ATL99s Role in Plant Defense Figure 3. Susceptibility of atl9 mutants to Golovinomycetes cichoracearum and hydrogen peroxide production. A) Quantification of Golovinomyces cichoracearum growth on Col-0 plants, three different atl9 T-DNA insertional mutants (atl9-1-3), 35S:ATL9, Kas-1, AtrbohD, AtrbohF, AtrbohD/F and the SA-compromised mutant sid2-1. The number of conidiophores per colony (c/c) were counted 6 dpi. Inoculations were performed at a low density (5–10 conidia/mm2; n = 36 plants). Data values represent one of at least three independent experiments with similar results. B) Production of hydrogen peroxide on plants infected with G. cichoracearum. Cleared leaves of infected plants were stained with trypan blue which stains the fungal hyphae and conidiophores on the leaf surface. Secondary staining with DAB was performed to indicate areas of hydrogen peroxide production (indicated by peroxidase activity). a, Kas-1, b, Col-0, c, atl9-1, d, AtrbohD, f, AtrbohD/F, g, sid2-1. Arrows indicate fungal penetration points in epidermal cells with detected hydrogen peroxide production. The images were taken at a final magnification of 40x. Bars.- 5 mm. C) Quantification of hydrogen peroxide production in mutants. The intensity of DAB staining was quantified at each inoculated plant and compared to the corresponding uninoculated control to obtain a relative DAB signal. The quantification was performed with the aid of the program Image J. doi:10.1371/journal.pone.0014426.g003 and inset), we were interested in determining the timing of ATL9 expression after fungal infection. Expression levels of ATL9, AtrbohD, AtrbohF, and the defense marker genes PR1 and PDF1.2 were monitored using qRT-PCR at several time points after infection with powdery mildew [37,38]. Wild-type plants were heavily inoculated with G. cichoracearum and tissue samples were PLoS ONE | www.plosone.org 5 December 2010 | Volume 5 | Issue 12 | e14426