Like many other positive strand RNA viruses, Turnip mosaic virus (TuMV) infection leads to the formation of viral replication vesicles that have their origin at endoplasmic reticulum (ER) exit sites. Once released from the ER, the viral replication vesicles mature and move to plasmodesmata (PDs) for cell-to-cell movement. While it is known that the membrane-associated viral protein 6K2 plays an important role in these processes, the contribution of the host proteins is poorly defined. Here, we observed a considerable delay in the development of the TuMV infection in the Arabidopsis thaliana mutant root hair defective3 (rhd3). RHD3 is an ER fusogen required for efficient fusion of different ER membrane tubules. Assessment of virus production and accumulation via Western Blot analyses indicated the significant role of RHD3 in the replication as well as the intercellular movement of TuMV. Time-course monitoring of the TuMV disease development using fluorescent and transmission electron microscopy revealed that RHD3 is required for the formation, maturation as well as intercellular movement of the viral replication vesicles. 6K2 physically interacted with RHD3 as demonstrated by yeast two-hybrid and Bimolecular Fluorescence Complementation analyses. On the other hand, 6K2GV, a Golgi-localized non-productive 6K2 mutant, failed to interact with RHD3. Furthermore, 6K2 relocated RHD3 from the ER to the viral replication vesicles, which was not the case with 6K2GV. We conclude that the formation, maturation and intercellular movement of TuMV replication vesicles require RHD3.
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