Bacterial growth conditions and RNA extraction P. syringae pv. phaseolicola NPS3121 was inoculated in 20 ml of M9 minimal media with glucose (0.8%) as carbon source and cultured overnight at 28°C. The cells were washed with minimal medium and inoculated into 200 ml of M9 minimal medium at OD600 nm 0.1. The bacteria were grown at 18°C until the mid-log phase (OD 600nm 0.6). The VX-680 culture was then split

into two equal parts. One of which was induced with 2% of bean leaf or pod extract Smad2 phosphorylation or apoplastic fluid and to the other an equal amount of minimal medium was added as control. Each culture was incubated for 6 h at 18°C, until the beginning of late-log phase and the cells were then recovered by centrifugation. Total RNA was isolated from these cultures using Trizol reagent as recommended by the manufacturer (Invitrogen, California, USA). A second step of purification was performed using RNeasy MinElute spin columns (Qiagen, Valencia, CA) to remove any contaminating DNA. RNAs were eluted in 50 μl of diethylpyrocarbonate (DEPC)-treated water selleck kinase inhibitor and their concentration was determined using the NanoDrop spectrophotometer. RNA integrity was checked by analytical agarose gel electrophoresis. Synthesis of fluorescently labelled cDNA from P. syringae pv.

phaseolicola NPS3121 total RNA First-strand cDNA was synthesized using the CyScribe First-Strand cDNA Labelling kit (Amersham Biosciences). Thirty μg of total RNA was mixed with 3 μl of random nonamers, 0.5 μl anchored oligo (dT), 1 μl score card Spike mix control or test, and 1 μl score card utility mix (in a final volume of 11 μl). The RNA sample was heated at 70°C for 5 min. Reactions were held at room temperature for 10 min to allow the primers and the RNA template to anneal. To each reaction, the following were added: 4 μl of 5× first strand buffer, 1 μl of 1 mM Cy5-dUTP or Cy3-dUTP, ADP ribosylation factor 2 μl of dithiothreitol 100 mM, 1 μl of dUTP nucleotide mix and 100 U of Superscript II reverse transcriptase.

cDNA synthesis was performed at 42°C for 2 h in the dark and then the RNA template was hydrolyzed by incubation with 2 μl of 2.5 N NaOH at 37°C for 15 min. The reaction was neutralized by adding 10 μl of 2 M HEPES. The labelled cDNA was purified using the CyScribe GFX purification Kit as recommended by the manufacturer (Amersham Biosciences). The incorporation of Cy3 or Cy5 nucleotides into first-strand cDNA was quantified with the NanoDrop equipment and samples were finally stored at -20°C before use. Microarray hybridizations Printed microarray slides were hydrated with distilled water steam and fixed with a UV cross linker at 1200 J, then denatured in boiling water for 2 min, immersed in 95% ethanol and dried. The slides were prehybridized at 45°C for 1 h in 5× SSC, 0.1% SDS, 1% BSA. They were then washed twice for 5 min in 0.1× SSC and 30 s in 0.01× SSC, dried and used directly for hybridization.