tomato  cultivar 7519 infected with Cg rep 2
Total RNA was extracted from tomato fruits according to Yang et al. (2008), with minor modifications: aliquots were taken from pooled samples of two inoculation areas on each tomato. The samples were ground to a fine powder in liquid nitrogen and transferred into 50-ml centrifuge tubes with 10 ml cetyltrimethylammonium bromide-RNA extraction buffer comprised of 100 mM Trisborate pH 8, 2 M NaCl, 25 mM ethylenediaminetetraacetic acid (EDTA) pH 8, 2% (w/v) CTAB, 2% (w/v) polyvinylpyrrolidone, and 2% (v/v) -mercaptoethanol. The mixture was shaken for 3 min and then incubated at 65 C for 15 min. Samples were extracted twice with an equal volume of 24:1 (v/v) chloroform:isoamyl alcohol, and the phases were separated by centrifugation at 10,000  g for 10 min. Following centrifugation, LiCl was added to a final concentration of 2.5 M and RNA was allowed to precipitate overnight at 4 C. The precipitated RNA was pelleted at 4 C for 30 min at 10,000 g, washed with 70% ethanol, and resuspended at 65 C for 3 min in SSTE buffer comprised of 10 mM Tris pH 8, 1 M NaCl, 1 mM EDTA pH 8, and 0.5% (w/v) SDS. Samples were extracted with equal volumes of 24:1 (v/v) chloroform:isoamyl alcohol, and with equal volumes of 24:1:25 (v/v) chloroform:isoamyl alcohol:water-saturated phenol, and the phases were separated by centrifugation at 10,000  g for 10 min. The RNA was ethanol-precipitated overnight, and resuspended in diethyl-pyrocarbonate-treated water (Yang et al. 2008). The RNA was further treated with Turbo DNAse (Ambion, Austin, TX, USA) and kept at 80 oC until further use.