Relative expression of <i>MtDefMd</i> and selected AM marker genes in mycorrhized RNAi:MtDefMd1/2 and RNAi:<i>gusA</i>int transgenic control roots of <i>M</i>. <i>truncatula</i>.

<p>Transcript amounts are shown relative to <i>MtTefα</i> (A) and were additionally normalized by building a ratio to <i>MtPt4</i>-expression (B). Measurements from RNAi:MtDefMd1/2 roots are colored in light grey, corresponding RNAi:<i>gusA</i>int control measurements in dark grey. Roots were harvested at 28 days post inoculation with <i>R</i>. <i>irregularis</i>. n = 12 biological replicates, depicted is the standard error of the mean. Statistical significances: _* p≤0.05, _** p≤0.005.</p

Sequence analyses of of AM-dependent defensins MtDefMd1-4 and AM-unrelated defensins.

<p>Secondary structures of MtDefMd1-4 and AM-unrelated defensin-like proteins of <i>M</i>. <i>truncatula</i> (A), a representation of their three-dimensional structures (B and C), as well as surface electrostatics (D and E) are shown. Predicted signal peptides were removed from the mature amino acid sequences. Consecutively, the defensins were aligned based on their secondary structures. Background colorisation of the amino acids (in A) indicate hydrophobicity in a scale from red to blue (red: high hydrophobicity). A conserved aspartic acid in the C-terminal region of MtDefMds is marked with a grey triangel. For the bi-domain defensin MtDef5, the domains MtDef5A (including a 7 amino acid linker towards the MtDef5B domain) and MtDef5B are shown. After modelling the three-dimensional structures of the MtDefMd1-4, MtDef4 and MtDef5A/B defensins, they were visualized (B and C) and their surface electrostatics were calculated (D and E). The region congruent to the γ-core motif is indicated with arrows. The following proteins were used for comparisons in addition to MtDefMd1-4: MtDef5 A [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref065" target="_blank">65</a>], MtDef5 B [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref065" target="_blank">65</a>]and MtDef4 [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref030" target="_blank">30</a>].</p

Size distribution of arbuscules in mycorrhized <i>M</i>. <i>truncatula MtDefMd1</i>-overexpression, <i>MtDefMd1/2</i>-knock-down, and pPT4:<i>gusA</i>int controls roots.

<p>Arbuscules were sorted into one of eleven size categories. In total, the size distribution of arbuscules in pUbi:MtDefMd1-overexpression (647 arbuscules), pPt4:MtDefMd1-overexpression (509 arbuscules), RNAi:MtDefMd1/2-knock-down (625 arbuscules), and pPt4:<i>gusA</i>int control roots (529 arbuscules) is depicted in orange, blue, green, and grey, respectively. Roots were harvested at 28 days post inoculation with <i>R</i>. <i>irregularis</i>. For each construct, three pools of root fragments, each pool being derived from four plants, were analysed. Depicted is the standard error of the mean.</p

Relative expression of MtDefMd1 and selected AM marker genes in mycorrhized MtDefMd1-overexpression and pPT4:<i>gusA</i>int-expressing transgenic control roots of <i>M</i>. <i>truncatula</i>.

<p>Transcript amounts are shown relative to <i>MtTefα</i> (A) and were additionally normalized by building a ratio to <i>MtPt4</i>-expression (B). Measurements of pPT4:MtDefMd1 overexpression roots are coloured in light grey, measurements of pUbi:MtDefMd1 overexpression roots in medium grey, and measurements of pPT4:<i>gusA</i>int control roots in dark grey. Roots were harvested at 28 days post inoculation with <i>R</i>. <i>irregularis</i>. n = 12 biological replicates, depicted is the standard error of the mean. Statistical significance: * p≤0.05.</p

Histochemical localization of <i>MtDefMd1</i> and <i>MtDefMd2</i> promoter activities.

<p>Activities of <i>MtDefMd1</i> (A-D) and <i>MtDefMd2</i> (E-H) promoters were studied in transgenic, mycorrhized roots of <i>M</i>. <i>truncatula</i> A17 wild type (A, B, E, and F) and <i>pt4-2</i> roots (C, D, G, and H). Representative images of roots after 18 (A, B, E, and F) or 56 (C, D, G, and H) days post inoculation with <i>R</i>. <i>irregularis</i>. The GUS-stainings (A, C, E, and G) as well as the Alexa-WGA Fluor 488 stainings (B, D, F, and H) were performed over night. Septa are denoted by arrows. Abbreviations: w, cells with weak promoter activity; s, cells with strong promoter activity.</p

Relative expression of <i>MtDefMd1-4</i> and selected AM marker genes in <i>M</i>. <i>truncatula</i> roots in a time course of mycorrhization.

<p>Expression of <i>MtDefMd1-4</i>, the fungal <i>α</i>-tubulin gene <i>GiTubα</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref036" target="_blank">36</a>] as well as the <i>M</i>. <i>truncatula</i> AM marker genes <i>MtPt4</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref066" target="_blank">66</a>] and <i>MtMyb1</i> [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0191841#pone.0191841.ref017" target="_blank">17</a>] is given in relation to the expression of <i>MtTefα</i>. Plant roots were harvested weekly from 7 to 42 days post inoculation with <i>R</i>. <i>irregularis</i>. Biological replicates were three pools of four plant roots per treatment. The standard deviation of the three biological replicates is given.</p