The triple gene block (TGB; consisting of proteins TGB1–3) and coat protein (CP) of white clover mosaic potexvirus (WClMV) are required for cell-to-cell movement of viral RNA. Cell-to-cell spread of WClMV mutants in which the TGB open reading frames had been mutated was rescued in transgenic plants expressing specific TGB proteins (TGBPs). This indicated that there are no requirements for the synthesis in cis of viral TGBPs. These transgenic plants provided an experimental framework to explore the roles performed by the TGBPs and CP in cell-to-cell movement of WClMV RNA. Microinjection experiments established that TGB1 functions as the WClMV cell-to-cell movement protein (MP). Furthermore, combined microinjection and dual-channel confocal laser scanning microscopy provided direct evidence that infectious transcripts of WClMV move cell to cell as a ribonucleoprotein complex, consisting of single-stranded RNA, TGB1, and CP. Movement of this ribonucleoprotein complex displayed an absolute requirement for the presence of both TGB2 and TGB3. A model consistent with these findings is presented.

SummaryPlasmodesmata and the nuclear pore complex (NPC) mediate the selective trafficking of proteins and protein-nucleic acid complexes. The events underlying the translocation of endogenous and viral proteins through plasmodesmata were investigated to further explore the parallels between these cell-to-cell and intracellular communication systems. Studies performed with crosslinked KNOTTED1 (KN1) revealed that a conformational change is required for the cell-to-cell movement of this protein. Microinjection of gold-conjugated KN1 established that, as with the NPC, a combination of protein unfolding and microchannel dilation appears to be involved in protein translocation. However, during this process the extent of microchannel dilation is much less than observed for the NPC, which may reflect a physical limitation imposed by the cell wall. Co-injection of KN1-gold with unbound KN1 or cucumber mosaic virus movement protein (CMV-MP) established that the KN1-gold probe is highly effective at blocking plasmodesmal transport of KN1 and CMV-MP. This result provided the foundation for competition experiments which demonstrated that KN1 and the viral movement proteins of CMV and tobacco mosaic virus likely utilize a common receptor in the pathway for cell-to-cell transport of proteins. A combination of biochemical fractionation methods, an in vitro binding assay founded on the high affinity between KN1-gold and the putative common plasmodesmal receptor, and microinjection techniques were used to isolate plasmodesmal constituents involved in cell-to-cell transport. A model describing the steps involved in protein transport through plasmodesmata is presented.