Mobile 24 nt Small RNAs Direct Transcriptional Gene Silencing in the Root Meristems of Arabidopsis thaliana  Charles W. Melnyk, Attila Molnar, Andrew Bassett, David C. Baulcombe  Current Biology  Volume 21, Issue 19, Pages 1678-1683 (October 2011) DOI: 10.1016/j.cub.2011.08.065 Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 1 TGS Is Associated with a DCL3-Dependent Graft-Transmissible Signal (A) Schematic diagram of the silencing inducer and the target transgene constructs (adapted from [12]). The target transgene (T) contains a meristem-active enhancer (shown in gray and in white) placed upstream of a minimal promoter (black) and GFP-coding region. The silencer transgene (S) harbors an inverted DNA repeat of distal enhancer sequences (gray) under the control of the 35S promoter. The hairpin RNA transcribed from the S locus is diced into sRNAs, which induce de novo methylation of the target enhancer in trans leading to transcriptional gene silencing of the GFP reporter gene. The star represents where the primers used for sRNA northern blotting hybridize (Figure 2B). (B–F) Transmission of the TGS signal by grafting. Grafts were made between wild-type and mutant plants containing the unlinked target (T) and silencer (S) homozygous transgenes to test the movement of a transcriptional gene silencing signal (right). TTSS dcl3 plants are impaired in the production of 24 nt sRNA due to an early stop mutation in DCL3 (dcl3-5 [12]). Root fluorescence (left) and brightfield (middle) images were taken 38 days after grafting. A TT root is presented in each fluorescent panel as an exposure control (white box). The scale bar in (B) represents 0.5 cm and applies to all panels. Current Biology 2011 21, 1678-1683DOI: (10.1016/j.cub.2011.08.065) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 2 Molecular Analyses of Mobile TGS (A) GFP mRNA levels in grafted plants. Transcript levels were quantified by qPCR for two independent biological replicates that were averaged using a weighted mean based on the levels of UBC9 transcript. Shoot/root notation is used, with the tissue sampled in bold. (B) Detection of sRNAs in grafted plants by northern hybridization. Grafting combinations are indicated on the top. The primary sRNA probe detects sRNAs that are generated from the silencer locus. AtREP2 sRNAs are DCL3 dependent and this probe tested for the accumulation of endogenous 24 nt sRNAs. Hybridization with miR319 and U6 probes are shown as loading controls. s, shoot; r, root. Current Biology 2011 21, 1678-1683DOI: (10.1016/j.cub.2011.08.065) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 3 Analysis of sRNAs Associated with Mobile TGS by Deep Sequencing sRNA libraries were generated from grafted Arabidopsis and aligned at the first nucleotide to 500 base pairs of the T enhancer where it is targeted by the S transgene. The positive or negative y axis shows the number of reads at each position on either the plus or minus strand. The line below the x axis represents portions of the T enhancer (white) and the region targeted by the S transgene (black). Shoot/root notation is used, with the tissue sampled in bold. See Figure S3 for an independent biological replicate. Table S2 summarizes the sRNA library details. (A) TT shoot grafted onto TT root. (B) TTSS shoot grafted onto TTSS root. (C) TTSS shoot grafted onto TT root. (D) TTSSd3 shoot grafted onto TTSSd3 root. (E) TTSSd3 shoot grafted onto TT root. Current Biology 2011 21, 1678-1683DOI: (10.1016/j.cub.2011.08.065) Copyright © 2011 Elsevier Ltd Terms and Conditions

Figure 4 24 nt sRNAs Direct DNA Methylation at the Target Locus DNA methylation analysis at the target (T) promoter in roots by bisulphite sequencing. 24–31 clones of each genotype were sequenced and analyzed. The percentage of methylated cytosines and their sequence context is indicated (black, CG; blue, CHG; red, CHH). (A) Total amount of DNA methylation in grafted roots at the target (T) enhancer region. (B–F) DNA methylation in grafted roots (in bold) across the target (T) enhancer region. The line below the x axis represents portions of the T enhancer (white) and the region targeted by the S transgene (black). Current Biology 2011 21, 1678-1683DOI: (10.1016/j.cub.2011.08.065) Copyright © 2011 Elsevier Ltd Terms and Conditions