Tobacco mosaic virus movement protein interacts with green fluorescent protein-tagged microtubule end-binding protein 1

Abstract

The targeting of the movement protein (MP) of Tobacco mosaic virus to plasmodesmata involves the actin/endoplasmic reticulum network and does not require an intact microtubule cytoskeleton. Nevertheless, the ability of MP to facilitate the cell-to-cell spread of infection is tightly correlated with interactions of the protein with microtubules, indicating that the microtubule system is involved in the transport of viral RNA. While the MP acts like a microtubule-associated protein able to stabilize microtubules during late infection stages, the protein was also shown to cause the inactivation of the centrosome upon expression in mammalian cells, thus suggesting that MP may interact with factors involved in microtubule attachment, nucleation, or polymerization. To further investigate the interactions of MP with the microtubule system in planta, we expressed the MP in the presence of green fluorescent protein (GFP)-fused microtubule end-binding protein 1a (EB1a) of Arabidopsis (Arabidopsis thaliana; AtEB1a:GFP). The two proteins colocalize and interact in vivo as well as in vitro and exhibit mutual functional interference. These findings suggest that MP interacts with EB1 and that this interaction may play a role in the associations of MP with the microtubule system during infection.

Figure 1.

Viral infection inhibits microtubule dynamics in AtEB1a:GFP-expressing N. benthamiana epidermal leaf cells. A, Expanding infection site (4 dpi) caused by TMV-MP:RFP. Zones for observation by high magnification video microscopy and confocal microscopy are indicated by white and yellow rectangles, respectively, referring to B to L. Scale bar = 200 μm. B, In cells outside the spreading infection site, microtubules are highly dynamic, and AtEB1a:GFP localizes to the tips of growing microtubule plus ends. The bottom segment shows dynamic GFP pixels within a time frame of 30 s. See also Supplemental Movie S2. Scale bar = 10 μm. C, In infected cells, AtEB1a:GFP is associated with microtubules along their length, and no dynamic behavior of the AtEB1a:GFP-associated microtubules can be detected by video time lapse microscopy. The bottom segment shows the absence of dynamic GFP pixels within a time frame of 30 s. See also Supplemental Movie S3. Scale bar = 10 μm. D to F, Dynamics of AtEB1a:GFP in cells at the front of infection, which express low levels of MP. The cell on the left shows AtEB1:GFP comets, whereas in the cell on the right, which is marked by a white rectangle, the dynamic behavior of microtubules and AtEB1a:GFP is inhibited (see Supplemental Movie S5). The area within the white rectangle is magnified in E and F showing that in the cell on the right, MP:RFP (E) colocalizes with AtEB1a:GFP (F) on microtubules and microtubule-associated spots (arrows). Scale bar = 10 μm. G, Confocal image of the leading front of a TMV-MP:RFP infection site. In the absence of AtEB1a:GFP, MP:RFP is localized to PD (arrows) and replication bodies and does not show any accumulation on microtubules. Differential interference contrast is applied to show the localization of cell walls. Scale bar = 20 μm. H, Dual-color confocal image of the leading front of infection in the presence of AtEB1a:GFP. The leading front cell is marked by an asterisk. An adjacent noninfected cell is marked by a double asterisk. The highlighted area shows parts of adjacent infected and noninfected cells and is magnified in I. Scale bar = 50 μm. I, Whereas in the lower, noninfected cell (double asterisk) microtubules are dynamic and show the typical comet-like staining pattern of AtEB1a:GFP (arrow), the dynamic behavior of microtubules and AtEB1a:GFP is inhibited in the upper, infected cell (asterisk; see also Supplemental Movie S6). In the infected cell, MP:RFP colocalizes with AtEB1a:GFP in microtubule-associated spots. The yellow color is due to colocalization of green AtEB1a:GFP and red MP:RFP signal (for individual red and green channels, see Supplemental Fig. S1). Scale bar = 10 μm. J to L, Split red (J), green (K), and merged (L) dual-color confocal image of a cell just behind the infection front. MP:RFP (J) colocalizes with AtEB1a:GFP (K) on microtubules. Scale bar = 10 μm.

Figure 2.

Expression of AtEB1a:GFP, but not expression of GFP, significantly reduces the efficiency of TMV-MP:RFP cell-to-cell movement. The statistical significance of the effect was confirmed by a Student's t test (P << 0.01).

Figure 3.

Transient expression of MP:RFP, AtEB1a:GFP, and both MP:RFP and AtEB1a:GFP. A to E4, Transient expression of MP:RFP. A, Upon expression by agroinfiltration, MP:RFP localizes to small and larger bodies as well as (weakly) to filaments. Scale bar = 10 μm. B, Transiently expressed MP:RFP also localizes to PD. Scale bar = 10 μm. C to D3, Expression of MP:RFP upon agroinfiltration in tua:GFP plants. The presence of MP:RFP (C) does not interfere with dynamic microtubule growth (yellow arrows, movie frames D1–3 and E1–4) or shrinkage (red arrows, movie frames E3 and 4). Scale bars = 5 μm. F to H7, Expression of AtEB1a:GFP. F, AtEB1a:GFP highlights growing plus ends of tua:GFP-labeled microtubules (see also Supplemental Movie S7). Scale bar = 10 μm. G1 to 3, Movie frames showing that AtEB1a:GFP also labels foci (arrow in G2) from which microtubules originate (arrow in G3) and, thus, marks the location of microtubule nucleation sites (arrowhead in G1). As the microtubule polymerizes, AtEB1a:GFP associates with the growing plus end (arrow in G3). Scale bar = 2.5 μm. H1 to 7, Loss of AtEB1a:GFP labeling is associated with microtubule shrinkage. Movie frames showing a growing microtubule with AtEB1a:GFP labeling at the tip (H1–3, arrow) that upon loss of the AtEB1a:GFP cap (H4, arrow) exhibits rapid shrinkage (H5–7, arrow). Scale bar = 2.5 μm. I to L, Transient coexpression of MP:RFP and AtEB1a:GFP. I to K, Green channel (I), red channel (J), and merged channel (K) movie frames showing transiently expressed MP:RFP and AtEB1a:GFP colocalizing to microtubules, which show no signs of dynamic activity. See also Supplemental Movie S8. Scale bar = 10 μm. L, Projection of green channel dynamic pixels within 30 s of the time-lapse movie. The absence of dynamic pixels indicates that the AtEB1a:GFP-labeled microtubules shown in I are dynamically inactive. M to Q, Protoplasts derived from an AtEB1a:GFP-transgenic BY2-cell suspension line infected with TMV-MP:RFP. M to O, Confocal images illustrating that AtEB1a:GFP and MP:RFP colocalize along the length of dynamically inhibited microtubules. Green (M) and red (N) channel images, showing the distribution of AtEB1:GFP and MP:RFP, respectively, as well as a merged image (O), are shown. Scale bar = 5 μm. P and Q, Movie data showing the lack of microtubule dynamics in TMV-MP:RFP-infected protoplasts.

Figure 4.

MP interacts with EB1a:GFP in vitro and in vivo. A, Pulldown assay with MP:His₆ as bait. Immobilized recombinant MP:His₆ binds AtEB1a:GFP as well as tubulin. MP:His₆ resin and control resin (control) were incubated with protein extracts derived from BY2-cells that were either wild type (WT) or transgenic for AtEB1a:GFP (EB1a). Following electrophoresis and blotting of the eluted proteins, the membrane was incubated with antibody against α-tubulin (left, lanes 1–4) or antibody against GFP (right, lanes 5–8; detection of AtEB1a:GFP). A slight cross reactivity of anti-GFP antibody with MP:His₆ is indicated by an asterisk. B, Compared to MP:His₆, MP^P81S:His₆ has reduced capacity to bind EB1 or tubulin in vitro. Pulldown assay with MP:His₆, MP^P81S:His₆, and control resin for binding of proteins from extracts derived from AtEB1a:GFP-transgenic BY2-cells. Eluted proteins were blotted and probed with antibody against α-tubulin (left, lanes 1–3) or antibody against GFP (right, lanes 4–6; detection of AtEB1a:GFP). The column chart displays the amount of eluted proteins averaged from four experiments. Amount of MP:His₆-bound α-tubulin and AtEB1a:GFP in the eluate was set to 100%. C, Far-western assay. Recombinant soluble MP:His₆, but not recombinant MP^P81S:His₆, binds to immobilized AtEB1a:GFP. Proteins derived from BY2-cells that were either wild type (WT) or transgenic for AtEB1a:GFP (EB1a) were separated by electrophoresis and blotted. Following denaturation and subsequent renaturation the blotted proteins were incubated with either soluble, recombinant MP^P81S:His₆ (lanes 1 and 2), MP:His₆ (lanes 3, 4, 5, 6, 13, and 14), heat-denatured MP^P81S:His₆ (lanes 7 and 8), heat-denatured MP:His₆ (lanes 9 and 10), or mock solution (lanes 11 and 12). Following extensive washing, the blot overlays were incubated with antibody against either MP (lanes 1 – 4 and 7–12) or GFP (lanes 5 and 6). Blot overlays were also incubated with secondary antibody only (lanes 13 and 14). *, Lanes 4 and 6, AtEB1a:GFP; **, all lanes except 5, 6, 13, and 14, unspecific cross reactivity with anti-MP antibody. D to G, FLIM assay. MP interacts with AtEB1a:GFP in vivo. Fluorescence intensity images acquired by FLIM are shown as gray scale pictures (left). Lifetime images (central) are represented as pseudo-color according to the color code ranging from 1 ns (blue) to 3 ns (orange). The respective lifetime values measured for AtEB1a:GFP (D) and GFP:MAP4 (F) alone or upon coexpression with MP:RFP (E) and (G), respectively, are indicated on the color scales. Coexpression with MP:RFP strongly reduces the fluorescence lifetime of AtEB1a:GFP but not of GFP:MAP4-MBD.