Figure 4.

PTGS transmission from L1 rootstocks to 6b4 scions requires siRNA amplification in L1 and triggers PTGS-induced DNA methylation in 6b4. (A) Kinetics of S-PTGS establishment in leaves of 6b4 shoots grafted onto L1 and L1 rdr6 roots and grown under short day conditions. GUS activity was measured in scion leaves every week. The graph represents the average of two independent experiments involving at least eight plants each (see Table 1). S-PTGS transmission efficiency is expressed as the percentage of silenced scions, i.e. scions exhibiting GUS activity <20 fluorescent units per minute per microgram of total proteins, whereas control 6b4 plants exhibits GUS activity ∼350 fluorescent units per minute per microgram of total proteins. (B) GUS activity and GUS siRNA accumulation in scion leaves of plants of the indicated genotypes grown under short day conditions for eight weeks after grafting. GUS activity is expressed as fluorescent units per minute per microgram of total proteins quantified by Bradford. Averages and standard deviations correspond to the number of plants indicated in Table 1. RNA was extracted from a bulk of four plants. Ethidium bromide staining is shown as loading control. (C) Percentage of DNA methylation at representative CHH and CHG sites in the 5′ and 3′ regions of the GUS coding sequence in scion leaves of plants of the indicated genotypes grown under short day conditions for eight weeks after grafting. Analyses were performed at HaeIII-49, MspI-126, HaeIII-1467 and MspI-1529 restriction sites (see Supplementary Figure S2). The percentage of DNA methylation was calculated as described in Figure 1D. Mean and standard deviation bars are based on two biological replicates.