Inhibition of TuMV intercellular movement by dominant negative mutants of secretory pathway factors.

<p><i>N. benthamiana</i> leaves were agroinfiltrated with <i>A. tumefaciens</i> containing plasmids pCambiaTuMV/6K₂:mCherry//GFP-HDEL alone (A) or with dominant negative mutant ARF1(NI) (B) or with RAB-E1d (NI) (C). All images were taken at 4 dpinf. Left panel, red fluorescence channel imaging TuMV producing 6K₂:mCherry; middle panel, green fluorescence channel imaging GFP-HDEL; and right panel, merged images. Scale bar = 200 µm. (D) Surface area of red-only fluorescent foci was calculated and expressed in fluorescence units. (E) Fluorescence intensity ratio of red over green foci was calculated and expressed in fluorescence units. Bars represent means and standard errors for 20 replicates per treatment. One-way analysis of variance calculation followed by Tukey's Multiple Comparison Test allowed analysis of differences between means: ***, P value<0.0001.</p

Myosin XI-2 is implicated in TuMV intercellular movement.

<p>(A) Quantitative RT-PCR was used to determine the relative expression ratio of target genes (myosin VIII-1, myosin VIII-2, myosin XI-2, and myosin XI-F) in <i>N. benthamiana</i> infected with the indicated TRV silencing constructs versus a TRV control not expressing a myosin fragment. (B) <i>N. benthamiana</i> leaves silenced for individual myosin genes (myosin VIII-1, myosinVIII-2, myosin XI-2, and myosin XI-F) were agroinfiltrated with pCambiaTuMV/6K₂:mCherry//GFP-HDEL and surface area of red-only fluorescent foci was calculated and expressed in fluorescence units at 4 dpinf. Wild-type TRV (TRV) or buffer (Mock) were used as controls. Bars represent means and standard errors for 10 replicates per treatment. One-way analysis of variance calculation followed by Tukey's Multiple Comparison Test allowed analysis of differences between means: NS, not significant; *, P<0.05. (C) Level of expression of non-target myosins in <i>N. benthamiana</i> leaves silenced for myosin XI-2. The internal loading control for each sample was actin-2. Expression analysis was performed on extracts from systemic leaves at 20 dpinf with TRV constructs. Bars represent means and standard errors for three replicates per treatment. One-way analysis of variance calculation followed by Tukey's Multiple Comparison Test allowed analysis of differences between means: = NS, not significant; *, P<0.05. The experiment was repeated twice for each TRV silencing construct. (D) images of pCambiaTuMV/6K₂:mCherry//GFP-HDEL in <i>N. benthamiana</i> leaves silenced for individual myosin genes (VIII1, VIII-2, XI-2, XI-F). Scale bar = 200 µm.</p

The secretory pathway is required for intercellular movement.

<p><i>N. benthamiana</i> leaves were infiltrated with DMSO (A), 10 µg/ml BFA (B) and 0.5 µM CMA (C) 4 hours before agroinfiltration with <i>A. tumefaciens</i> containing pCambiaTuMV/6K₂:mCherry//GFP-HDEL. All images were taken at 4 dpinf. Left panel, red fluorescence channel imaging TuMV producing 6K₂:mCherry; middle panel, green fluorescence channel imaging GFP-HDEL; and right panel, merged images. Scale bar = 200 µm. (D) Surface area of red-only fluorescent foci was calculated and expressed in fluorescence units. (E) Fluorescence intensity ratio of red over green foci was calculated and expressed in fluorescence units. Bars represent means and standard errors for 20 replicates per treatment. One-way analysis of variance calculation followed by Tukey's Multiple Comparison Test allowed analysis of differences between means: **, 0.001</p><p></p

Microfilament network is required for TuMV intercellular movement.

<p><i>N. benthamiana</i> leaves were infiltrated with DMSO (A), 5 µM LatB (B), and 10 µM CytD (C) 4 hours before agroinfiltration with <i>A. tumefaciens</i> containing pCambiaTuMV/6K₂:mCherry//GFP-HDEL. Images were taken at 4 dpinf. Left panel, red fluorescence channel imaging TuMV producing 6K₂:mCherry; middle panel, green fluorescence channel imaging GFP-HDEL; and right panel, merged images. Scale bar = 200 µm. (D) Surface area of red-only fluorescent foci was calculated and expressed in fluorescence units. (E) Fluorescence intensity ratio of red over green foci was calculated and expressed in fluorescence units. Bars represent means and standard errors for 20 replicates per treatment. One-way analysis of variance calculation followed by Tukey's Multiple Comparison Test allowed analysis of differences between means: NS, not significant, **, 0.001</p><p></p

TuMV 6K₂-tagged vesicles do not colocalize with endocytic markers.

<p>Three-dimensional rendering of 22 1 µm thick confocal images that overlap by 0.5 µm of <i>N. benthamiana</i> agroinfiltrated leaves. (A) shows the distribution of TuMV-induced 6K₂:mCherry-tagged structures and YFP labeled Ara7 dots. Left panels; red fluorescence channel imaging TuMV producing 6K₂:mCherry, middle panel; green fluorescence channel imaging YFP-RabF2b, and right panel; merged images. (B) shows the distribution of TuMV-induced 6K₂:GFP-tagged structures and FM4-64 labeled vesicles. Left panel shows green fluorescence channel from TuMV producing 6K₂:GFP, middle panel red fluorescence channel from FM4-64, and right panel merged images. Images were taken at 4 dpinf. Scale bar = 20 µm.</p

TuMV intercellular movement does not depend on the endocytic pathway.

<p><i>N. benthamiana</i> leaves were infiltrated with DMSO (A), 20 µM Wortmannin (B), 30 µM Tyrphostin A51 (C) and 30 µM Tyrphostin A23 (D) 4 hours before agroinfiltration with <i>A. tumefaciens</i> containing pCambiaTuMV/6K₂:mCherry//GFP-HDEL. Images were taken at 4 dpinf. Left panel, red fluorescence channel imaging TuMV producing 6K₂:mCherry; middle panel, green fluorescence channel imaging GFP-HDEL; and right panel, merged images. Scale bar = 200 µm. (E) Surface area of red-only fluorescent foci was calculated and expressed in fluorescence units. Bars represent means and standard errors for 15 replicates per treatment. One-way analysis of variance calculation followed by Tukey's Multiple Comparison Test allowed analysis of differences between means: NS, not significant.</p