Transport of TMV movement protein particles associated with the targeting of RNA to plasmodesmata

The cell-to-cell movement of Tobacco mosaic virus through plasmodesmata (PD) requires virus-encoded movement protein (MP). The MP targets PD through the endoplasmic reticulum (ER)/actin network, whereas the intercellular movement of the viral RNA genome has been correlated with the association of the MP with mobile, microtubule-proximal particles in cells at the leading front of infection as well as the accumulation of the protein on the microtubule network during later infection stages. To understand how the associations of MP with ER and microtubules are functionally connected, we applied multiple marker three-dimensional confocal and time-lapse video microscopies to Nicotiana benthamiana cells expressing fluorescent MP, fluorescent RNA and fluorescent cellular markers. We report the reconstitution of MP-dependent RNA transport to PD in a transient assay. We show that transiently expressed MP occurs in association with small particles as observed during infection. The same MP accumulates in PD and mediates the transport of its messenger RNA transcript to the pore. In the cellular cortex, the particles occur at microtubuleproximal sites and can undergo ER-associated and latrunculin-sensitive movements between such sites. These and other observations suggest that the microtubule network performs anchorage and release functions for controlling the assembly and intracellular movement of MP-containing RNA transport particles in association with the ER.