Additional file 5 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 5. Table S

Additional file 4 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 4. Table S

Additional file 3 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 3. Table S

Additional file 1 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 1. Figures S1 to Figures S

Additional file 2 of Comparative analysis of stress-induced calcium signals in the crop species barley and the model plant Arabidopsis thaliana

Additional file 2. Table S

Growth of <i>C</i>. <i>graminicola</i> colonies on different carbon sources.

<p>Growth was assessed on mLCM agar, PDA, and minimal media administered with 2% of the respective carbon source. All values were normalized to the growth of the wild type on the respective medium. Colony diameter was measured 117 hours post inoculation. Data are means ± SE (N = 3).</p

Expression profiles of the <i>CgTRPF</i> genes in axenic culture and during infection of <i>C</i>. <i>graminicola</i> on maize.

<p><b>(A)</b> Full-length cDNA of <i>CgTRPF</i> genes was amplified from RNA extracted from <i>in vitro</i> grown mycelium and spores. Products were expected at 2098, 2152, 3522, and 2101 bp for <i>CgTRPF1</i>, <i>CgTRPF2</i>, <i>CgTRPF3</i>, and <i>CgTRPF4</i>, respectively. <b>(B, C)</b> Expression during the infection process relative to the expression in spores (0 hours post infection, hpi); black: <i>CgTRPF1</i>, dark grey: <i>CgTRPF2</i>, light grey: <i>CgTRPF3</i>, white: <i>CgTRPF4</i>. <b>(B)</b> Detached third leaves of 2.5-week-old drop-infected plants (cv. Mikado); assayed by qRT-PCR. Data are means ± SE (N = 3). <b>(C)</b> Third leaf of intact two-week-old drop-infected plants (cv. Golden Jubilee); assayed by RNA-Seq. Data are means ± SE (N = 3).</p

Yellow Cameleon-based measurements of [Ca²⁺]_cyt in individual hyphae.

<p>Kymographs of individual hyphae of wild type and deletion mutants. Each horizontal pixel line represents the mean [Ca²⁺]_cyt of a 5-pixel-wide ROI in the middle of each hypha. ROIs of subsequent images, acquired every 2 sec, were plotted one below the other. The slope of the kymographs, read from top to bottom, thus indicates the growth rate. Relative [Ca²⁺]_cyt is displayed in false-colour using the RGB rainbow scale. All measurements were performed on hyphae growing at an mLCM agar-glass interface [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158561#pone.0158561.ref045" target="_blank">45</a>].</p

Detached-leaf infection assay.

<p>Sections of the third leaf of two-week-old maize plants (cv. Golden Jubilee) were infected with 10⁴ spores of <i>C</i>. <i>graminicola</i> wild type or <i>Cgtrpf1</i> through <i>4</i> deletion mutants per 10-μL drop, or mock-infected with 10 μL of 0.02% Tween 20 in bidistilled water. Representative images of three biological replicates are shown; the experiment was repeated twice with similar results.</p

Response of mycelial growth of <i>C</i>. <i>graminicola</i> and [Ca²⁺]_cyt of <i>C</i>. <i>graminicola</i> and yeast to hyperosmotic stress.

<p><b>(A, B)</b> Mycelial growth of <i>C</i>. <i>graminicola</i> osmotically stressed with glycerol. <b>(A)</b> Colony diameter of wild type stressed with varying concentrations of glycerol, normalized to unstressed colonies. <b>(B)</b> Wild type and deletion strains stressed with 0.5 M glycerol, normalized to the wild type. Colony diameters were determined 122 hours post inoculation. Data are means ± SE (N = 3). <b>(C, D)</b> [Ca²⁺]_cyt response of yeast and <i>C</i>. <i>graminicola</i> to NaCl measured by aequorin luminescence. <b>(C)</b> Response of <i>trpy1</i>Δ yeast mutant cells transformed with the indicated vectors to a solution (pH 7.0) containing 1.5 M NaCl, 50 mM MES-KOH, and 25 mM EGTA. Treatment was started at 1 min. Red line: empty pFL61 vector (negative control); green line: pFL61-ScTRPY1 (positive control); blue lines: yeast strains transformed with pFL61 containing <i>CgTRPF1</i> through <i>4</i>. Data are means ± SE (N = 3). <b>(D)</b> [Ca²⁺]_cyt measurements on <i>C</i>. <i>graminicola</i> wild type colonies. Whole colonies were pre-treated with 50 mM MES-KOH (pH 7.0) for 30 min prior to recording, followed by treatment with a solution (pH 7.0) containing 50 mM MES-KOH and no NaCl (grey line) or 1.5 M NaCl (final concentration; red line). To abolish the influx of extracellular Ca²⁺, colonies were pre-treated with a solution (pH 7.0) containing 50 mM MES-KOH and 25 mM EGTA for 30 min prior to measurement, followed by treatment with a solution (pH 7.0) containing 50 mM MES-KOH, 25 mM EGTA, and no NaCl (green line) or 1.5 M NaCl (final concentration) (blue line). Treatment solutions were added after 1 min of recording. Traces show individual measurements in order to demonstrate [Ca²⁺]_cyt spikes in the MES-KOH control treatment. Replicate measurements can be found in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0158561#pone.0158561.s004" target="_blank">S4 Fig</a>.</p