, PCR primers and the probes used in the Southern blots are indicated. <i>Hyg</i><sup><i>R</i></sup>, hygromycin resistance gene. <i>Neo</i><sup><i>R</i></sup>, neomycin resistance gene. <b>(B, D, E)</b> Southern blot analysis of putative <i>ctr3</i>Δ <b>(B)</b>, <i>ctr1a</i>Δ <b>(D)</b> and <i>ctr3</i>Δ<i>ctr1a</i>Δ <b>(E)</b> deletion mutants. Genomic DNA of the wt strain and independent hygromycin or neomycin resistant transformants was treated with <i>Eco</i>RI <b>(B)</b> or <i>Bam</i>HI <b>(D, E)</b>, separated on 0.7% agarose gels, transferred to nylon membranes and hybridized with a DNA probe corresponding to the 5’ flanking region of <i>ctr3</i> <b>(B, indicated in A)</b> or to the 3’ flanking region of <i>ctr1a</i> <b>(D, E indicated in C)</b>. Molecular sizes of the hybridizing bands are indicated on the left. <b>(F)</b> Colony phenotypes of the indicated strains after 2 d growth on MM+TE<sup>-Cu</sup>, supplemented with the indicated concentrations of CuSO<sub>4</sub>. Scale bar, 1 cm. The colonies of wt and <i>mac1</i>Δ are the same as those shown in <a href="http://www.plospathogens.org/article/info:doi/10.1371/journal.ppat.1012671#ppat.1012671.g001" target="_blank">Fig 1A</a> and are repeated here for clarity. Scale bar, 1 cm. <b>(G)</b> Kaplan-Meier plot showing survival of groups of 10 tomato plants (cv. Momotaro) inoculated by dipping the roots into a suspension of 5x10<sup>6</sup> microconidia/ml of the indicated fungal strains or water (Mock). Data shown are from one representative experiment. Experiments were performed at least two times with similar results. <i>p</i>-value: ***<0.001 versus the wt according to Log-rank (Mantel-Cox) test.</p> <p>(PDF)</p>