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

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

Gene structures of <i>C</i>. <i>graminicola TRPF</i> genes and predicted membrane topologies of yeast TRPY1 and <i>C</i>. <i>graminicola</i> TRPF proteins.

<p><b>(A)</b> Gene structures of <i>CgTRPF</i> genes. Boxes: exons, lines: introns; grey: match of the initial tBLASTn search against TRPY1, black: regions identified by RACE-PCR and verified by cloning PCR. <b>(B)</b> Artistic representation (forged steel) of the TRP channel core structure containing six transmembrane (TM) domains and a pore loop between TM domain 5 and 6. <b>(C)</b> Predicted membrane topology of TRPY1 and CgTRPFs. Cytosolic amino acid residues are indicated in light green, TM domains are shown in yellow, luminal amino acid residues are depicted in light blue, and the predicted pore loop is marked in dark blue. Acidic amino acid residues [Asp (D) or Glu (E)] are indicated by a red edge, which is boldfaced in motifs of 4 or more consecutive acidic amino acid residues. One circle represents one amino acid residue. The first and the last amino acid of the whole protein, as well as of each TM domain, are enumerated.</p

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

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

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