Descriptive statistics of six newly identified and four classical tomato reference genes based on their crossing point values in all samples combined (n = 48) as calculated by BestKeeper.

<p>^a) New reference gene candidates are indicated in bold. [Ct], cycle threshold; Geo Mean [Ct], geometric mean of Ct; Min [Ct] and Max [Ct], the extreme values of Ct; SD [± Ct], standard deviation of the Ct; CV [% Ct], CV expressed as a percentage on the Ct level; Min [x-fold] and Max [x-fold], the extreme values of expression levels expressed as an absolute x-fold over- or under-regulation coefficient; SD [± x-fold], standard deviation of the absolute regulation coefficients.</p><p>Descriptive statistics of six newly identified and four classical tomato reference genes based on their crossing point values in all samples combined (n = 48) as calculated by BestKeeper.</p

Expression pattern of traditional reference genes in healthy and <i>Xcv</i>-infected tomato plants.

<p>Leaves of <i>S</i>. <i>lycopersicum</i> cv. MM plants were untreated or infiltrated with 10 mM MgCl₂ (mock) and 85–10Δ<i>hrcN</i>, respectively. To analyze mRNA accumulation of selected genes leaf material was harvested at 0, 4, 8 and 16 hours post infiltration (hpi). Relative RNA levels of housekeeping genes traditionally employed as references [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136499#pone.0136499.ref030" target="_blank">30</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136499#pone.0136499.ref061" target="_blank">61</a>–<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0136499#pone.0136499.ref064" target="_blank">64</a>] were determined by microarray hybridization analysis. Expression values of analyzed plant samples are plotted separately, i.e., three biological replicates per time-point per infiltration. For each gene, expression values were obtained as the mean of the intensities of all probes representing the respective gene. Blue curves represent housekeeping genes that were also analyzed in this study together with novel reference genes. In addition, the most stably expressed gene in the microarray experiment is shown (red curve). <i>ACT</i> (<i>actin</i>), TC194780a; <i>EF-1α</i> (<i>elongation factor 1α</i>), SGN-U212845; <i>GAPDH</i> (<i>glyceraldehyde 3-phosphate dehydrogenase</i>), TC198136a; <i>UBI</i> (<i>ubiquitin</i>), TC193502a; <i>TBP</i> (<i>TATA binding protein</i>), SGN-U329249; <i>RPL8</i> (<i>ribosomal protein L8</i>), X64562; <i>TUA (α-tubulin</i>), AC122540; <i>CYP</i> (<i>cyclophilin</i>), AK326854; <i>TAF6</i> (<i>TFIID subunit 6</i>), Solyc10g006100.2.1.</p

Expression stability of candidate reference genes in <i>Xcv</i>-infected and mock-treated pepper plants evaluated by NormFinder.

<p>Ranking of <i>C</i>. <i>annuum</i> reference genes based on expression stability calculated by NormFinder (n = 48). New reference gene candidates are indicated in bold. The cut-off value for reliable reference genes is indicated by a dashed line. Sample groups were defined based on (a) treatment [MgCl₂, <i>Xcv</i> 85–10, 85–10Δ<i>hrcN</i> and 85-10(p<i>avrBs3</i>)] and (b) time-points of harvesting (0, 6, 10 and 24 hpi).</p

The 50 most stable tomato genes during <i>Xcv</i> infection based on microarray analyses.

<p>^a) Coefficient of variation (CV) values for the second microarray study defined as standard deviation (SD) of expression levels of a specific gene across all experiments (treatments, time points, and replicates) divided by its mean expression level (ME). Only genes with a CV value ≤ 0.12 in the first microarray study are listed.</p><p>^b) Based on the annotation by the international tomato annotation group”(ITAG, version 2.3).</p><p>The 50 most stable tomato genes during <i>Xcv</i> infection based on microarray analyses.</p

Expression stability of candidate reference genes in <i>Xcv</i>-infected and mock-treated tomato plants evaluated by geNorm.

<p>Tomato reference genes were ranked based on expression stability calculated by geNorm. New reference gene candidates are indicated in bold. <i>M</i> values represent the average expression stability of each gene (n = 48). The cut-off value for reliable reference genes is indicated by a dashed line.</p

Expression profiles of new candidate reference genes and classical housekeeping genes from pepper.

<p>Box plot graphs of Ct values for each reference gene tested in all samples (n = 48). Ct values are inversely proportional to the amount of template. The boxes indicate the 25/75 percentiles, median values are represented by black lines. Whisker caps indicate the value range, dots represent outliers. New reference gene candidates are indicated in bold.</p

Relative expression of PTI and ETI marker genes in <i>Xcv</i>-infected and mock-treated pepper leaves.

<p>Expression patterns of <i>CaLRR22</i> (a) and <i>CaTFT4</i> (b) in <i>C</i>. <i>annuum</i> ECW-30R leaves treated with 10 mM MgCl₂ (mock) or 5×10⁸ cfu/ml of <i>Xcv</i> 85–10, 85–10Δ<i>hrcN</i> and 85-10(p<i>avrBs3</i>), respectively, six hpi. qRT-PCR data were normalized with different reference gene pairs. Values are mean fold changes in mRNA levels in <i>Xcv</i>-infected relative to mock-inoculated leaves for three biological replicates. Error bars indicate SD. Letters denote statistically significant differences (Student´s <i>t</i>-test, <i>P</i> < 0.05).</p