Myosins VIII and XI Play Distinct Roles in Reproduction and Transport of Tobacco Mosaic Virus

Viruses are obligatory parasites that depend on host cellular factors for their replication as well as for their local and systemic movement to establish infection. Although myosin motors are thought to contribute to plant virus infection, their exact roles in the specific infection steps have not been addressed. Here we investigated the replication, cell-to-cell and systemic spread of Tobacco mosaic virus (TMV) using dominant negative inhibition of myosin activity. We found that interference with the functions of three class VIII myosins and two class XI myosins significantly reduced the local and long-distance transport of the virus. We further determined that the inactivation of myosins XI-2 and XI-K affected the structure and dynamic behavior of the ER leading to aggregation of the viral movement protein (MP) and to a delay in the MP accumulation in plasmodesmata (PD). The inactivation of myosin XI-2 but not of myosin XI-K affected the localization pattern of the 126k replicase subunit and the level of TMV accumulation. The inhibition of myosins VIII-1, VIII-2 and VIII-B abolished MP localization to PD and caused its retention at the plasma membrane. These results suggest that class XI myosins contribute to the viral propagation and intracellular trafficking, whereas myosins VIII are specifically required for the MP targeting to and virus movement through the PD. Thus, TMV appears to recruit distinct myosins for different steps in the cell-to-cell spread of the infection.     

Myosin XI-K Is required for rapid trafficking of Golgi stacks, peroxisomes, and mitochondria in leaf cells of Nicotiana benthamiana

A prominent feature of plant cells is the rapid, incessant movement of the organelles traditionally defined as cytoplasmic streaming and attributed to actomyosin motility. We sequenced six complete Nicotiana benthamiana cDNAs that encode class XI and class VIII myosins. Phylogenetic analysis indicates that these two classes of myosins diverged prior to the radiation of green algae and land plants from a common ancestor and that the common ancestor of land plants likely possessed at least seven myosins. We further report here that movement of Golgi stacks, mitochondria, and peroxisomes in the leaf cells of N. benthamiana is mediated mainly by myosin XI-K. Suppression of myosin XI-K function using dominant negative inhibition or RNA interference dramatically reduced movement of each of these organelles. When similar approaches were used to inhibit functions of myosin XI-2 or XI-F, only moderate to marginal effects were observed. Organelle trafficking was virtually unaffected in response to inhibition of each of the three class VIII myosins. Interestingly, none of the tested six myosins appears to be involved in light-induced movements of chloroplasts. Taken together, these data strongly suggest that myosin XI-K has a major role in trafficking of Golgi stacks, mitochondria, and peroxisomes, whereas myosins XI-2 and XI-F might perform accessory functions in this process. In addition, our analysis of thousands of individual organelles revealed independent movement patterns for Golgi stacks, mitochondria, and peroxisomes, indicating that the notion of coordinated cytoplasmic streaming is not generally applicable to higher plants.     

Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells

Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP). The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown. To study intracellular GFLV MP trafficking, a green fluorescent protein-MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter. We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules. During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction. In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation. However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent. Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein. These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized.     

Class VIII myosins are required for plasmodesmatal localization of a closterovirus Hsp70 homolog

The Hsp70 homolog (Hsp70h) of Beet yellows virus (BYV) functions in virion assembly and cell-to-cell movement and is autonomously targeted to plasmodesmata in association with the actomyosin motility system (A. I. Prokhnevsky, V. V. Peremyslov, and V. V. Dolja, J. Virol. 79:14421-14428, 2005). Myosins are a diverse category of molecular motors that possess a motor domain and a tail domain involved in cargo binding. Plants have two classes of myosins, VIII and XI, whose specific functions are poorly understood. We used dominant negative inhibition to identify myosins required for Hsp70h localization to plasmodesmata. Six full-length myosin cDNAs from the BYV host plant Nicotiana benthamiana were sequenced and shown to encode apparent orthologs of the Arabidopsis thaliana myosins VIII-1, VIII-2, VIII-B, XI-2, XI-F, and XI-K. We found that the ectopic expression of the tail domains of each of the class VIII, but not the class XI, myosins inhibited the plasmodesmatal localization of Hsp70h. In contrast, the overexpression of the motor domains or the entire molecules of the class VIII myosins did not affect Hsp70h targeting. Further mapping revealed that the minimal cargo-binding part of the myosin VIII tails was both essential and sufficient for the inhibition of the proper Hsp70h localization. Interestingly, plasmodesmatal localization of the Tobacco mosaic virus movement protein and Arabidopsis protein RGP2 was not affected by myosin VIII tail overexpression. Collectively, our data implicate class VIII myosins in protein delivery to plasmodesmata and suggest that more than one mechanism of such delivery exist in plants.     

Two class XI myosins function in organelle trafficking and root hair development in Arabidopsis

Multigene families encoding class XI myosins are conserved in higher plants, however, little information is available on specific functions of these ubiquitous molecular motors. We isolated gene knockout mutants for all 13 class XI myosins present in Arabidopsis (Arabidopsis thaliana) genome. Inactivation of 11 myosin genes resulted in no discernible phenotypes under the normal growth conditions. In contrast, the knockouts of the remaining two myosin genes, XI-2 (formerly MYA2) and XI-K, exhibited similar defects in root hair elongation suggesting that the myosin-driven motility plays a significant role in a polar tip growth. Strikingly, inactivation of each of these myosins also reduced trafficking of Golgi stacks, peroxisomes, and mitochondria in root hairs and in leaf epidermal cells. These results indicate that myosins XI-K and XI-2 play major and overlapping roles in the cell dynamics in Arabidopsis and highlight the redundant nature of myosin function in plants.     

Nanobody‐mediated resistance to Grapevine fanleaf virus in plants

Since their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy‐chain‐only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode‐transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell‐to‐cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs.     

Class XI Myosins Are Required for Development, Cell Expansion, and F-Actin Organization in Arabidopsis

The actomyosin system is conserved throughout eukaryotes. Although F-actin is essential for cell growth and plant development, roles of the associated myosins are poorly understood. Using multiple gene knockouts in Arabidopsis thaliana, we investigated functional profiles of five class XI myosins, XI-K, XI-1, XI-2, XI-B, and XI-I. Plants lacking three myosins XI showed stunted growth and delayed flowering, whereas elimination of four myosins further exacerbated these defects. Loss of myosins led to decreased leaf cell expansion, with the most severe defects observed in the larger leaf cells. Root hair length in myosin-deficient plants was reduced ∼10-fold, with quadruple knockouts showing morphological abnormalities. It was also found that trafficking of Golgi and peroxisomes was entirely myosin dependent. Surprisingly, myosins were required for proper organization of F-actin and the associated endoplasmic reticulum networks, revealing a novel, architectural function of the class XI myosins. These results establish critical roles of myosin-driven transport and F-actin organization during polarized and diffuse cell growth and indicate that myosins are key factors in plant growth and development.     

Is 2,3-butanedione monoxime an effective inhibitor of myosin-based activities in plant cells?

The effectiveness of 2,3-butanedione monoxime (BDM) as an inhibitor of plant myosins has been investigated. Three myosin-dependent motility phenomena in plants, namely cytoplasmic streaming in Chara corallina, light-dependent chloroplast repositioning in Elodea sp., and brefeldin A(BFA)-induced Golgi membrane dynamics in wheat (Triticum aestivum L. cv. Kite) root-tip cells were investigated. All three processes were inhibited by the sulfhydryl-modifying agent N-ethylmalemide (NEM), indicating the probable involvement of myosin as the motor protein in each case. However, none of these myosin-dependent processes were inhibited by BDM at concentrations as high as 20 mM in some instances. These results therefore question the general usefulness of BDM as an inhibitor of myosin-based activities in plant cells.     

Is 2,3-butanedione monoxime an effective inhibitor of myosin-based activities in plant cells?

Summary The effectiveness of 2,3-butanedione monoxime (BDM) as an inhibitor of plant myosins has been investigated. Three myosin-dependent motility phenomena in plants, namely cytoplasmic streaming inChara corallina, light-dependent chloroplast repositioning inElodea sp., and brefeldin A(BFA)-induced Golgi membrane dynamics in wheat (Triticum aestivum L. cv. Kite) roottip cells were investigated. All three processes were inhibited by the sulfhydryl-modifying agent N-ethylmalemide (NEM), indicating the probable involvement of myosin as the motor protein in each case. However, none of these myosin-dependent processes were inhibited by BDM at concentrations as high as 20 mM in some instances. These results therefore question the general usefulness of BDM as an inhibitor of myosin-based activities in plant cells.     

ANK, a host cytoplasmic receptor for the Tobacco mosaic virus cell-to-cell movement protein, facilitates intercellular transport through plasmodesmata

Plasmodesma (PD) is a channel structure that spans the cell wall and provides symplastic connection between adjacent cells. Various macromolecules are known to be transported through PD in a highly regulated manner, and plant viruses utilize their movement proteins (MPs) to gate the PD to spread cell-to-cell. The mechanism by which MP modifies PD to enable intercelluar traffic remains obscure, due to the lack of knowledge about the host factors that mediate the process. Here, we describe the functional interaction between Tobacco mosaic virus (TMV) MP and a plant factor, an ankyrin repeat containing protein (ANK), during the viral cell-to-cell movement. We utilized a reverse genetics approach to gain insight into the possible involvement of ANK in viral movement. To this end, ANK overexpressor and suppressor lines were generated, and the movement of MP was tested. MP movement was facilitated in the ANK-overexpressing plants, and reduced in the ANK-suppressing plants, demonstrating that ANK is a host factor that facilitates MP cell-to-cell movement. Also, the TMV local infection was largely delayed in the ANK-suppressing lines, while enhanced in the ANK-overexpressing lines, showing that ANK is crucially involved in the infection process. Importantly, MP interacted with ANK at PD. Finally, simultaneous expression of MP and ANK markedly decreased the PD levels of callose, β-1,3-glucan, which is known to act as a molecular sphincter for PD. Thus, the MP-ANK interaction results in the downregulation of callose and increased cell-to-cell movement of the viral protein. These findings suggest that ANK represents a host cellular receptor exploited by MP to aid viral movement by gating PD through relaxation of their callose sphincters.     

Intracellular trafficking of Potato leafroll virus movement protein in transgenic Arabidopsis

Intracellular trafficking of viral movement proteins (MPs) in plants has mainly been studied using Tobacco mosaic virus MP30 (TMV MP30) as a model system. Because of the limitations of TMV MP30 expression in Arabidopsis thaliana, these studies have mostly been restricted to tobacco plants. Here we present data on the analysis of transgenic Arabidopsis plants expressing Potato leafroll virus 17-kDa movement protein (MP17) fused to green fluorescent protein. MP17 localizes to secondary branched plasmodesmata (PD) in source but not to simple PD in sink tissues, where MP17 is believed to be degraded by proteolysis. To unravel the intracellular transport path of MP17, we analyzed the relevance of the cytoskeleton and of the secretory pathway on MP17 targeting. To this end, a new incubation system for in vivo analysis of immediate and long-term responses of whole Arabidopsis plants to inhibitor treatments was established. Microscopic and histochemical analysis showed that MP17 is targeted to PD in an actin- and endoplasmic reticulum-Golgi-dependent manner. In contrast, degradation of MP17 in sink tissues required intact microtubules and occurred at 26S proteasomes. Interestingly, inhibition of the 26S proteasome led to aggregation of MP17 in aggresome-like structures. Formation of these structures could be inhibited by colchicine, as was shown for aggresomes in mammalian cells.     

Myosin-Powered Membrane Compartment Drives Cytoplasmic Streaming,Cell Expansion and Plant Development

Using genetic approaches, particle image velocimetry and an inert tracer of cytoplasmic streaming, we have made a mechanistic connection between the motor proteins (myosins XI), cargo transported by these motors (distinct endomembrane compartment defined by membrane-anchored MyoB receptors) and the process of cytoplasmic streaming in plant cells. It is shown that the MyoB compartment in Nicotiana benthamiana is highly dynamic moving with the mean velocity of ~3 μm/sec. In contrast, Golgi, mitochondria, peroxisomes, carrier vesicles and a cytosol flow tracer share distinct velocity profile with mean velocities of 0.6–1.5 μm/sec. Dominant negative inhibition of the myosins XI or MyoB receptors using overexpression of the N. benthamiana myosin cargo-binding domain or MyoB myosin-binding domain, respectively, resulted in velocity reduction for not only the MyoB compartment, but also each of the tested organelles, vesicles and cytoplasmic streaming. Furthermore, the extents of this reduction were similar for each of these compartments suggesting that MyoB compartment plays primary role in cytosol dynamics. Using gene knockout analysis in Arabidopsis thaliana, it is demonstrated that inactivation of MyoB1-4 results in reduced velocity of mitochondria implying slower cytoplasmic streaming. It is also shown that myosins XI and MyoB receptors genetically interact to contribute to cell expansion, plant growth, morphogenesis and proper onset of flowering. These results support a model according to which myosin-dependent, MyoB receptor-mediated transport of a specialized membrane compartment that is conserved in all land plants drives cytoplasmic streaming that carries organelles and vesicles and facilitates cell growth and plant development.     

Contribution of Host Intracellular Transport Machineries to Intercellular Movement of Turnip Mosaic Virus

The contribution of different host cell transport systems in the intercellular movement of turnip mosaic virus (TuMV) was investigated. To discriminate between primary infections and secondary infections associated with the virus intercellular movement, a gene cassette expressing GFP-HDEL was inserted adjacent to a TuMV infectious cassette expressing 6K₂:mCherry, both within the T-DNA borders of the binary vector pCambia. In this system, both gene cassettes were delivered to the same cell by a single binary vector and primary infection foci emitted green and red fluorescence while secondarily infected cells emitted only red fluorescence. Intercellular movement was measured at 72 hours post infiltration and was estimated to proceed at an average rate of one cell being infected every three hours over an observation period of 17 hours. To determine if the secretory pathway were important for TuMV intercellular movement, chemical and protein inhibitors that blocked both early and late secretory pathways were used. Treatment with Brefeldin A or Concanamycin A or expression of ARF1 or RAB-E1d dominant negative mutants, all of which inhibit pre- or post-Golgi transport, reduced intercellular movement by the virus. These treatments, however, did not inhibit virus replication in primary infected cells. Pharmacological interference assays using Tyrphostin A23 or Wortmannin showed that endocytosis was not important for TuMV intercellular movement. Lack of co-localization by endocytosed FM4-64 and Ara7 (AtRabF2b) with TuMV-induced 6K₂-tagged vesicles further supported this conclusion. Microfilament depolymerizing drugs and silencing expression of myosin XI-2 gene, but not myosin VIII genes, also inhibited TuMV intercellular movement. Expression of dominant negative myosin mutants confirmed the role played by myosin XI-2 as well as by myosin XI-K in TuMV intercellular movement. Using this dual gene cassette expression system and transport inhibitors, components of the secretory and actomyosin machinery were shown to be important for TuMV intercellular spread.     

Getting it together in plant virus movement: cooperative interactions between bipartite geminivirus movement proteins

To move cell-to-cell and systemically infect the host, plant viruses must cross the barrier posed by the plant cell wall. Plant viruses accomplish this through strategies that alter the architecture of the infected cell, eliminating this barrier through the action of viral-encoded 'movement proteins'. Detailed studies of a number of cytoplasmically replicating viruses suggest that movement proteins interact with components of the cytoskeleton and transport systems of the plant cell to allow passage of progeny into adjacent cells. Recent work on the two movement proteins encoded by the phloem-restricted geminivirus squash leaf curl virus has defined unique aspects of nuclear transport and protein protein interaction in the movement of this nuclear-replicating virus, and suggests that post-translational phosphorylation may be important in the regulation of movement protein function.     

Inhibition of Tobacco Mosaic Virus Movement by Expression of an Actin-Binding Protein

The tobacco mosaic virus (TMV) movement protein (MP) required for the cell-to-cell spread of viral RNA interacts with the endoplasmic reticulum (ER) as well as with the cytoskeleton during infection. Whereas associations of MP with ER and microtubules have been intensely investigated, research on the role of actin has been rather scarce. We demonstrate that Nicotiana benthamiana plants transgenic for the actin-binding domain 2 of Arabidopsis (Arabidopsis thaliana) fimbrin (AtFIM1) fused to green fluorescent protein (ABD2:GFP) exhibit a dynamic ABD2:GFP-labeled actin cytoskeleton and myosin-dependent Golgi trafficking. These plants also support the movement of TMV. In contrast, both myosin-dependent Golgi trafficking and TMV movement are dominantly inhibited when ABD2:GFP is expressed transiently. Inhibition is mediated through binding of ABD2:GFP to actin filaments, since TMV movement is restored upon disruption of the ABD2:GFP-labeled actin network with latrunculin B. Latrunculin B shows no significant effect on the spread of TMV infection in either wild-type plants or ABD2:GFP transgenic plants under our treatment conditions. We did not observe any binding of MP along the length of actin filaments. Collectively, these observations demonstrate that TMV movement does not require an intact actomyosin system. Nevertheless, actin-binding proteins appear to have the potential to exert control over TMV movement through the inhibition of myosin-associated protein trafficking along the ER membrane.     

Identification of Myosin XI Receptors in Arabidopsis Defines a Distinct Class of Transport Vesicles

To characterize the mechanism through which myosin XI-K attaches to its principal endomembrane cargo, a yeast two-hybrid library of Arabidopsis thaliana cDNAs was screened using the myosin cargo binding domain as bait. This screen identified two previously uncharacterized transmembrane proteins (hereinafter myosin binding proteins or MyoB1/2) that share a myosin binding, conserved domain of unknown function 593 (DUF593). Additional screens revealed that MyoB1/2 also bind myosin XI-1, whereas myosin XI-I interacts with the distantly related MyoB7. The in vivo interactions of MyoB1/2 with myosin XI-K were confirmed by immunoprecipitation and colocalization analyses. In epidermal cells, the yellow fluorescent protein–tagged MyoB1/2 localize to vesicles that traffic in a myosin XI–dependent manner. Similar to myosin XI-K, MyoB1/2 accumulate in the tip-growing domain of elongating root hairs. Gene knockout analysis demonstrated that functional cooperation between myosin XI-K and MyoB proteins is required for proper plant development. Unexpectedly, the MyoB1-containing vesicles did not correspond to brefeldin A–sensitive Golgi and post-Golgi or prevacuolar compartments and did not colocalize with known exocytic or endosomal compartments. Phylogenomic analysis suggests that DUF593 emerged in primitive land plants and founded a multigene family that is conserved in all flowering plants. Collectively, these findings indicate that MyoB are membrane-anchored myosin receptors that define a distinct, plant-specific transport vesicle compartment.     

Differential localization of Acanthamoeba myosin I isoforms

Acanthamoeba myosins IA and IB were localized by immunofluorescence and immunoelectron microscopy in vegetative and phagocytosing cells and the total cell contents of myosins IA, IB, and IC were quantified by immunoprecipitation. The quantitative distributions of the three myosin I isoforms were then calculated from these data and the previously determined localization of myosin IC. Myosin IA occurs almost exclusively in the cytoplasm, where it accounts for approximately 50% of the total myosin I, in the cortex beneath phagocytic cups and in association with small cytoplasmic vesicles. Myosin IB is the predominant isoform associated with the plasma membrane, large vacuole membranes and phagocytic membranes and accounts for almost half of the total myosin I in the cytoplasm. Myosin IC accounts for a significant fraction of the total myosin I associated with the plasma membrane and large vacuole membranes and is the only myosin I isoform associated with the contractile vacuole membrane. These data suggest that myosin IA may function in cytoplasmic vesicle transport and myosin I-mediated cortical contraction, myosin IB in pseudopod extension and phagocytosis, and myosin IC in contractile vacuole function. In addition, endogenous and exogenously added myosins IA and IB appeared to be associated with the cytoplasmic surface of different subpopulations of purified plasma membranes implying that the different myosin I isoforms are targeted to specific membrane domains through a mechanism that involves more than the affinity of the myosins for anionic phospholipids.     

The Ins and Outs of Nondestructive Cell-to-Cell and Systemic Movement of Plant Viruses

Propagation of viral infection in host plants comprises two distinct and sequential stages: viral transport from the initially infected cell into adjacent neighboring cells, a process termed local or cell-to-cell movement, and a chain of events collectively referred to as systemic movement that consists of entry into the vascular tissue, systemic distribution with the phloem stream, and unloading of the virus into noninfected tissues. To achieve intercellular transport, viruses exploit plasmodesmata, complex cytoplasmic bridges interconnecting plant cells. Viral transport through plasmodesmata is aided by virus-encoded proteins, the movement proteins (MPs), which function by two distinct mechanisms: MPs either bind viral nucleic acids and mediate passage of the resulting movement complexes (M-complexes) between cells, or MPs become a part of pathogenic tubules that penetrate through host cell walls and serve as conduits for transport of viral particles. In the first mechanism, M-complexes pass into neighboring cells without destroying or irreversibly altering plasmodesmata, whereas in the second mechanism plasmodesmata are replaced or significantly modified by the tubules. Here we summarize the current knowledge on both local and systemic movement of viruses that progress from cell to cell as M-complexes in a nondestructive fashion. For local movement, we focus mainly on movement functions of the 30 K superfamily viruses, which encode MPs with structural homology to the 30 kDa MP of Tobacco mosaic virus, one of the most extensively studied plant viruses, whereas systemic movement is primarily described for two well-characterized model systems, Tobacco mosaic virus and Tobacco etch potyvirus. Because local and systemic movement are intimately linked to the molecular infrastructure of the host cell, special emphasis is placed on host factors and cellular structures involved in viral transport.