| Potato virus X (PVX) encodes a 166 kDa replicase. RNA virus replication typically occurs along cellular membranes. Since recent studies showed that PVX TGBp2 and TGBp3 proteins associate with endoplasmic reticulum(ER), experiments were conducted to find out whether PVX replicase is also ER associated. Protoplasts and plants were inoculated with PVX.GFP-TGBp2 and PVX.TGBp3-GFP. Following immunolabeling and confocal or electron microscopy, the PVX replicase, TGBp2, and TGBp3 were seen in vesicles and along the ER. Furthermore replicase, BiP (ER resident protein) and GFP labelling were mainly observed in ER and ER-derived vesicles. The ER-derived vesicles were novel virus-induced structures containing virions. Sucrose-gradient-fractionation, immunoblot analysis, and densitometry were conducted to determine if the PVX replicase, TGBp3-GFP and BiP co-fractionate with other components of the endomembrane network. PVX replicase, TGBp3-GFP, and BiP were found in the ER-containing fraction, providing further support that these PVX proteins target to the ER or ER-derived vesicles. |
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