Fig. 1. Short-range movement of GFP silencing. (A–D) Analysis of Agrobacterium-infiltrated tissues in line 16c. (A) Principle of the experiment. LB and RB, left and right borders of the pBin19 T-DNA, respectively; 35S, CaMV 35S promoter; Nos, nopaline synthase terminator. (B) Upon expression of transgenic and ectopic GFPs, a thin border of red tissue is visible at 5 d.p.i. (arrow). (C) At 11 d.p.i., the silenced patch accumulates both GFP siRNAs (inlay). The red border is now clearly visible (arrow). (D) Microscopic inspection of the red border (arrow) at 5 d.p.i. Bar, 1 cm. (E and F) Systemic silencing in a developing leaf of line 16c, imaged at a 6 day interval. (G–J) The red border results from movement of a silencing signal. (G) Principle of the experiment. OCS, octopine synthase terminator. (H) The red border (arrow) triggered by the construct in (G) at 10 d.p.i. (I) GUS histochemical staining of the leaf in (H). (J) Overlay of the images in (H) and (I). S, silenced; NS, non-silenced.

Fig. 3. Short-range silencing movement in wt plants. The two sequential infiltrations with 35S-GFP are performed at a 5 day interval (numbered 1 and 2, respectively). In the middle panel, the first infiltration was performed with the P19 protein, as in Figure 2A. Arrows, short-range movement of silencing. Bar, 5 mm.

Fig. 5. Extensive, but not limited, movement of silencing is conditioned by SDE1 in transgenic Arabidopsis. (A) Experimental set up. (B) A GFP142 plant transformed with atSUC2-GF-FG. (C) Typical phenotype of GFP142/sde1 plants transformed with atSUC2-GF-FG. (D and E) A leaf of a seedling from the plant in (C) imaged under UV without (D) or with (E) a band-pass filter. (F) Confocal imaging of vein-centred, silenced tissues as seen in (E). G, guard cells. Bar, 300 µm. (G–I) Silencing in a mature leaf of the plant in (C). (H) Acetic acid clearing of the leaf in (G). (I) Overlay of (G) and (H): silencing expands outside the vein network. (J) Transversal section of a mature leaf from a GFP142 plant. Right/middle split channels: GFP and chlorophyll fluorescence, respectively. Left channel: overlay. (K) Transversal section of the stem of a GFP142 plant. Phl, phloem. (L and M) Same as (J) and (K) but in a GFP142/sde1 plant transformed with atSUC2-GF-FG. (N and O) same as (J) and (K) but in a GFP142 plant transformed with atSUC2-GF-FG. v.I, class I vein; v.II, class II vein.

Fig. 7. Model for cell-to-cell movement of RNA silencing in plants. (A) Silencing can spread over 10–15 cells in the absence of amplification through movement of 21 nt primary siRNAs. (B) Extensive cell-to-cell movement requires 21 nt siRNA-induced de novo synthesis of dsRNA by the action of SDE1 and SDE3 using transgene mRNA as template. This leads to production of secondary 21 nt siRNAs that spread over a further 10–15 cells. P: plasmodesmata.

Fig. 2. The effect of viral-encoded silencing suppressors on short distance movement of RNA silencing. (A) The effect of the P1, AC2 and P19 proteins on the onset of localized signalling (arrows) at 7 d.p.i. (B) Molecular analysis of GFP, GFP mRNA and GFP siRNA levels in the co-infiltrated tissues depicted in (A). rRNA, ethidium bromide staining of ribosomal RNA provides a control for RNA loading. Coomassie staining of total protein provides a control for protein loading. Bar, 5 mm.

Fig. 4. Vein-limited recombinant PVX triggers short-range movement of RbcS silencing. (A) Viral constructs. The PVX genome has four ORFs. The RdRp is required for replication. The 25, 12 and 8 kDa and the coat (CP) proteins ensure cell-to-cell and systemic movement. Expression of GFP is from a duplicated CP promoter (red bar). (B) The viruses in (A) were inoculated in one leaf (1). At 15 d.p.i., systemic leaves were infiltrated with the Rx Agrobacterium strain (2). (C) Extensive necrosis (red outline) elicited by the Rx-CP interaction in a PVX–GFP-infected leaf. (D) Same as in (C), but on a PVX–GFP-Δ25-infected leaf. (E) Enlarged view of the inlay in (D). Necrosis is mainly restricted to the class III veins (v.III). (F) RbcS silencing due to PVX-GF:RbcS:P-Δ25 on a systemic, mature leaf. (G) Same as (F) but on a developing leaf. (H) Enlarged view of the inlay in (G). (I) Accumulation of RbcS siRNAs in PVX-RbcS- and PVX-GF:RbcS:P-Δ25-infected plants. M, mock. (J) Wt plants were first inoculated with PVX-GF:RbcS:P-Δ25 (1). The inoculated leaf was removed and, at 15 d.p.i., plants were used as rootstocks in grafts involving shoots of line 16c as scions (2). (K) Silencing of RbcS and GFP in a leaf of a 16c scion, 10 days post-grafting. Left: RbcS silencing around a few class III veins (arrows). Middle: UV imaging of the same leaf. GFP silencing is much more extensive. Right: 1 week later, GFP silencing affects the whole lamina, whereas RbcS silencing remains the same. Chlorosis due to RbcS silencing alters the red fluorescence of chlorophyll under UV, providing an internal control in these images.

Fig. 6. Effect of SDE3 on extensive signalling and northern analyses. (A) Experimental set up. (B) Typical phenotype of GFP142/sde3 plants transformed with atSUC2-GF-FG. GFP silencing is initially centred around the main vein (1) and expands in the lamina as the leaf age increase (2, 3, 4). (C and D) Mature leaves of the plant in (B). (E) Northern analysis of low and high molecular weight RNA extracted from the plants depicted in Figure 5 and this figure, 3 weeks post-germination. (F) Wt, sde1 and sde3 mutant Arabidopsis transformed with the atSUC2-SUL-LUS construct. (G) Wt Arabidopsis transformed with the atSUC2-RUB-BUR construct. The vein-centred chlorosis is similar in extent to that in (F).