Plasmodesmal targeting and intercellular movement of potato mop-top pomovirus is mediated by a membrane anchored tyrosine-based motif on the lumenal side of the endoplasmic reticulum and the C-terminal transmembrane domain in the TGB3 movement protein

Live-cell fluorescence microscopy was used to investigate the third triple gene block protein (TGB3) of potato mop-top pomovirus and its role in assisted targeting of TGB2 to plasmodesmata (PD). Wild-type and mutant TGB3 proteins were expressed under the control of the 35 S promoter or from a virus reporter clone. Assisted targeting of TGB2 to PD was optimal when the proteins were expressed from a bicistronic plasmid in the relative ratios expected in a virus infection, suggesting that excess TGB3 inhibited PD localisation. Contrary to the generally accepted view, bimolecular fluorescence complementation showed that the TGB3 N terminus is located in the cytosol. Mutational analysis to dissect TGB3 sub domain functions showed that PD targeting was mediated by a composite signal comprising an ER-lumenal tyrosine-based motif and the C-terminal transmembrane domain. Mutation of either of these domains also abolished cell-to-cell movement of the virus. The results are discussed in the context of TGB3 membrane topology. (C) 2010 Elsevier Inc. All rights reserved.