Fig. 3. Suppression of the intercellular spread of GUS transgene silencing in N.tabacum plants. Total low (top) and high (middle) molecular weight RNAs were extracted either from individual 8-week-old tobacco plants of the genotypes WTx6b5 (lanes 1–4), C2bx6b5 (lanes 5–8) and T19 (lanes 9–12) or from scions of the genotypes T19 (lanes 15–18) and C2bx6b5 (lanes 13 and 14) 6 weeks after they were grafted (Figure 4A–C) on rootstocks of Wtx6b5 (lanes 13–16) or C2bx6b5 (lanes 17 and 18). Equal RNA loading was monitored by methylene blue staining of the 28S RNA and the blot was probed sequentially for the mRNA of GUS (second panel) and Cmv2b (third panel). The blot of sRNAs was hybridized with a ³²P-labeled riboprobe corresponding to the minus strand of the full-length GUS mRNA. The position of a 25 nt oligonucleotide is indicated by an arrow. Note that RNA samples were prepared from the individually labeled T19 (1–4) and C2bx6b5 (I and II) plants before they were used as scions on Wtx6b5 or C2bx6b5 rootstocks, as indicated at the top of each lane. Hence, the accumulation level of Cmv2b mRNA was consistently lower in plant II of C2bx6b5 (lanes 6 and 14) than in plant I of C2bx6b5 (lanes 5 and 13) before and after grafting.