After scanning, images were analysed in Genepix Pro v. 4.1 software (Axon Instruments, Inc., USA). Signal intensities of spots, that were lower than 2-fold of the overall mean slide background intensity were excluded from further analysis. Files were processed with EXPRESSCONVERTER ver. 1.7 and Lowess (Locfit) normalisation was performed with TIGR-MIDAS ver. 2.19.
Hybridization was carried out with the 3DNA ARRAY 50 (version 2)' labelling kit (Genisphere Inc., Hatfield, PA, USA) following the manufacturer's instructions.
(Parameters: Chamber type = OTHER: ArrayIt® Hybridization Cassette, Quantity of label target used = 100, Mass unit = Micro gram, time = 16, Tiny time unit = hours, Volume = 50, Volume unit = Micro litre, temperature = 55)
Array slides were scanned using an ‘Affymetrix 428 Array Scanner' (Affymetrix UK Ltd, High Wycombe, Buckinghamshire, UK) with the provided acquisition software (version 1.1; Affymetrix UK Ltd, High Wycombe, Buckinghamshire, UK); Cy3 was excited at 550nm and fluorescence emission was collected at 570 nm; Cy5 was excited at 649nm and signal was collected at 670 nm; Gain was adjusted according to signal intensity and was 54-60.
(Parameters: Scanning hardware = 428 [Affymetrix], Scanning software = GenePix Pro [Axon Instruments])
For each of the four biological replicates the youngest fully expanded leaves from about 40 4-week-old plants were pooled and cut into 2-mm diameter leaf slices and distributed into petri dishes containing the effector solutions for vacuum infiltration. All solutions were buffered with 1mM MES pH 5.5 (KOH) to maintain the pH of the apoplast. After vacuum infiltration (3 x 5 min) the leaf slices were incubated at 80 µmol
quanta m-2 s-1 for 8h. After 8h the leaf slices were removed from the effector solutions, quickly dried on filter paper and and flash frozen in liquid nitrogen and stored at -80°C until further use.
control: 1mM MES pH 5.5 (KOH)
citrate: 1mM citrate, 1mM MES pH 5.5 (KOH)
malate: 1mM malate, 1mM MES pH 5.5 (KOH)
50 µg total RNA was used for cDNA synthesis according to the manufacturer's instructions of the ‘3DNA ARRAY 50 (version 2)' labelling kit (Genisphere Inc., Hatfield, PA, USA); cDNA was synthesized using SuperScriptTM II Reverse Transcriptase (Invitrogen, Ltd, Paisley, Renfrewshire, UK) at 42°C; labelling was carried out with the 3DNA ARRAY 50 (version 2)' labelling kit (Genisphere Inc., Hatfield, PA, USA) following the manufacturer's instructions.
(Parameters: Amplification = none, Mass unit = Micro gram)
RNA was extracted from 200 mg frozen leaf tissue (fresh weight of tissue) using TRIzol® Invitrogen Ltd, Paisley, Renfrewshire, UK) followed by LiCl precipitation; RNA was quantified and quality controlled spectroscopically using a ‘GeneQuant pro' spectro-photometer (Amersham Pharmacia Ltd, Bucks, UK). Furthermore, RNA integrity was checked by gel electrophoresis.
(Parameters: Extracted product = total_RNA, Amplification = none)
Arabidopsis plants (ecotype Colombia 0) were grown on compost supplemented with Vermiculite at 22°C with a photoperiod of 14 h and a light intensity of 80 µE m-2s-1 for 4 weeks.
(Parameters: time unit = seconds, min temperature = 22, temperature unit = C)