Analysis of mobile TMV Movement Protein particles and their role in PD targeting of RNA

EDUARDO JOSÉ PEÑA; ADRIAN SAMBADE; MANFRED HEINLEIN

Sydney

Congreso; Plasmodesmata 2010; 2010

The symplastic pathway allows the direct intercellular exchange of macromolecules trough plasmodesmata (PD). Several viruses use this characteristic plant communication system in order to move cell-to-cell and to spread the infection. To elucidate the cellular mechanism of RNA transport, our laboratory uses the spread of the Tobacco mosaic virus (TMV) RNA genome (vRNA) as a model. Cell-to-cell movement of 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 if expressed outside the viral context. MP also binds nucleic acids in vitro and since viral Coat Protein is dispensable for virus movement, it is believed that the particle that spreads the infection between cells contains MP in a complex with non-encapsidated vRNA.At early stages of infection, MP accumulates in mobile ER-associated particles. At later infection stages, i.e. after the viral RNA has been transported between cells, MP accumulates in large ER-associated inclusion bodies and on 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, the subcellular distribution of MPNi2519 is similar to that of wild-type MP. However, at restrictive temperature, at which TMV movement is halted, MT association is inhibited and MP 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 distribute throughout the cytoplasm along distinct tracks.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. 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. Thus, ectopically expressed MPNi2519 fails to form cytoplasmic particles at restrictive temperature and recovers wild-type distribution after 4 h at the permissive temperature. Using FRAP techniques, we investigated the targeting of MP:GFP and MPNi2519:GFP to PD. These experiments revealed that the efficiency of recovery at PD seen with MP is maintained for MPNi2519, even at the restrictive temperature, thus confirming that the Ni2519 mutation does not affect the capacity of MP to target PD.Using a previously described method for in vivo visualization of RNA molecules, we show that transiently expressed MP co-localizes with its own mRNA to 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. Current studies focus on determining whether the mobile cytoplasmic MP, mRNA, or MP/mRNA particles are targeted to PD, and on further identifying the specific MP activity compromised in MPNi2519.