Analysis of mobile TMV movement protein particles and their role in the targeting of RNA to plasmodesmata

EDUARDO JOSÉ PEÑA; MANFRED HEINLEIN

Aussois

Congreso; 13 encuentro de Virología Vegetal; 2011

INRA

In plants, cell-to-cell transport of viruses and of other RNA and protein macromolecules occurs through plasmodesmata (PD), dynamic channels in the cell wall that provide cytoplasmic continuity between cells and tissues. Tobacco mosaic virus (TMV) moves between cells in a non-encapsidated form and thus provides a key to the cellular mechanisms that support the intercellular trafficking of RNA and ribonucleoprotein complexes.The cell-to-cell movement of the TMV RNA genome (vRNA) is assisted by virus-encoded movement protein (MP). The MP targets PD, modifies their size exclusion limit, and has the ability to move between cells. MP also binds nucleic acids in vitro and may thus form a vRNP complex with vRNA in vivo. At early stages of infection in Nicotiana benthamiana, MP occurs in mobile ER-associated particles. At later infection stages, i.e. after the viral RNA has been transported between cells, further accumulated MP usually localizes to large ER-associated inclusion bodies and along microtubules (MT). A thermosensitive mutation in MP known as Ni2519 interferes with TMV movement at 32ºC whereas movement at permissive temperature (e.g. 22ºC) is normal. At permissive temperature, MPNi2519 subcellular distribution is similar to that of wild-type MP. However, at restrictive temperature, at which TMV movement is halted, MT association is inhibited and MPNi2519 is trapped in growing inclusion bodies. Despite these effects, MPNi2519 still localises to PD. Upon restoration of MP function at permissive temperature for 3-4 h, the inclusion bodies start to generate cytoplasmic particles that move throughout the cytoplasm along distinct tracks (1).In order to understand the identity and role of these particles in the movement of vRNA we transiently expressed fluorescent protein-tagged MP and MPNi2519. Both proteins were able to complement a movement defective TMV at 22ºC but only the wild type MP complements at restrictive temperature. We show that the accumulation and localization patterns of MP and MPNi2519 are independent of infection or other viral factors. Moreover, the temperature-sensitive behaviour of MPNi2519 is maintained upon ectopic expression (2). Fluorescence recovery after photobleaching analysis revealed that the efficiency of recovery at PD of MPNi2519 is only affected after incubation at restrictive temperature. Using a previously described method for in vivo visualization of RNA molecules, we show that transiently expressed MP co-localizes with its own mRNA in PD, suggesting that the protein mediates targeting of its mRNA to the pore. Time-lapse imaging analyses reveal mobile mRNA particles in the cytoplasm and first evidence suggests that these particles also contain MP. The sequence specificity of this targeting is being investigated. Current studies also focus on determining whether the mobile cytoplasmic particles are targeted to PD, and on further identifying the specific MP activity compromised in MPNi2519