The role of cytoskeletal motility in viral infection is a rapidly progressing albeit relatively young field of research at the frontiers of virology and cell biology. The microtubule-dependent transport of retroviruses to nucleus 
and Herpesvirus to axon endings 
, actin-dependent formation of the virological synapses through which HIV moves between cells 
, and an actin-tail propelled transport of poxviruses 
are a few illuminating discoveries in this field. Animal and plant viruses share multiple replication mechanisms that rely on conserved features of eukaryotic cells 
. In contrast, virus cell-to-cell movement in plants occurs via
the plant-specific PD, channel-like organelles providing symplasitc continuity between adjacent cells 
. To accomplish movement through PD, plant viruses have evolved dedicated MPs that target and modify PD to mediate virus passage. One of the principal mechanisms of MP action is a tubule-guided PD transport used by a wide variety of the RNA and retroid DNA viruses 
whereby MP modifies PD by assembly into multimeric tubules through which virion movement occurs.
Most of the previous work on plant virus-cytoskeleton relationships involved chemical inhibitors 
. Although useful for an initial insight, this approach is not unlike a sledgehammer because global disruption of microtubules or microfilaments causes dramatic changes in cell physiology that are difficult to associate with specific mechanisms of virus replication or transport. Even in the cases like TMV, where genetic and other more subtle approaches were used 
, the picture is less than clear. In a large part, difficulties in reconciling work from different labs stem from the incomplete understanding of the cellular partners required for the MP function. Our recent discovery of PDLPs as host receptors 
that mediate PD targeting of the tubule-forming MPs of the nepovirus GFLV and the caulimovirus CaMV provided a unique opportunity to address the role of actomyosin motility in virus transport using both the chemical and the more specific dominant negative inhibition of myosins 
Combining these approaches, we revealed critical contributions of the myosin motors in the GFLV transport between the cells. We identified myosin XI-K as a principal driver of this process with additional contributions provided by other class XI, but not class VIII myosins. Furthermore, we obtained important new insight into myosin-driven endomembrane transport in plants by showing that myosin XI-K acts in a specific pathway within a general ER-to-Golgi-to-PM transport network.
Because GFLV transport is tubule-dependent, it was important to determine if myosin inactivation interfered with tubule formation or PD localization. Our previous work using suspension cell culture has shown that tubule assembly requires ER-to-Golgi pathway, whereas cytoskeletal systems appeared to contribute to tubule targeting 
. Here, we found that the inhibition of myosin XI-K resulted in a conspicuous nucleo-cytosolic redistribution of the GFP:2B with no detectable PD-associated tubules. Thus, tubule formation was specifically affected by myosin inactivation.
As was demonstrated recently, 2B assembles tubules at PD via
interaction with the host PDLP receptors 
that, in turn, are transported to PD along the ER-to-Golgi pathway 
. Therefore, both GFLV movement and tubule formation at PD require proper PDLP targeting. To determine if PDLP targeting was actomyosin dependent, we investigated PDLP1:GFP transport pathway using cytoskeletal inhibitors and dominant negative inhibition of the individual myosins. We found that PDLP1:GFP was present in mobile bodies whose rapid trafficking was abolished by application of LatB or BDM similarly to Golgi stacks whose transport in plants relies entirely on myosins XI 
Furthermore, we showed that the myosins XI-K and XI-2, but not XI-F, VIII-1, VIII-2, and VIII-B are required for PDLP1 delivery to PD. Inactivation of the two former myosins resulted in PDLP1:GFP redistribution in the cortical cytoplasm and inclusion bodies that were never observed in the cells where other myosins were inhibited. Given the strong correlation between disruption of PDLP targeting and GFLV movement by interference with myosins XI-K and XI-2 ( and ), we propose that the primary contribution of these myosins to virus transport is the delivery of PDLP-receptors to PD. It is important to stress that this result is also the first indication of myosin XI function in the trafficking of secretory vesicles to the PM/PD compartment.
The next question to ask was if PDLP transport occurred along a common post-Golgi secretory pathway, or represented a specialized route within this pathway driven primarily by myosins XI-K and XI-2. To address this question, we assessed a role of myosin XI-K in the targeting of markers differentially localized to: i) entire PM; ii) lipid raft subdomains within PM and PD; iii) PD neck or iv) vacuolar membrane (tonoplast). We found that proper targeting of the former three markers was not affected by myosin XI-K inhibition suggesting that the myosin XI-K-dependent PDLP targeting represents a specific route within a broad endomembrane transport network. In addition, we found that myosin XI-K is required for the normal tonoplast reshaping via transient invaginations.
It was previously demonstrated that PD targeting of the closteroviral Hsp70 homolog requires myosins VIII 
, although significance of this process for virus movement was not addressed. It was also found that myosin XI-2 knockdown reduced TMV movement 
, but this effect was not linked to a specific mechanism. Together with our previous work 
, this study provides a basis for an advanced mechanistic model of myosin-dependent virus movement.
According to this model (), the GFLV MP and its host receptor, PDLP, traffic to the cell periphery along distinct pathways. 2B reaches PD by diffusion or by association with microtubules 
. The transport route employed by PDLP is dependent on the myosins XI with XI-K playing the principal role. At PD, MP binds PDLP for anchorage and tubule assembly. Because transient inhibition of PDLP traffic to PD reduces virus movement (), it seems that steady-state supply of this receptor is required for the formation of tubules that restructure PD. Finally, assembled GFLV virions enter tubules and translocate into adjacent cells. It remains to be determined if virion transport to and through tubules involves cytoskeleton-dependent motility.
Model for PDLP and GFLV MP 2B targeting to PD and tubule formation.
The emerging picture of the plant-virus interactions with myosin motors is complex and nuanced. It appears that closteroviral Hsp70 homolog directly recruits myosins VIII for virion delivery to PD 
, whereas tenuiviral MP uses myosin VIII-assisted vesicular transport for the same task 
. Currently, the PD-directed transport of these viral proteins remains the only experimentally supported function of the class VIII myosins. On the other hand, TMV MP targeting to PD does not require myosins 
, whereas myosin XI-2 facilitates TMV movement likely via
delivering the ER-associated viral replication complexes to PD 
. This latter hypothesis resonates well with the role of myosins XI-2 and XI-K in ER transport 
. In the case of GFLV presented here, the virus relies on the myosins XI-K and XI-2 for the trafficking of the host MP receptor PDLP to PD.
In addition to important insight into virus-cytoskeleton interactions, our work suggests novel functions of the myosins XI-K and XI-2 in vesicle trafficking and vacuole remodelling. These myosins were previously shown to drive the trafficking of Golgi stacks, peroxisomes, and mitochondria 
, as well as the ER flow 
. Here we show that these same myosins are also involved in PDLP delivery to PD via
a specific endomembrane transport pathway, as well as in remodelling of the vacuolar membrane. Further inquiries into the mechanisms of myosin-dependent transport are certain to deepen our understanding of the cell interior dynamics and the importance of these processes for virus movement.