Tobacco mosaic virus movement protein enhances the spread of RNA silencing

Abstract

Eukaryotic cells restrain the activity of foreign genetic elements, including viruses, through RNA silencing. Although viruses encode suppressors of silencing to support their propagation, viruses may also exploit silencing to regulate host gene expression or to control the level of their accumulation and thus to reduce damage to the host. RNA silencing in plants propagates from cell to cell and systemically via a sequence-specific signal. Since the signal spreads between cells through plasmodesmata like the viruses themselves, virus-encoded plasmodesmata-manipulating movement proteins (MP) may have a central role in compatible virus:host interactions by suppressing or enhancing the spread of the signal. Here, we have addressed the propagation of GFP silencing in the presence and absence of MP and MP mutants. We show that the protein enhances the spread of silencing. Small RNA analysis indicates that MP does not enhance the silencing pathway but rather enhances the transport of the signal through plasmodesmata. The ability to enhance the spread of silencing is maintained by certain MP mutants that can move between cells but which have defects in subcellular localization and do not support the spread of viral RNA. Using MP expressing and non-expressing virus mutants with a disabled silencing suppressing function, we provide evidence indicating that viral MP contributes to anti-viral silencing during infection. Our results suggest a role of MP in controlling virus propagation in the infected host by supporting the spread of silencing signal. This activity of MP involves only a subset of its properties implicated in the spread of viral RNA.

Figure 1. MP enhances the spread of GFP silencing in systemic leaves.

(A) Systemic GFP silencing in N. benthamiana line 16c was induced by agroinfiltration of a GFP expressing construct into lower leaves. Silencing in upper, non-infiltrated leaves was analyzed. (B) A plant under UV illumination in which GFP silencing has spread from infiltrated leaf (arrow) into non-infiltrated, upper leaves (asterisks). An example of an upper leaf showing the pattern of GFP silencing spreading from class I–III veins into adjacent tissue is shown on the right. (C) Efficiency of cell-to-cell spread of GFP silencing during 36 h in segments of upper, non-infiltrated leaves that where heterozygous for GFP and MP (MP/-; GFP/-, top row) or heterozygous for GFP alone (-/-; GFP/-, second row). The first and second panels in each row show the silencing pattern at 8 dpi and 36 h later (10 dpi), respectively. The third panels show overlays of the first two panels. Blue false color represents the silenced area at 8 dpi (as shown in first panels) and red enhanced color indicates the increase of silenced areas after the 36 h incubation period (as shown in second panels). Panels four and five in each row show similar overlays made from different source images. At 10 dpi the area of newly silenced tissue (shown in red artificial color) in these plants was considerably greater in the presence than in the absence of MP. (D) and (E) Quantification of GFP silencing in upper leaves of 10 plants each of either MP-expressing plants (MP/-; GFP/-) and control plants (-/-; GFP/-). (D) Percentage of silenced leaf area as revealed by the number of green pixels (representing non-silenced area) and red pixels (representing silenced area) in digital leaf images. (E) Percentage of GFP fluorescence in leaf extracts (compared to GFP fluorescent control leaves = 100%).

Figure 2. MP enhances cell-to-cell spread of local GFP silencing.

(A) Examples showing the rim of GFP-silenced cells surrounding the agroinfiltrated leaf areas. Magnifications of the highlighted areas (dashed boxes) are shown at the lower right. (B) Relative average width of the silenced rim in plants expressing no MP (-/-; GFP/-) or MP (MP/-; GFP/-). The relative average widths of the silenced area around 20 agroinfiltrated patches each are shown. Error bars show the standard deviations. The mean value for the control infiltration (-/-; GFP/-) was set to 100%. The statistical significance of the MP effect was confirmed by ANOVA with a Tukey's HSD (P<0.01).

Figure 3. MP enhances cell-to-cell spread of local GFP silencing upon transient expression in agroinfiltrated leaves.

(A) Transiently expressed MP enhances cell-to-cell spread of local GFP silencing. A positive effect on the spread of local silencing is also seen upon transient expression of dMP. The relative average widths of the silenced area around agroinfiltrated patches are shown. Error bars show the standard deviations. The value derived from the control infiltration (empty vector) was set to 100%. The statistical significance of the measured differences was confirmed by ANOVA with a Tukey's HSD test (Empty vector (n = 18) vs. MP (n = 36), P<0.01; Empty vector vs. dMP (n = 36), P<0.01). (B) The enhancing effect of transiently expressed MP on the short-distance cell-to-cell spread of GFP silencing is not affected by mutations that interfere with accumulation at microtubules (TAD5) or with accumulation at both microtubules and plasmodesmata (PS1). The relative average widths of the locally silenced areas surrounding agroinfiltrated patches are shown. All tested mutant MP variants enhance the short-distance spread of GFP silencing as wild type MP. Error bars show the standard deviations. The statistical significance of the measured differences was confirmed by ANOVA with a Tukey's HSD test (Empty vector (n = 48) vs. MP (n = 54), P<0.01; Empty vector vs. TAD5 (n = 42), P<0.01; Empty vector vs. PS1 (n = 48), P<0.01). (C) Expression of CP does not enhance the local spread of silencing. Relative widths of the leaf area surrounding agroinfiltrated patches are shown. Error bars show the standard deviations. ANOVA with a Tukey's HSD was performed (empty vector (n = 36) vs. CP (n = 36); P = 0.41).

Figure 4. Level of MP expression in transgenic plants and upon agroinfiltration.

(A) Westernblot analysis of protein extracts produced at 3 dpi. MP is detected with specific antibody. The plant genotype and the proteins expressed by transient expression are stated above each lane. Coomassie staining of the same membrane reveals the amount of Rubisco, which was used for normalization in (B). Transiently expressed MP and MP expressed from the transgene accumulate to similar levels in the cells. PS1 and TAD5 expression levels are lower. (B) Quantification of MP levels based on the Westernblot shown in (A). The differences in gel loading as revealed by Coomassie staining (A, lower panel) were used for normalization. The normalized level of MP produced from the transgene was set to 100.

Figure 5. Expression of MP does not interfere with the silencing pathway.

(A), The levels of GFP mRNA, GFP siRNA, and miR166 (loading control) at different time points (0, 2, 5, and 8 dpi) in agroinfiltrated tissues. The patterns of GFP mRNA and siRNA in MP- or dMP-expressing tissue are similar as in tissues expressing empty vector. GFP mRNA levels peaks at 2 dpi due to the expression of GFP from the silencing inducer construct. Between 2 and 5 dpi a strong decrease below the original levels (0 dpi) of GFP mRNA is observed. This indicates that in addition to the GFP expressed from the silencing inducer construct the GFP expressed from the transgene is silenced. Hc-Pro, a known silencing suppressor, prevents degradation of GFP mRNA, which peaks at 5 dpi. GFP siRNAs appear at 5 dpi. Expression of Hc-Pro delays the accumulation of siRNAs. (B), Quantification of mRNA levels. mRNA patterns reveal no effects of both MP and dMP on GFP silencing. The values for 0 dpi were set to 100%. Black bars: 0 dpi; dark grey bars: 2 dpi; light gray bars: 5 dpi; white bars: 8 dpi. (C) Quantification of siRNA levels. In the presence of MP, the GFP siRNA levels are reduced by about 50% compared to the empty vector control, whereas dMP causes a slight increase. The values for 0 dpi were set to 0. Dark grey bars: 2 dpi; light gray bars: 5 dpi; white bars: 8 dpi.

Figure 6. PS1:GFP moves cell-to-cell.

Single epidermal cells from N. benthamiana leaves were infiltrated with agrobacteria co-transformed with plasmids for specific co-expression in the same cells of the red fluorescent cell-autonomous nuclear marker RMS2 together with either MP:GFP (A to F) or PS1:GFP (G to L). (Left panels: DIC; middle panels: RFP channel; right panels: GFP channel). (A–F), Movement of MP:GFP. Movement is indicated by the presence of punctate MP:GFP fluorescence (plasmodesmata) in the cell wall of a cell distant to the transfected cell (arrowheads). (A) DIC image of epidermis. The transfected cell is indicated by a yellow, dotted line. Area delimited by dashed line is magnified in (D). (B) RFP channel image showing transfected cell as indicated by presence of the red fluorescent nuclear protein RMS2. Area delimited by dashed line is magnified in (E). (C) GFP channel image. Area delimited by dashed line is magnified in (F), indicating the presence of MP:GFP at plasmodesmata of non-transfected cells (arrowheads). (G–L) Movement of PS1. Movement is indicated by the presence of diffuse PS1:GFP fluorescence in the cell adjacent to the transfected cell (arrowheads). (G) DIC image of epidermis. The transfected cell is indicated by a yellow, dotted line. Area delimited by dashed line is magnified in (J). (H) RFP channel image showing transfected cell as indicated by presence of the red fluorescent marker RMS2. Area delimited by dashed line is magnified in (K). (I) Green channel image showing presence of PS1:GFP. Area delimited by dashed line is magnified in (L) indicating the presence of PS1:GFP in a non-transfected cell (arrowheads). Size bars: C, 50 µm; F, 10 µm; I, 50 µm; L, 10 µm.

Figure 7. MP enhances silencing during infection.

Green fluorescent infection sites of GFP-expressing TMV-derivatives in inoculated leaves of wild type and MP-transgenic (MP+) N. benthamiana plants. (A) Anti-viral silencing is exposed in infection sites caused by silencing suppressor-defective virus TMV-126k^m-GFP as seen by the disappearance of GFP fluorescence in the center of the infection sites. Scale bars: 5 mm. (B) Deletion of the MP gene from the virus (ΔM) abolishes induced silencing. In contrast to infection sites of TMV-126k^m-GFP (A), infection sites of TMV-126k^m-ΔM-GFP do not show any silencing. Scale bars: 5 mm.