Phosphorylation of viral movement proteins--regulation of cell-to-cell trafficking

In plants, proteins and nucleoprotein complexes can traffic from cell to cell, via plasmodesmata. Studies based on viral movement proteins (MP) have revealed that such trafficking events are likely to be regulated at the level of protein phosphorylation. Plasmodesmal-associated protein kinases could play a central role in plant defense, in addition to regulating the translatability of endogenous MP-mRNA complexes that function at a supracellular level.     

Simple, but not branched, plasmodesmata allow the nonspecific trafficking of proteins in developing tobacco leaves.

Leaves undergo a sink-source transition during which a physiological change occurs from carbon import to export. In sink leaves, biolistic bombardment of plasmids encoding GFP-fusion proteins demonstrated that proteins with an Mr up to 50 kDa could move freely through plasmodesmata. During the sink-source transition, the capacity to traffic proteins decreased substantially and was accompanied by a developmental switch from simple to branched forms of plasmodesmata. Inoculation of sink leaves with a movement protein-defective virus showed that virally expressed GFP, but not viral RNA, was capable of trafficking between sink cells during infection. Contrary to dogma that plasmodesmata have a size exclusion limit below 1 kDa, the data demonstrate that nonspecific "macromolecular trafficking" is a general feature of simple plasmodesmata in sink leaves.     

Plasmodesmata: composition,structure and trafficking

Plasmodesmata are highly specialized gatable trans-wall channels that interconnect contiguous cells and function in direct cytoplasm-to-cytoplasm intercellular transport. Computer-enhanced digital imaging analysis of electron micrographs of plasmodesmata has provided new information on plasmodesmatal fine structure. It is now becoming clear that plasmodesmata are dynamic quasi-organelles whose conductivity can be regulated by environmental and developmental signals. New findings suggest that signalling mechanisms exist which allow the plasmodesmatal pore to dilate to allow macromolecular transport. Plant viruses spread from cell to cell via plasmodesmata. Two distinct movement mechanisms have been elucidated. One movement mechanism involves the movement of the complete virus particle along virus-induced tubular structures within a modified plasmodesma. Apparently two virus-coded movement proteins are involved. A second movement mechanism involves the movement of a non-virion form through existing plasmodesmata. In this mechanism, the viral movement protein causes a rapid dilation of existing plasmodesmata to facilitate protein and nucleic acid movement. Techniques for the isolation of plasmodesmata have been developed and information on plasmodesma-associated proteins is now becoming available. New evidence is reviewed which suggests that plasmodesmatal composition and regulation may differ in different cells and tissues.     

Plasmodesmal-associated protein kinase in tobacco and Arabidopsis recognizes a subset of non-cell-autonomous proteins

Cell-to-cell communication in plants involves the trafficking of macromolecules through specialized intercellular organelles, termed plasmodesmata. This exchange of proteins and RNA is likely regulated, and a role for protein phosphorylation has been implicated, but specific components remain to be identified. Here, we describe the molecular characterization of a plasmodesmal-associated protein kinase (PAPK). A 34-kD protein, isolated from a plasmodesmal preparation, exhibits calcium-independent kinase activity and displays substrate specificity in that it recognizes a subset of viral and endogenous non-cell-autonomous proteins. This PAPK specifically phosphorylates the C-terminal residues of tobacco mosaic virus movement protein (TMV MP); this posttranslational modification has been shown to affect MP function. Molecular analysis of purified protein established that tobacco (Nicotiana tabacum) PAPK is a member of the casein kinase I family. Subcellular localization studies identified a possible Arabidopsis thaliana PAPK homolog, PAPK1. TMV MP and PAPK1 are colocalized within cross-walls in a pattern consistent with targeting to plasmodesmata. Moreover, Arabidopsis PAPK1 also phosphorylates TMV MP in vitro at its C terminus. These results strongly suggest that Arabidopsis PAPK1 is a close homolog of tobacco PAPK. Thus, PAPK1 represents a novel plant protein kinase that is targeted to plasmodesmata and may play a regulatory role in macromolecular trafficking between plant cells.     

Replication and trafficking of a plant virus are coupled at the entrances of plasmodesmata

Plant viruses use movement proteins (MPs) to modify intercellular pores called plasmodesmata (PD) to cross the plant cell wall. Many viruses encode a conserved set of three MPs, known as the triple gene block (TGB), typified by Potato virus X (PVX). In this paper, using live-cell imaging of viral RNA (vRNA) and virus-encoded proteins, we show that the TGB proteins have distinct functions during movement. TGB2 and TGB3 established endoplasmic reticulum–derived membranous caps at PD orifices. These caps harbored the PVX replicase and nonencapsidated vRNA and represented PD-anchored viral replication sites. TGB1 mediated insertion of the viral coat protein into PD, probably by its interaction with the 5′ end of nascent virions, and was recruited to PD by the TGB2/3 complex. We propose a new model of plant virus movement, which we term coreplicational insertion, in which MPs function to compartmentalize replication complexes at PD for localized RNA synthesis and directional trafficking of the virus between cells.     

Peptide antagonists of the plasmodesmal macromolecular trafficking pathway

In plants, cell-to-cell transport of endogenous and viral proteins and ribonucleoprotein complexes (RNPCs) occurs via plasmodesmata. Specificity of this transport pathway appears to involve interaction between such proteins/RNPCs and plasmodesmal chaperones/receptors. Here, KN1 and the cucumber mosaic virus movement protein (CMV-MP) were used, in a modified phage-display screening system, to identify peptides capable of interacting with proteins present in a plasmodesmal-enriched cell wall fraction. Binding/competition assays and microinjection experiments revealed that these phage-displayed peptides and homologous synthetic oligopeptides function as ligand-specific antagonists of macromolecular trafficking through plasmodesmata. A KN1 peptide antagonist had the capacity to interact with a motif involved in the dilation of plasmodesmal microchannels. Although KN1 could still achieve limited movement through plasmodesmata when this SEL motif was blocked, KN1-mediated transport of KN1-sense RNA was fully inhibited. These findings provide direct support for the hypothesis that KN1 requires, minimally, two physically separated signal motifs involved in the dilation of, and protein translocation through, plasmodesmal microchannels, and provide direct proof that plasmodesmal dilation is a prerequisite for the cell-to-cell transport of an RNPC.     

Getting the message across: how do plant cells exchange macromolecular complexes?

A major pathway for macromolecular exchange in plants involves plasmodesmata (PD), the small pores that connect adjoining cells. This article considers the nature of macromolecular complexes (MCs) that pass through PD and the pathways and mechanisms that guide them to the PD pore. Recent cell-biological studies have identified proteins involved in the directional trafficking of MCs to PD, and yeast two-hybrid studies have isolated novel host proteins that interact with viral movement proteins. Collectively, these studies are yielding important clues in the search for components that compose the plant intercellular MC trafficking pathway. Here, they are placed in the context of a functional model that links the cytoskeleton, chaperones and secretory pathway in the intercellular trafficking of MCs.     

A novel function for a ubiquitous plant enzyme pectin methylesterase: the host-cell receptor for the tobacco mosaic virus movement protein

Plant virus-encoded movement proteins promote viral spread between plant cells via plasmodesmata. The movement is assumed to require a plasmodesmata targeting signal to interact with still unidentified host factors presumably located on plasmodesmata and cell walls. The present work indicates that a ubiquitous cell wall-associated plant enzyme pectin methylesterase of Nicotiana tabacum L. specifically binds to the movement protein encoded by tobacco mosaic virus. We also show that pectin methylesterase is an RNA binding protein. These data suggest that pectin methylesterase is a host cell receptor involved in cell-to-cell movement of tobacco mosaic virus.     

Intracellular targeting of a hordeiviral membrane-spanning movement protein: sequence requirements and involvement of an unconventional mechanism

The membrane-spanning protein TGBp3 is one of the three movement proteins (MPs) of Poa semilatent virus. TGBp3 is thought to direct other viral MPs and genomic RNA to peripheral bodies located in close proximity to plasmodesmata. We used the ectopic expression of green fluorescent protein-fused TGBp3 in epidermal cells of Nicotiana benthamiana leaves to study the TGBp3 intracellular trafficking pathway. Treatment with inhibitors was used to reveal that the targeting of TGBp3 to plasmodesmata does not require a functional cytoskeleton or secretory system. In addition, the suppression of endoplasmic reticulum-derived vesicle formation by a dominant negative mutant of small GTPase Sar1 had no detectable effect on TGBp3 trafficking to peripheral bodies. Collectively, these results suggested the involvement of an unconventional pathway in the intracellular transport of TGBp3. The determinants of targeting to plasmodesmata were localized to the C-terminal region of TGBp3, including the conserved hydrophilic and terminal membrane-spanning domains.     

Effects of calreticulin on viral cell-to-cell movement

Cell-to-cell tobacco mosaic virus movement protein (TMV MP) mediates viral spread between the host cells through plasmodesmata. Although several host factors have been shown to interact with TMV MP, none of them coresides with TMV MP within plasmodesmata. We used affinity purification to isolate a tobacco protein that binds TMV MP and identified it as calreticulin. The interaction between TMV MP and calreticulin was confirmed in vivo and in vitro, and both proteins were shown to share a similar pattern of subcellular localization to plasmodesmata. Elevation of the intracellular levels of calreticulin severely interfered with plasmodesmal targeting of TMV MP, which, instead, was redirected to the microtubular network. Furthermore, in TMV-infected plant tissues overexpressing calreticulin, the inability of TMV MP to reach plasmodesmata substantially impaired cell-to-cell movement of the virus. Collectively, these observations suggest a functional relationship between calreticulin, TMV MP, and viral cell-to-cell movement.     

Developmental regulation and significance of KNOX protein trafficking in Arabidopsis

Intercellular communication delivers critical information for position-dependent specification of cell fate. In plants, a novel mechanism for cell-to-cell communication involves the intercellular trafficking of regulatory proteins and mRNAs. The maize KNOTTED1 (KN1) gene acts non cell-autonomously in the maize leaf, and KN1 was the first plant protein shown to traffic cell-to-cell, presumably through plasmodesmata. We have compared the intercellular trafficking of green fluorescent protein (GFP) fusions of KN1 and Arabidopsis KN1-related homeobox proteins to that of the viral movement protein from turnip vein clearing tobamovirus. We show that there is specific developmental regulation of GFP approximately KN1 trafficking. GFP -- KN1 was able to traffic from the inner layers of the leaf to the epidermis, but not in the opposite direction, from epidermis to mesophyll. However, GFP or the GFP -- movement protein fusion moved readily out of the epidermis. GFP -- KN1 was however able to traffic out of the epidermal (L1) layer in the shoot apical meristem, indicating that KN1 movement out of the L1 was developmentally regulated. GFP -- KNAT1/BREVIPEDICELLUS and GFP -- SHOOTMERISTEMLESS fusions could also traffic from the L1 to the L2/L3 layers of the meristem. In a test for the functional significance of trafficking, we showed that L1-specific expression of KN1 or of KNAT1 was able to partially complement the strong shootmeristemless-11 (stm-11) mutant. However, a cell-autonomous GUS fusion to KN1 showed neither trafficking ability nor complementation of stm-11 when expressed in the L1. These results suggest that the activity of KN1 and related homeobox proteins is maintained following intercellular trafficking, and that trafficking may be required for their normal developmental function.     

Cell-to-cell transport of proteins: requirement for unfolding and characterization of binding to a putative plasmodesmal receptor

Plasmodesmata 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.     

Viral infection enables phloem loading of GFP and long-distance trafficking of the protein

It is generally accepted that viral systemic infection follows the source-to-sink symplastic pathway of sugar translocation. In plants that are classified as apoplastic loaders, the boundary between the companion cell-sieve element (CC-SE) complex and neighboring cells is symplastically restricted, and the potential passage of macromolecules between the two domains has yet to be explored. Transgenic tobacco plants expressing green fluorescence protein (GFP) and cucumber mosaic virus (CMV)-encoded proteins fused to GFP under the control of the fructose-1,6-bisphosphatase (FBPase) promoter were produced in order to localize the encoded proteins in mesophyll and bundle sheath cells and to explore the influence of viral infection on the functioning of plasmodesmata interconnecting the two domains. GFP produced outside the vascular tissue could overcome the symplastic barrier between the CC-SE complex and the surrounding cells to enter the vasculature in CMV-infected plants. Grafting of control (non-transgenic) tobacco scions to CMV-infected FBPase-GFP-expressing root stocks confirmed that GFP could move long distances in the phloem. No movement of the gfp mRNA was noticeable in this set of experiments. The ability of GFP to enter the vasculature and move long distances was also evident upon infection of the grafting plants with other viruses. These results provide experimental evidence for alteration of the functioning of plasmodesmata interconnecting the CC-SE complex and neighboring cells by viral infection to enable non-selective trafficking of macromolecules from the mesophyll into the sieve tube.     

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.     

Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.     

How do plant virus nucleic acids move through intercellular connections?

In addition to their function in transport of water, ions, small metabolites, and growth factors in normal plant tissue, the plasmodesmata presumably serve as routes for cell-to-cell movement of plant viruses in infected tissue. Virus cell-to-cell spread through plasmodesmata is an active process mediated by specialized virus encoded movement proteins; however, the mechanism by which these proteins operate is not clear. We incorporate recent information on the biochemical properties of plant virus movement proteins and their interaction with plasmodesmata in a model for transport of nucleic acids through plasmodesmatal channels. We propose that only single stranded (ss) nucleic acids can be transported efficiently through plasmodesmata, and that movement proteins function as molecular chaperones for ss nucleic acids to form unfolded movement protein-ss nucleic acid complexes. These complexes are targeted to plasmodesmata. Plasmodesmatal permeability is then increased following interaction with movement protein and the entire movement complex or its nucleic acid component is translocated across the plasmodesmatal channel.     

胞间连丝：共质体运输的动态通道

胞间连丝作为一种细胞质结构将相邻的细胞连系起来而形成植物的共质体。胞间连丝通过调控许多离子和分子的共质体运输而广泛地参与植物的生命活动。胞间连丝的主要构成部分是细胞质膜、连丝小管、以及位于二之间的环层细胞质。这三都很容易在电子显微镜下观察到。细胞骨架的成分（肌动蛋白和肌球蛋白）起到稳定胞间连丝的作用。同时,钙结合蛋白可能具有调节间连丝功能的作用。在胞间连丝里,环层细胞质为大多数溶质提供共质体运输的通道,而有些 共质体运输则可能是通过连丝小管的内腔、连丝小管的壳层、甚或是细胞质膜来实现的。共质体可以细分为数个区块,它们各自允许不同大小的分子（从低于1000到高于10000道尔顿）通过。从发生上看,胞间连丝可以是初生的,也可以是次生的。前是伴随着新细胞壁的形成则产生的,而后则是在已有的细胞壁上产生的。胞间连丝的动态性质还表现在它们的频率是处于变化之中,这是由于组织或植物整体的发育和生理状态决定的。虽然共质体运输的基本形式是扩散,但胞间连丝对于某些离子和分子却是选择性的。在病毒感染细胞时,病毒的移动蛋白作用于胞间连丝的受体蛋白,结果,胞间连丝被显地扩张（其机理尚不清楚）。于是,病毒的移动蛋白连同与之结合在一起的病毒基因组进入毗邻的健康细胞。一些植物源性的蛋白质也能够通过胞间连丝来运输;推测其方式类似于病毒的移动蛋白。有些植物蛋白质本身就是信号分子,它们调节分化和其他活动。与此相反,还有一些植物蛋白质的共质体运输并不是通过特异的方式来实现的。     

Actin cytoskeleton is involved in targeting of a viral Hsp70 homolog to the cell periphery

The cell-to-cell movement of plant viruses involves translocation of virus particles or nucleoproteins to and through the plasmodesmata (PDs). As we have shown previously, the movement of the Beet yellows virus requires the concerted action of five viral proteins including a homolog of cellular approximately 70-kDa heat shock proteins (Hsp70h). Hsp70h is an integral component of the virus particles and is also found in PDs of the infected cells. Here we investigate subcellular distribution of Hsp70h using transient expression of Hsp70h fused to three spectrally distinct fluorescent proteins. We found that fluorophore-tagged Hsp70h forms motile granules that are associated with actin microfilaments, but not with microtubules. In addition, immobile granules were observed at the cell periphery. A pairwise appearance of these granules at the opposite sides of cell walls and their colocalization with the movement protein of Tobacco mosaic virus indicated an association of Hsp70h with PDs. Treatment with various cytoskeleton-specific drugs revealed that the intact actomyosin motility system is required for trafficking of Hsp70h in cytosol and its targeting to PDs. In contrast, none of the drugs interfered with the PD localization of Tobacco mosaic virus movement protein. Collectively, these findings suggest that Hsp70h is translocated and anchored to PDs in association with the actin cytoskeleton.