Age: 14 day old Tissue: stem and leaves Variety: ILL6002
Treatment protocol
Plants were inoculated by spraying approximately 5 mL Ascochyta lentis (strain: AL4) spore suspension (1 x 106 spores mL-1 ) per plant with a plastic sprayer until run-off.
Growth protocol
Seeds were sown in 8cm diameter pots (five seeds pot-1) in sterile soil. All plants were grown in a climate controlled growth cabinet (20 ± 2ºC) for 14 days before inoculation with A. lentis.
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from pooled stem and leaf samples for each genotype at each time-point (including control samples) using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA). The quantity, quality and integrity of the total RNA samples were assessed by OD260/OD280 ratios and by gel electrophoresis.
Label
Cy3, Cy5
Label protocol
Fifty μg of total RNA from each sample was reverse transcribed using Superscript II (Invitrogen) with incorporation of amino-allyl dUTP (Sigma) into the cDNA for post labeling with Cy3- or Cy5-NHS esters (Amersham). Briefly, the dried down RNA and 5 μg of oligo(dT) 18mer primer was heated at 70 °C for 10 min and chilled on ice before adding first strand buffer to a final concentration of 1×, aa-dUTP/dNTPs mix (final concentrations of 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.2 mM dTTP, 0.3 mM aa-dUTP), DTT to a final concentration of 10 mM, and 150 units Superscript II in a total reaction volume of 30 μL. Reverse transcription was performed at 42 °C for 2 h before hydrolysis of RNA template with NaOH for 15 min at 65 °C followed by neutralisation with HEPES (pH 7.0). cDNA samples were purified and labeled by applying them to QIAquick™ PCR purification columns (QIAGEN™) and washed/dried according to manufacturers instructions, before adding the appropriate volume of Cy3/Cy5 dye (resuspended in 0.1 M Na2CO3 ,pH 9.0) to the column membrane and incubating for 1 h at room temperature in the dark. After incubation, Cy3/Cy5 labeled samples were eluted, appropriate (control and treatment) Cy3 and Cy5 probes were combined, and purification was repeated.
Channel 2
Source name
leaf and stem tissue of five plants of ILL6002, 24 hour after mock inoculated wit water
Age: 14 day old Tissue: stem and leaves Variety: ILL6002
Extracted molecule
total RNA
Extraction protocol
Total RNA was extracted from pooled stem and leaf samples for each genotype at each time-point (including control samples) using the RNeasy® Plant Mini Kit (Qiagen, Valencia, CA). The quantity, quality and integrity of the total RNA samples were assessed by OD260/OD280 ratios and by gel electrophoresis.
Label
Cy3, Cy5
Label protocol
Fifty μg of total RNA from each sample was reverse transcribed using Superscript II (Invitrogen) with incorporation of amino-allyl dUTP (Sigma) into the cDNA for post labeling with Cy3- or Cy5-NHS esters (Amersham). Briefly, the dried down RNA and 5 μg of oligo(dT) 18mer primer was heated at 70 °C for 10 min and chilled on ice before adding first strand buffer to a final concentration of 1×, aa-dUTP/dNTPs mix (final concentrations of 0.5 mM dATP, 0.5 mM dGTP, 0.5 mM dCTP, 0.2 mM dTTP, 0.3 mM aa-dUTP), DTT to a final concentration of 10 mM, and 150 units Superscript II in a total reaction volume of 30 μL. Reverse transcription was performed at 42 °C for 2 h before hydrolysis of RNA template with NaOH for 15 min at 65 °C followed by neutralisation with HEPES (pH 7.0). cDNA samples were purified and labeled by applying them to QIAquick™ PCR purification columns (QIAGEN™) and washed/dried according to manufacturers instructions, before adding the appropriate volume of Cy3/Cy5 dye (resuspended in 0.1 M Na2CO3 ,pH 9.0) to the column membrane and incubating for 1 h at room temperature in the dark. After incubation, Cy3/Cy5 labeled samples were eluted, appropriate (control and treatment) Cy3 and Cy5 probes were combined, and purification was repeated.
Hybridization protocol
Microarray slides were pre-hybridised by blocking in 5X SSC, 0.1% SDS, 25% Formamide, 1% BSA for 45 min at 42ºC, rinsed in distilled water and air dried. ). A complete description of the microarray features can be found in the supplementary table. Purified combined Cy3 and Cy5 probes were resuspended in 2× hybridisation buffer (5× SSC, 0.2% SDS, 50% formamide (Sigma), 25 μg Cot1 DNA (Invitrogen), 0.4 mg polyA (Sigma), 0.5 mg salmon sperm DNA (Sigma), made up to 60.0 μL with sterile water) and was denaturated at 100 °C for 2 min before applied for hybridization to the array under a 60×25 mm Lifter slip (Grale Scientific). The slides were then incubated in a 42°C water bath for 16–20 h in a waterproof and humidified hybridisation chamber (Corning) in the dark. Hybridized slides were slides were washed for 5 min in each of 1× SSC/0.2% SDS and 0.1× SSC/0.2% SDS, and twice for 2 min in 0.1× SSC followed by a gentle rinse in distill water and drying in air flow. For each time point after inoculation, hybridisations were performed with six technical replicates (corresponding to the six complete grids on each microarray slide) and three biological replicates and included a dye-swapping (i.e. reciprocal labeling of Cy3 and Cy5) to eliminate any dye bias. In total 180 images were analysed from 30 slides, resulting in 18 data points for each time-point of each genotype.
Scan protocol
An Affymetrix® 428™ array scanner (Santa Clara, CA) was used to scan hybridized microarray slides at 660 nm for Cy5 (red laser) and 532 nm for Cy3 (green laser) at 10 µm resolution and the image was captured with Affymetrix® Jaguar™ software (v. 2.0, Santa Clara, CA). Scanned image was analysed and quantified using Imagene™ 5 (BioDiscovery, Marina Del Rey, CA) software, Images were quantified using Agilent Feature Extraction Software (version A.7.5).
Description
data table contains condensed/normalized data from three biological replicates
Data processing
Transformation of quantified spot data with GeneSight™ 3 (BioDiscovery, Marina Del Rey, CA) consisted of a local background correction (subtracting mean background intensity from mean signal intensity for each spot), omitting flagged spots (i.e. empty spots, negative spots (signal mean<background mean), poor spots and spots with mean signal intensity less than two times the local background), LOWESS normalisation of the entire population, creating a test/reference mean signal ratio, taking a shifted log (base 2), and combination of duplicated spot data. For identifying differentially expressed (DE) genes, expression ratios were filtered to eliminate genes whose mean fold change (FC) was less than 1.83-fold up or down at 95% confidence interval followed by Students t test and False Discovery Rate (FDR) multiple testing correction to retain only genes in which expression ratio changes were significant at P < 0.05. Datasets for ILL7537 and ILL6002 was built from mean expression ratios of a non-redundant list of genes differentially expressed at any time-point for any genotype at This dataset was then used to perform Figure of Merit (FOM) and k-means clustering using euclidean distance metrics with the MeV software.
