Tubule-guided cell-to-cell movement of a plant virus requires class XI myosin motors.

Cell-to-cell movement of plant viruses occurs via plasmodesmata (PD), organelles that evolved to facilitate intercellular communications. Viral movement proteins (MP) modify PD to allow passage of the virus particles or nucleoproteins. This passage occurs via several distinct mechanisms one of which is MP-dependent formation of the tubules that traverse PD and provide a conduit for virion translocation. The MP of tubule-forming viruses including Grapevine fanleaf virus (GFLV) recruit the plant PD receptors called Plasmodesmata Located Proteins (PDLP) to mediate tubule assembly and virus movement. Here we show that PDLP1 is transported to PD through a specific route within the secretory pathway in a myosin-dependent manner. This transport relies primarily on the class XI myosins XI-K and XI-2. Inactivation of these myosins using dominant negative inhibition results in mislocalization of PDLP and MP and suppression of GFLV movement. We also found that the proper targeting of specific markers of the Golgi apparatus, the plasma membrane, PD, lipid raft subdomains within the plasma membrane, and the tonoplast was not affected by myosin XI-K inhibition. However, the normal tonoplast dynamics required myosin XI-K activity. These results reveal a new pathway of the myosin-dependent protein trafficking to PD that is hijacked by GFLV to promote tubule-guided transport of this virus between plant cells.

A family of plasmodesmal proteins with receptor-like properties for plant viral movement proteins.

Plasmodesmata (PD) are essential but poorly understood structures in plant cell walls that provide symplastic continuity and intercellular communication pathways between adjacent cells and thus play fundamental roles in development and pathogenesis. Viruses encode movement proteins (MPs) that modify these tightly regulated pores to facilitate their spread from cell to cell. The most striking of these modifications is observed for groups of viruses whose MPs form tubules that assemble in PDs and through which virions are transported to neighbouring cells. The nature of the molecular interactions between viral MPs and PD components and their role in viral movement has remained essentially unknown. Here, we show that the family of PD-located proteins (PDLPs) promotes the movement of viruses that use tubule-guided movement by interacting redundantly with tubule-forming MPs within PDs. Genetic disruption of this interaction leads to reduced tubule formation, delayed infection and attenuated symptoms. Our results implicate PDLPs as PD proteins with receptor-like properties involved the assembly of viral MPs into tubules to promote viral movement.

Is the 6 kDa tobacco etch viral protein a bona fide ERES marker?

The claim that the 6 kDa viral protein (VP) of Tobacco Etch Virus is a marker for ER exit sites (ERES) has been investigated. When transiently expressed as a CFP tagged fusion construct in tobacco mesophyll protoplasts, this integral membrane protein co-localizes with both the COPII coat protein YFP-SEC24 and the Golgi marker Man1-RFP. However, when over-expressed the VP locates to larger spherical structures which co-localize with neither ER nor Golgi markers. Nevertheless, deletion of the COPII interactive N-terminal D(X)E motif causes it to be broadly distributed throughout the ER, supporting the notion that this protein could be an ERES marker. Curiously, whereas brefeldin A (BFA) caused a typical Golgi-stack response (redistribution into the ER) of the VP in leaf epidermal cells, in protoplasts it resulted in the formation of structures identical to those formed by over-expression. However, anomalous results were obtained with protoplasts: when co-expressed with the non-cycling cis-Golgi marker Man1-RFP, a BFA-induced redistribution of the VP-CFP signal into the ER was observed, but, in the presence of the cycling Golgi marker ERD2-YFP, this did not occur. High resolution images of side-on views of Golgi stacks in epidermal cells showed that the 6 kDa VP-CFP signal overlapped considerably more with YFP-SEC24 than with Man1-RFP, indicating that the VP is proportionately more associated with ERES. However, based on a consideration of the structure of its cytoplasmic tail, the scenario that the VP collects at ERES and is transported to the cis-Golgi before being recycled back to the ER, is supported.

The Golgi ribbon structure facilitates anterograde transport of large cargoes.

In mammalian cells, individual Golgi stacks fuse laterally to form the characteristic perinuclear ribbon structure. Yet the purpose of this remarkable structure has been an enigma. We report that breaking down the ribbon of mammalian cells strongly inhibits intra-Golgi transport of large cargoes without altering the rate of transport of smaller cargoes. In addition, insect cells that naturally harbor dispersed Golgi stacks have limited capacity to transport artificial oversized cargoes. These results imply that the ribbon structure is an essential requirement for transport of large cargoes in mammalian cells, and we suggest that this is because it enables the dilated rims of cisternae (containing the aggregates) to move across the stack as they transfer among adjacent stacks within the ribbon structure.

ER Import Sites and Their Relationship to ER Exit Sites: A New Model for Bidirectional ER-Golgi Transport in Higher Plants.

Per definition, ER exit sites are COPII vesiculation events at the surface of the ER and in higher plants are only visualizable in the electron microscope through cryofixation techniques. Fluorescent COPII labeling moves with Golgi stacks and locates to the interface between the ER and the Golgi. In contrast, the domain of the ER where retrograde COPI vesicles fuse, i.e., ER import sites (ERIS), has remained unclear. To identify ERIS we have employed ER-located SNAREs and tethering factors. We screened several SNAREs (SYP81, the SYP7 family, and USE1) to find a SNARE whose overexpression did not disrupt ER-Golgi traffic and which gave rise to discrete fluorescent punctae when expressed with an XFP tag. Only the Qc-SNARE SYP72 fulfilled these criteria. When coexpressed with SYP72-YFP, both the type I-membrane protein RFP-p24δ5 and the luminal marker CFP-HDEL whose ER localization are due to an efficient COPI-mediated recycling, form nodules along the tubular ER network. SYP72-YFP colocalizes with these nodules which are not seen when RFP-p24δ5 or CFP-HDEL is expressed alone or when SYP72-YFP is coexpressed with a mutant form of RFP-p24δ5 that cannot exit the ER. SYP72-YFP does not colocalize with Golgi markers, except when the Golgi stacks are immobilized through actin depolymerization. Endogenous SYP7 SNAREs, also colocalize with immobilized COPII/Golgi. In contrast, XFP-tagged versions of plant homologs to TIP20 of the Dsl1 COPI-tethering factor complex, and the COPII-tethering factor p115 colocalize perfectly with Golgi stacks irrespective of the motile status. These data suggest that COPI vesicle fusion with the ER is restricted to periods when Golgi stacks are stationary, but that when moving both COPII and COPI vesicles are tethered and collect in the ER-Golgi interface. Thus, the Golgi stack and an associated domain of the ER thereby constitute a mobile secretory and recycling unit: a unique feature in eukaryotic cells.