Linear regression analysis of TCP expression depending on phytoplasma quantity or <i>SAP11</i>_<i>CaPm</i> expression.

95% confidence interval (CI), goodness of fit and significance level (alpha = 0.05) of linear regression, calculated with GraphPad Prism 7.05 (GraphPad Software Inc.), between normalized phytoplasma quantity and SAP11CaPm, MdTCP16, MdTCP25 and MdTCP24 expression (left panel) and between normalized SAP11CaPm expression and MdTCPs expression in leaves from greenhouse (middle panel) and from foil-tunnel, respectively (right panel).</p

SAP11_CaPm interacts with MdTCP16 <i>in planta</i>.

Nicotiana benthamiana mesophyll protoplasts were co-transformed with a BiFC expression vector encoding SAP11CaPm and MdTCP16. SAP11CaPm and MdTCP16 interact in the nucleus and in the cytoplasm as indicated by the occurrence of a YFP signal in these two cellular compartments. The co-expression of SAP11CaPm and MdNYC1 did not show any YFP signal. The RFP signal (depicted in magenta) indicates a successful transformation of protoplasts with the BiFC vector. Chl (depicted in blue) shows the autofluorescence of chlorophyll within the chloroplasts. Microscopic analysis was performed with a Zeiss LSM 800 confocal microscope. Bars represent 20 μm for all micrographs.</p

<i>MdTCP16</i> expression increases from spring to autumn, is slightly higher in infected samples and strongly correlates to phytoplasma levels in leaves from infected <i>Malus × domestica</i>.

MdTCP expression in spring and autumn of non-infected (green) and naturally infected (black) apple leaves (A) or graft inoculated and grown in greenhouse plants (B). Naturally infected samples (A) comprise one leaf pool from non-infected trees, and three leaf pools from infected trees. Graft inoculated greenhouse plants (B) comprise leaf samples from 5 non-infected and 5 infected trees, each represented by a data point. Correlation of MdTCP and SAP11CaPm expression (C, D, E) in infected Malus × domestica leaf-samples from spring (grey, n = 8) and autumn (black, n = 8). Lines in graphs C, D, E and F show linear regression of the respective samples either from the greenhouse (dashed line) or from the foil tunnel (solid line). Samples in C, D and E were grouped regarding their growing conditions and plotted to different y-axis due to the differences in the concentration ranges of the different sample subsets. Correlation of MdTCP, SAP11CaPm expression and phytoplasma levels (F) in graft-inoculated Malus × domestica leaf-samples from spring (grey, n = 3) and autumn (black, n = 4). Statistical analysis was performed with multiple t-test. Statistical differences were determined using the Holm-Sidak method, with alpha = 0.05 and linear regression analysis, using GraphPad Prism 7.05 (GraphPad Software Inc.). For pools comprising only one biological replicate, technical replicates accounted to the statistical analysis. Significant differences between groups are indicated with asterisks (* P≤0.05, ** P≤0.01, *** P≤0.001). Data points with very similar values might overlap in the graph and can thus misleadingly appear as a single data point. In only seven out of the ten samples from the greenhouse phytoplasma concentration could be determined (F). This was due to a lack of sample material for the DNA preparation which is necessary for phytoplasma detection.</p

Primers used in this study.

Lowercase letters indicate bases for Gateway-attB site overhangs or SfiI restriction site overhangs. (PDF)</p

SAP11_CaPm interacts with MdTCP16 but not with MdNYC1 in yeast.

Interaction between the bait SAP11CaPm fused to a DNA binding domain (BD) and the prey protein from Malus × domestica fused to an activation domain (AD) is indicated by growth of the Saccharomyces cerevisiae reporter strain NMY51 on SD minimal medium lacking the amino acids histidine (his) and adenine (ade). The white color of BD-SAP11CaPm + AD-MdTCP16 grown on full medium is an additional indication for a strong interaction, since the ADE2 reporter gene is activated upon interaction, while in absence of a protein-protein interaction and thus no ADE2 activation, a red colored intermediate accumulates in the adenine metabolic pathway.</p

Reference Sequence of XM_008376500.2.

The sequence includes the CDS for XP_008374722.1 (MdTCP16) with TCP domain and the identified part in the Y2H screen. (PDF)</p

Phytoplasma concentration of leaf and root samples.

The normalized phytoplasma concentration is given as the ratio of the ’Ca. P. mali’ specific 16S gene copies and the Malus x domestica single-copy gene ACO. Phytoplasma concentration was quantified in seven infected leaf samples from greenhouse plants (three from spring and four from autumn), in three naturally infected pooled leaf samples (one from spring, two from autumn) and in one naturally infected pooled root sample (autumn). (PDF)</p

Harvest- and wound-induced transcript accumulation of in roots and shoots of 5-week-old plants

Relative transcript levels were determined by real-time RT-PCR analysis using TaqMan probes. Plants were carefully removed from expanded clay and stayed untreated (white columns) or were wounded on roots (cross-hatched columns). From a third batch of plants, shoots and roots were separated from each other and both were wounded (dark gray columns). At each time point, up to five plants were pooled, and total RNA was extracted from roots and shoots separately. Real-time RT-PCR was carried out in triplicate for each sample. The mean transcript level of plants directly after harvest (time point 0) was set to 1. Data are presented as mean values ±SD of at least three biological replicates. Different letters designate statistically different values separately for each treatment (ANOVA with Tukey HSD test,  <p><b>Copyright information:</b></p><p>Taken from "Mechanostimulation of leads to enhanced levels of jasmonic acid"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2847-2856.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2486479.</p><p></p

Immunolocalization of AOC protein in cross-sections of stem of untreated plants

AOC was immunolabelled by an antibody against tomato AOC followed by a secondary antibody coupled to AlexaFluor488. The occurrence of AOC is visible by the green fluorescence signal. To visualize DNA-containing organelles, sections were counterstained with DAPI (C, D). (A) Bright-field image of a cross-section. (B) The same section as in (A) illuminated for green fluorescence. The green label indicative of AOC is visible in the pith, phloem tissue, and cortex. (C, D) Confocal laser scanning micrographs of the immunolabelled sections showing the occurrence of AOC in plastids of parenchymatic cells of pith, phloem, and cortex. The blue staining visualizes the nuclei due to the fluorescence of DAPI. Scale bars represent 500 μm in A and B, 100 μm in C, and 50 μm in D.<p><b>Copyright information:</b></p><p>Taken from "Mechanostimulation of leads to enhanced levels of jasmonic acid"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2847-2856.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2486479.</p><p></p

Harvest- and wound-induced accumulation of JA in roots and shoots of 5-week-old plants

Plants were carefully removed from expanded clay and stayed untreated (white columns) or were wounded on roots (cross-hatched columns). From a third batch of plants shoots and roots were separated from each other and both were wounded (dark grey columns). At each time point, up to five plants were pooled, and roots and shoots were separately extracted for quantification of JA. The means ±SD are given for five biological replicates and are tested for each treatment with one-way ANOVA followed by Tukey HSD test ( <p><b>Copyright information:</b></p><p>Taken from "Mechanostimulation of leads to enhanced levels of jasmonic acid"</p><p></p><p>Journal of Experimental Botany 2008;59(10):2847-2856.</p><p>Published online 6 Jun 2008</p><p>PMCID:PMC2486479.</p><p></p