Subcellular localization and in vivo identification of the putative movement protein of olive latent virus 2

The gene encoding the 36.5 kDa ('36K') nonstructural protein located on RNA 3 of olive latent virus 2 (OLV-2) was cloned, expressed with the Escherichi a coli pGEX-2T system and the purified protein used to raise a polyclonal a ntiserum. Immunoblot analysis of OLV-2-infected Nicotiana benthamiana plant s showed that the 36K protein accumulated in the early stages of infection and was associated with a subcellular fraction enriched in cytoplasmic memb ranes. In infected cells there were tubular structures, some containing vir us-like particles, scattered in the cytoplasm or protruding from or penetra ting the cell wall at the plasmodesmata, Immunogold labelling localized the 36K protein in the plasmodesmata of OLV-2-infected cells and showed it to be associated with virus-containing tubules, Leaf trichome cells of N. taba cum plants, transformed with a 36K-green fluorescent protein (GFP) fusion c onstruct, revealed localized fluorescence in the cell walls, possibly due t o association of the fusion protein with plasmodesmata, When the same 36K-G FP fusion protein was expressed in N. tabacum protoplasts, long tubular flu orescent structures protruded from the protoplast surface, suggesting that the 36K protein is responsible for tubule induction. The conclusion is draw n that this protein is likely to be the OLV-2 movement protein, mediating c ell-to-cell virus movement, and that movement is by a tubule-guided mechani sm.