ID_REF = VALUE = normalized log2 intensities
The signals were scanned using GenePix 4200 AL microarray scanner and image analysis was done using GenePixPro 6.1 software (both Molecular devices (Axon), Sunnyvale, CA).
Fluorescence intensities were normalized using quantile normalization function. Differences in log-intensities were tested with moderated t-test and the resulting p-values were transferred to FDR-values using limma R/Bioconductor package (Smyth, 2004). To annotate the probe targets, probe sequences were blasted against tobacco unigene database (SOL genomics network, http://solgenomics.net/) using BLAST+ command-line applications (National Institutes of Health). The log2 mutant/wt data and additional annotations for all of the Samples combined are linked on the Series record.
Labeled samples were combined pairwise, fragmented and hybridised to subarrays of tobacco 4x44K 60mer oligo array (Agilent, design ID 021113) according to instructions of Gene expression hybridisation kit (Agilent). During hybridisation, the array was rotated at 10rpm at +65C for 17h. Slide was washed with Gene expression wash buffers 1 and 2 (Agilent) with triton-X102 added.
20μg of the amino-allyl-UTP RNA were labelled with Cy3 or Cy5 (GE Healthcare), and purified (Amino allyl MessageAmp kit, Life Technologies). Incorporation of dyes to samples were assessed with Nanodrop. 825μg of each labeled RNA was used in hybridisation.
Leaf samples were ground with mortal and pestle in liquid nitrogen. RNA was extracted using home-made Trizol (Caldo et al., 2004, Plant Cell 16, 2514–2528) and purifed with LiCl precipitation. Equal amounts of RNA from 6 plants were pooled and further purified with Rneasy plant mini kit (Qiagen, Hilden, Germany) and quality of total RNA was assessed with Bioanalyzer (Agilent). mRNA was captured with oligo(dT)-primers, first and second strands of cDNA synthesized and resulting cDNA transcribed to antisense RNA to incorporate amino allyl-dUTP using Amino allyl MessageAmp kit (Life Technologies, Carlsbad, California). The quality of the resulting RNA was analysed with Bioanalyzer prior to labeling.
Tobacco plants were sap-inoculated with sap from healthy, mutPVA or wtPVA-infected N. benthamiana leaves. Samples were collected at 12 days post inoculation from the first systemically infected leaves and frozen in liquid nitrogen.
Nicotiana tabacum cv. Samsun nn were grown from seeds and planted in soil prepared by mixing peat (Kekkilä Horticulture Peat, Kekkilä Oyj, Finland) and washed sand at 5:1 ratio (v/v) . Plants were grown in growth rooms (photoperiod 16 h, temperature 18/22°C, relative humidity 70%; light intensity 200 μE m–2s–1; or temperature 20/24°C, relative humidity 40%, light intensity 250 μE m–2s–1), with. fertilizer (N:P:K = 16:9:22, Yara, Espoo, Finland) mixed in water (0.3g/l) and given in every watering.