Seedling Materials and Elicitor Treatment for the Microarray Experiments

Supplementary Methods

Experimental design: seedling materials and elicitor treatment for the microarray experiments. After a 48-hour treatment at 4°C, A. thaliana Landsberg erecta (Ler-0) and fls2-17 seeds were grown for 12 days on plates containing 1x MS medium (Duchefa), 1% sucrose and 1% agar under continuous light (60 µE m-2 sec-1, Biolux lamps) at 22°C. Seedlings were then transferred to liquid MS medium (two seedlings per 500 µl of medium in wells of 24-well-plates). Two days after transfer, the medium was supplied with flg22 peptide (10 µM final concentration). Plantlets were collected 30 min after treatment, frozen in liquid nitrogen and stored at -80°C.

Samples used. Samples from 4 different wells were pooled. Total RNA was extracted using RNeasy Plant Mini Kit (Qiagen). Two independent experiments were performed in an interval of two weeks.

Extract labeling. Microarray analysis was performed using ATH1 GeneChips™ (Affymetrix). 10 µg of total RNA was reverse transcribed using the SuperScript Choice system for cDNA synthesis (Life Technologies) according to the protocol recommended by Affymetrix. The oligonucleotide used for priming was 5’-ggccagtgaattgtaatacgactcactatagggaggcgg-(t)24-3’ (Genset Oligo). Double-stranded cDNA was cleaned by phenol:chloroform extraction and the aqueous phase removed by centrifugation through Phase-lock Gel (Eppendorf). In vitro transcription was performed on 1 µg of cDNA using the Enzo BioArray High Yield RNA transcript labelling kit (Enzo Diagnostics). The cRNA was cleaned using RNAeasy clean-up columns (Qiagen). The cRNA was fragmented by heating in 40 mM Tris-acetate pH 8.1, 100 mM KOAc, 30 mM MgOAc. The Affymetrix eukaryotic hybridization controls were added to the sample prior to hybridization as per manufacturer’s instructions.

Hybridization conditions. 10 µg of fragmented cRNA were hybridised (45°C, 16 hours). Hybridization was controlled by use of the GeneChip™ Eukaryotic Hybridization Control Kit (Affymetrix). Washing and staining was performed in a Fluidics Station 400 (Affymetrix) using the protocol EukGE-WS2v4 and scanned in an Affymetrix GeneChip scanner.

Microarray analysis. Chip analysis was performed using the Affymetrix Microarray Suite v5 (with a median target intensity of 500) and GeneSpring 5.1 (Silicon Genetics). Changes in gene expression were assessed using a signed Wilcoxon rank test. Genes were required to show the same direction of change in all replicate comparisons with a p-value cutoff of < 0.003 in each comparison. Any gene whose detection p-value was > 0.05 in all experimental conditions was discarded from the analysis as being unreliable data. The data were then further filtered using a one-way ANOVA (p-value < 0.05) with a Benjamini and Hochberg false discovery multiple testing correction.

Array design. Affymetrix ATH1 GeneChip