Fully-expanded leaves at the third or fourth position from the top of each shoot were used for treatment
third or forth leaveswere mock inoculated
14 hours day/10 hours night. Light intensity: 500 umol m-2. Light source: high pressure sodium and metal halide. Temperature: 25°C. Relative humidity: 85%.
Total RNA was extracted from each replicate sample individually. The leaves were ground in liquid nitrogen and homogenized in extraction buffer (2% Hexadecyltrimethyl Ammonium Bromide 1% sodium dodecyl sulfate, 2.5M NaCl, 0.5M Tris, 50mM EDTA, 5% beta-mercaptoethanol, and 3% polyvinyl poly-pyrrolidone. Following a 30-min incubation at -80°C, the samples were thawed and centrifuged. The supernatant was supplemented with 1/30 volume of 3 M sodium acetate (pH5.2) and 1/10 volume of ethanol, incubated on ice and centrifuged three times at 7000g, 30 min 4 ºC, each time transferring the supernatant into a clean tube. The supernatant was supplemented with 1/9 volume of 3 M sodium acetate (pH5.2) and iso-propanol to a final concentration of 33%. Following 60-min incubation at -20°C, the RNA was collected by centrifugation at 12,000g, 30 min, 4 ºC. The RNA was then supplemented with 1/3 volume of 8 M LiCl and incubated overnight at 4 °C. RNA was then collected by centrifugation at 12,000g, 30 min, 4 ºC, washed with 75% ethanol, and resuspended in 40 ul DEPC water. Ten micrograms of total RNA were treated with Turbo DNase I (Ambion) and purified with RNA purification kit (Qiagen) according to manufacturer’s instructions.
The cDNA synthesis and antisense cRNA amplification/biotin labeling was performed using the One-Cycle Target Labeling Kit (Catalog number: 900493, Affymetrix, Santa Clara, California)
cRNA was cleaned up by the Affymetrix sample cleanup module which in included in the One-Cycle Target Labeling kit. Fifteen ug of cRNA at concentration of 1.301ug/ul were used for hybridization. Prior to hybridization, cRNA was heated at 94ºC for 35 min, and was fragmented to 35 to 200 nucleotides. After adding hybridization buffer to the fragmented samples, hybridization cocktails were boiled for 5 min, incubated for 5 min at 45ºC, then centrifuged for 5 minutes prior to loading per standard one-cycle protocol. Hybridization took place in an Affy hybridization oven model 640, for 16h at 45ºC. All washing took place on the fluidics station (as per the recommended fluidics protocol) with non-stringent wash buffer (6X SSPE and 0.01% Tween-20) and Stringent wash buffer (100mM MES, 0.1M NaCl, 0.01% Tween-20). Fluorescence was amplified by first adding streptavidin-phycoerythrin (SAPE) stain, then adding a biotinylated antibody (anti-streptavidin) solution, followed by another SAPE staining (as per standard one-cycle protocol). The fluidics protocol Midi_Euk2v3 was used (as per V. vinifera microarray package insert) on fluidics station model 450.
Affymetrix Scanner 3000 7G was used to collect at resolution of 1.56um with emission filter at a wavelength of 570nm and excitation wavelength of 532nm.
Gene expression data from V. aestivalis 'Norton ' at 0 hour post mock inoculation
Raw intensity data were processed using the GeneChip Operating Software Version 1.2 (GCOS 1.2; Affymetrix, 2001). Background correction and expression value calculation were performed as described by Affymetrix (2002). Normalization was done by global scaling, with a target intensity value of 150 (Affymetrix, 2001). Probe sets with missing values were deleted across all replicates and species to allow comparisons among datasets