Plant virus transport: motions of functional equivalence

Plant virus cell-to-cell movement and subsequent systemic transport are governed by a series of mechanisms involving various virus and plant factors. Specialized virus encoded movement proteins (MPs) control the cell-to-cell transport of viral nucleoprotein complexes through plasmodesmata. MPs of different viruses have diverse properties and each interacts with specific host factors that also have a range of functions. Most viruses are then transported via the phloem as either nucleoprotein complexes or virions, with contributions from host and virus proteins. Some virus proteins contribute to the establishment and maintenance of systemic infection by inhibiting RNA silencing-mediated degradation of viral RNA. In spite of all the different movement strategies and the viral and host components, there are possible functional commonalities in virus-host interactions that govern viral spread through plants.     

Citrus tristeza virus: survival at the edge of the movement continuum

Systemic invasion of plants by viruses is thought to involve two processes: cell-to-cell movement between adjacent cells and long-distance movement that allows the virus to rapidly move through sieve elements and unload at the growing parts of the plant. There is a continuum of proportions of these processes that determines the degrees of systemic infection of different plants by different viruses. We examined the systemic distribution of Citrus tristeza virus (CTV) in citrus species with a range of susceptibilities. By using a "pure" culture of CTV from a cDNA clone and green fluorescent protein-labeled virus we show that both cell-to-cell and long-distance movement are unusually limited, and the degree of limitation varies depending on the citrus host. In the more-susceptible hosts CTV infected only a small portion of phloem-associated cells, and moreover, the number of infection sites in less-susceptible citrus species was substantially decreased further, indicating that long-distance movement was reduced in those hosts. Analysis of infection foci in the two most differential citrus species, Citrus macrophylla and sour orange, revealed that in the more-susceptible host the infection foci were composed of a cluster of multiple cells, while in the less-susceptible host infection foci were usually single cells, suggesting that essentially no cell-to-cell movement occurred in the latter host. Thus, CTV in sour orange represents a pattern of systemic infection in which the virus appears to function with only the long-distance movement mechanism, yet is able to survive in nature.     

Potato virus X movement in Nicotiana benthamiana: new details revealed by chimeric coat protein variants

Potato virus X coat protein is necessary for both cell-to-cell and phloem transfer, but it has not been clarified definitively whether it is needed in both movement phases solely as a component of the assembled particles or also of differently structured ribonucleoprotein complexes. To clarify this issue, we studied the infection progression of a mutant carrying an N-terminal deletion of the coat protein, which was used to construct chimeric virus particles displaying peptides selectively affecting phloem transfer or cell-to-cell movement. Nicotiana benthamiana plants inoculated with expression vectors encoding the wild-type, mutant and chimeric viral genomes were examined by microscopy techniques. These experiments showed that coat protein-peptide fusions promoting cell-to-cell transfer only were not competent for virion assembly, whereas long-distance movement was possible only for coat proteins compatible with virus particle formation. Moreover, the ability of the assembled PVX to enter and persist into developing xylem elements was revealed here for the first time.     

Systemic movement of a tobamovirus requires host cell pectin methylesterase

Systemic movement of plant viruses through the host vasculature, one of the central events of the infection process, is essential for maximal viral accumulation and development of disease symptoms. The host plant proteins involved in this transport, however, remain unknown. Here, we examined whether or not pectin methylesterase (PME), one of the few cellular proteins known to be involved in local, cell-to-cell movement of tobacco mosaic virus (TMV), is also required for the systemic spread of viral infection through the plant vascular system. In a reverse genetics approach, PME levels were reduced in tobacco plants using antisense suppression. The resulting PME antisense plants displayed a significant degree of PME suppression in their vascular tissues but retained the wild-type pattern of phloem loading and unloading of a fluorescent solute. Systemic transport of TMV in these plants, however, was substantially delayed as compared to the wild-type tobacco, suggesting a role for PME in TMV systemic infection. Our analysis of virus distribution in the PME antisense plants suggested that TMV systemic movement may be a polar process in which the virions enter and exit the vascular system by two different mechanisms, and it is the viral exit out of the vascular system that involves PME.     

Cell-to-cell movement of potato potexvirus X is dependent on suppression of RNA silencing

RNA silencing in transgenic and virus-infected plants involves a mobile silencing signal that can move cell-to-cell and systemically through the plant. It is thought that this signal can influence long-distance movement of viruses because protein suppressors of silencing encoded in viral genomes are required for long-distance virus movement. However, until now, it was not known whether the mobile signal could also influence short-range virus movement between cells. Here, through random mutation analysis of the Potato Potexvirus X (PVX) silencing suppressor P25, we provide evidence that it does. All mutants that were defective for silencing suppression were also non-functional in viral cell-to-cell movement. However, we identified mutant P25 proteins that were functional as silencing suppressors but not as movement proteins and we conclude that suppression of silencing is not sufficient to allow virus movement between cells: there must be a second P25 function that is independent of silencing but also required for cell-to-cell movement. Consistent with this hypothesis, we identified two classes of suppressor-inactive P25 mutants. One class of these mutants is proposed to be functional for the accessory function because their failure to support PVX movement could be complemented by heterologous suppressors of silencing. The second class of P25 mutants is considered defective for both the suppressor and second functions because the heterologous silencing suppressors did not restore virus movement. It is possible, based on analyses of short interfering RNA accumulation, that P25 suppresses silencing by interfering with either assembly or function of the effector complexes of RNA silencing.     

NTH201, a novel class II KNOTTED1-like protein, facilitates the cell-to-cell movement of Tobacco mosaic virus in tobacco

NTH201, a novel class II KNOTTED1-like protein gene, was cloned from tobacco (Nicotiana tabacum cv. Xanthi) and its role in Tobacco mosaic virus (TMV) infection was analyzed. Virus-induced gene silencing of NTH201 caused a delay in viral RNA accumulation as well as virus spread in infected tobacco plants. Overexpression of the gene in a transgenic tobacco plant (N. tabacum cv. Xanthi nc) infected by TMV showed larger local lesions than those of the nontransgenic plant. NTH201 exhibited no intercellular trafficking ability but did exhibit colocalization with movement protein (MP) at the plasmodesmata. When NTH201-overexpressing tobacco BY-2 cultured cells were infected with TMV, the accumulation of MP but not of viral genomic and subgenomic RNA clearly was accelerated compared with those in nontransgenic cells at an early infection period. The formation of virus replication complexes (VRC) also was accelerated in these transgenic cells. Conversely, NTH201-silenced cells showed less MP accumulations and fewer VRC formations than did nontransgenic cells. These results suggested that NTH201 might indirectly facilitate MP accumulation and VRC formation in TMV-infected cells, leading to rapid viral cell-to-cell movement in plants at an early infection stage.     

Turnip mosaic virus Moves Systemically through Both Phloem and Xylem as Membrane-Associated Complexes

Plant viruses move systemically in plants through the phloem. They move as virions or as ribonucleic protein complexes, although it is not clear what these complexes are made of. The approximately 10-kb RNA genome of Turnip mosaic virus (TuMV) encodes a membrane protein, known as 6K₂, that induces endomembrane rearrangements for the formation of viral replication factories. These factories take the form of vesicles that contain viral RNA (vRNA) and viral replication proteins. In this study, we report the presence of 6K₂-tagged vesicles containing vRNA and the vRNA-dependent RNA polymerase in phloem sieve elements and in xylem vessels. Transmission electron microscopy observations showed the presence in the xylem vessels of vRNA-containing vesicles that were associated with viral particles. Stem-girdling experiments, which leave xylem vessels intact but destroy the surrounding tissues, confirmed that TuMV could establish a systemic infection of the plant by going through xylem vessels. Phloem sieve elements and xylem vessels from Potato virus X-infected plants also contained lipid-associated nonencapsidated vRNA, indicating that the presence of membrane-associated ribonucleic protein complexes in the phloem and xylem may not be limited to TuMV. Collectively, these studies indicate that viral replication factories could end up in the phloem and the xylem.Plant viruses use the host preexisting transport routes to propagate infection to the whole plant. After replication in the initially infected cells, viruses move cell to cell through plasmodesmata (PD) and start a new round of replication in the newly infected cells. This cycle is repeated until viruses reach vascular tissues, where they enter into the conducting tubes for systemic movement. Several studies have indicated that plant viruses are passively transported along the source-to-sink flow of photoassimilates and thus are believed to move systemically through the phloem (for review, see Hipper et al., 2013).The conducting tube of the phloem is the sieve element. The mature sieve element is enucleated and relies on the associated companion cells for the maintenance of its physiological function (Fisher et al., 1992). The specialized PD connecting one sieve element with one companion cell is called the pore plasmodesmal unit (PPU). Different from the other PDs, PPUs are always branched on the companion cell side but have only one channel on the sieve element side (Oparka and Turgeon, 1999). It is believed that the loading and uploading of viral material during phloem transport are through PPUs. Even though the size exclusion limit of PPUs (Kempers and Bel, 1997) is larger than that of the other PDs (Wolf et al., 1989; Derrick et al., 1990), PPUs should not allow, in their native state, virions or viral ribonucleoprotein (vRNP) complexes to pass through. It is thus believed that specific interactions between virus and host factors are required to allow the viral entity to go through. For instance, the movement protein of Cucumber mosaic virus (CMV) is targeted to PPUs (Blackman et al., 1998), suggesting that this viral protein modifies the size exclusion limit of PPUs and helps viral entry into sieve elements.Most plant viruses are assumed to move systemically through the phloem as virions. This assumption is based on the observation that Coat Protein (CP) deletions debilitating virus assembly prevent systemic infection (Brault et al., 2003; Zhang et al., 2013; Hipper et al., 2014). Some investigations showed the actual presence of virions in sieve elements. This is the case for the icosahedral Tobacco ringspot virus (Halk and McGuire, 1973), Carrot red leaf virus (Murant and Roberts, 1979), Potato leaf roll virus (Shepardson et al., 1980), and Beet western yellows virus (Hoefert, 1984). In addition, virions also were observed in phloem sap, such as the icosahedral CMV (Requena et al., 2006) and the rigid rod-shaped Cucumber green mottle mosaic virus (Simón-Buela and García-Arenal, 1999). Some viruses also are believed to move as ribonucleic protein (RNP) complexes, since systemic movement was observed in CP mutants where virion assembly was hindered. For instance, Tobacco rattle virus, Potato mop-top virus, Brome mosaic virus, and Tomato bushy stunt virus can still move systemically when the CP gene has been deleted from the viral genome (Swanson et al., 2002; Savenkov et al., 2003; Gopinath and Kao, 2007; Manabayeva et al., 2013). For potyviruses, it is still not clear if long-distance transport involves exclusively viral particles or if vRNP complexes also are implicated (Dolja et al., 1994, 1995; Cronin et al., 1995; Schaad et al., 1997; Kasschau and Carrington, 2001; Rajamaki and Valkonen, 2002). But whether virions or vRNP complexes are involved in viral movement, the full nature of the viral entity being implicated has not been defined.Xylem also is used for systemic infection of viruses, but its importance in viral transport generally has been overlooked. Vessel elements are the building blocks of xylem vessels, which constitute the major part of the water-upward-transporting system in a plant. The side walls of mature vessel elements contain pits, which are areas lacking a secondary cell wall; the end walls of the mature vessel elements are removed, and the openings are called perforation plates (Roberts and McCann, 2000). CP or virions of some plant viruses of all different shapes have been detected in the xylem vessels and/or guttation fluid, suggesting that these viruses may move systemically through xylem vessels. For example, the CP of the icosahedral Tomato bushy stunt virus (Manabayeva et al., 2013) and Rice yellow mottle virus (Opalka et al., 1998), the CP of the rigid rod-shaped Soilborne wheat mosaic virus (Verchot et al., 2001) and Cucumber green mottle mosaic virus (Moreno et al., 2004), and the flexuous rod-shaped Potato virus X (PVX; Betti et al., 2012) were detected in xylem vessels. Colocalization of anti-Rice yellow mottle virus antibodies and a cell wall marker for cellulosic β-(1-4)-d-glucans over vessel pit membranes suggests that the pit membranes might be a pathway for virus migration between vessels (Opalka et al., 1998). Moreover, flexuous rod-shaped virions of Zucchini yellow mosaic virus were found in both xylem vessels of root tissue and the guttation fluid (French and Elder, 1999). Finally, icosahedral Brome mosaic virus (Ding et al., 2001) and rigid rod-shaped Tomato mosaic virus and Pepper mild mottle virus (French et al., 1993) virions were found in guttation fluid. Guttation fluid originates from xylem exudate, indicating that these plant viruses can move through xylem within the infected plant. The above studies, however, mainly relied on electron microscopy and infection assays and may have missed the presence of other viral components that might be involved in transport.Turnip mosaic virus (TuMV) is a positive-strand RNA virus belonging to the family Potyviridae, genus Potyvirus, which contains around 30% of the currently known plant viruses and causes serious diseases in numerous crops (Shukla et al., 1994). Potyviruses are nonenveloped, flexuous rod-shaped particles of 680 to 900 nm in length and 11 to 13 nm in diameter. The genomic approximately 10-kb RNA encodes a polyprotein, which is processed into at least 11 mature proteins. TuMV remodels cellular membranes into viral factories, which are intracellular compartments involved in viral replication and movement. These compartments take the form of vesicles of approximately 100 nm in diameter originating from the endoplasmic reticulum (Grangeon et al., 2012). These vesicles contain viral RNA (vRNA) and viral and host proteins involved in vRNA replication (Beauchemin et al., 2007; Beauchemin and Laliberté, 2007; Dufresne et al., 2008; Huang et al., 2010; Grangeon et al., 2012). The viral membrane 6K₂ protein is involved in the membrane alterations and vesicle production (Beauchemin et al., 2007). The membrane-bound replication complexes can move intracellularly and cell to cell (Grangeon et al., 2013) at a rate of one cell being infected every 3 h (Agbeci et al., 2013). Intercellular trafficking of the replication complex is likely mediated by the PD-localized potyviral proteins Cytoplasmic Inclusion (CI) and P3N-PIPO (for N-terminal Half of P3 fused to the Pretty Interesting Potyviridae ORF; Carrington et al., 1998; Wei et al., 2010; Vijayapalani et al., 2012) as well as CP (Dolja et al., 1994, 1995), Viral Protein genome-linked (VPg; Nicolas et al., 1997; Rajamaki and Valkonen, 1999, 2002), and Helper Component-Proteinase (HC-Pro; Cronin et al., 1995; Kasschau et al., 1997; Rojas et al., 1997; Kasschau and Carrington, 2001), which are involved in both cell-to-cell and vascular movement.It is expected that, ultimately, TuMV reaches the vascular tissues of the plant, but how and under what form it is released into the conducting tubes are not known. To further understand viral spread and systemic movement, we investigated the distribution of 6K₂-tagged TuMV factories in all of the leaf and stem tissues other than the epidermal cells. We found TuMV factories in all tissues. Interestingly, we observed 6K₂-tagged vesicles, containing vRNA and viral replication proteins, in both phloem sieve elements and xylem vessels. We confirmed that TuMV could move systemically through xylem by a so-called stem-girdling assay, which induces cell death of the phloem without affecting xylem integrity. Hence, our study indicates that membrane-associated TuMV replication complexes are involved in the systemic movement of the virus.     

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.     

Conversion in the requirement of coat protein in cell-to-cell movement mediated by the cucumber mosaic virus movement protein

Plant viruses have movement protein (MP) gene(s) essential for cell-to-cell movement in hosts. Cucumber mosaic virus (CMV) requires its own coat protein (CP) in addition to the MP for intercellular movement. Our present results using variants of both CMV and a chimeric Brome mosaic virus with the CMV MP gene revealed that CMV MP truncated in its C-terminal 33 amino acids has the ability to mediate viral movement independently of CP. Coexpression of the intact and truncated CMV MPs extremely reduced movement of the chimeric viruses, suggesting that these heterogeneous CMV MPs function antagonistically. Sequential deletion analyses of the CMV MP revealed that the dispensability of CP occurred when the C-terminal deletion ranged between 31 and 36 amino acids and that shorter deletion impaired the ability of the MP to promote viral movement. This is the first report that a region of MP determines the requirement of CP in cell-to-cell movement of a plant virus.     

Identification and study of tobacco mosaic virus movement function by complementation tests

The phenomenon of trans-complementation of cell-to-cell movement between plant positive-strand RNA viruses is discussed with an emphasis on tobamoviruses. Attention is focused on complementation between tobamoviruses (coding for a single movement protein, MP) and two groups of viruses that contain the triple block of MP genes and require four (potato virus X) or three (barley stripe mosaic virus) proteins for cell-to-cell movement. The highlights of complementation data obtained by different experimental approaches are given, including (i) double infections with movement-deficient (dependent) and helper viruses; (ii) infections with recombinant viral genomes bearing a heterologous MP gene; (iii) complementation of a movement-deficient virus in transgenic plants expressing the MP of a helper virus; and (iv) co-bombardment of plant tissues with the cDNAs of a movement-dependent virus genome and the MP gene of a helper virus.     

Interaction of the host protein NbDnaJ with Potato virus X minus-strand stem-loop 1 RNA and capsid protein affects viral replication and movement

Plant viruses must interact with host cellular components to replicate and move from cell to cell. In the case of Potato virus X (PVX), it carries stem-loop 1 (SL1) RNA essential for viral replication and movement. Using two-dimensional electrophoresis northwestern blot analysis, we previously identified several host proteins that bind to SL1 RNA. Of those, we further characterized a DnaJ-like protein from Nicotiana benthamiana named NbDnaJ. An electrophoretic mobility shift assay confirmed that NbDnaJ binds only to SL1 minus-strand RNA, and bimolecular fluorescence complementation (BiFC) indicated that NbDnaJ interacts with PVX capsid protein (CP). Using a series of deletion mutants, the C-terminal region of NbDnaJ was found to be essential for the interaction with PVX CP. The expression of NbDnaJ significantly changed upon infection with different plant viruses such as PVX, Tobacco mosaic virus, and Cucumber mosaic virus, but varied depending on the viral species. In transient experiments, both PVX replication and movement were inhibited in plants that over-expressed NbDnaJ but accelerated in plants in which NbDnaJ was silenced. In summary, we suggest that the newly identified NbDnaJ plays a role in PVX replication and movement by interacting with SL1(-) RNA and PVX CP.     

The chloroplast ribosomal protein large subunit 1 interacts with viral polymerase and promotes virus infection

Chloroplasts play an indispensable role in the arms race between plant viruses and hosts. Chloroplast proteins are often recruited by plant viruses to support viral replication and movement. However, the mechanism by which chloroplast proteins regulate potyvirus infection remains largely unknown. In this study, we observed that Nicotiana benthamiana ribosomal protein large subunit 1 (NbRPL1), a chloroplast ribosomal protein, localized to the chloroplasts via its N-terminal 61 amino acids (transit peptide), and interacted with tobacco vein banding mosaic virus (TVBMV) nuclear inclusion protein b (NIb), an RNA-dependent RNA polymerase. Upon TVBMV infection, NbRPL1 was recruited into the 6K2-induced viral replication complexes in chloroplasts. Silencing of NbRPL1 expression reduced TVBMV replication. NbRPL1 competed with NbBeclin1 to bind NIb, and reduced the NbBeclin1-mediated degradation of NIb. Therefore, our results suggest that NbRPL1 interacts with NIb in the chloroplasts, reduces NbBeclin1-mediated NIb degradation, and enhances TVBMV infection.     

Immunocapture of dsRNA-bound proteins provides insight into Tobacco rattle virus replication complexes and reveals Arabidopsis DRB2 to be a wide-spectrum antiviral effector

Plant RNA viruses form organized membrane-bound replication complexes to replicate their genomes. This process requires virus- and host-encoded proteins and leads to the production of double-stranded RNA (dsRNA) replication intermediates. Here, we describe the use of Arabidopsis thaliana expressing GFP-tagged dsRNA-binding protein (B2:GFP) to pull down dsRNA and associated proteins in planta upon infection with Tobacco rattle virus (TRV). Mass spectrometry analysis of the dsRNA-B2:GFP-bound proteins from infected plants revealed the presence of viral proteins and numerous host proteins. Among a selection of nine host candidate proteins, eight showed relocalization upon infection, and seven of these colocalized with B2-labeled TRV replication complexes. Infection of A. thaliana T-DNA mutant lines for eight such factors revealed that genetic knockout of dsRNA-BINDING PROTEIN 2 (DRB2) leads to increased TRV accumulation and DRB2 overexpression caused a decrease in the accumulation of four different plant RNA viruses, indicating that DRB2 has a potent and wide-ranging antiviral activity. We propose B2:GFP-mediated pull down of dsRNA to be a versatile method to explore virus replication complex proteomes and to discover key host virus replication factors. Given the universality of dsRNA, development of this tool holds great potential to investigate RNA viruses in other host organisms.

Double-stranded RNA immunoprecipitation allows the identification of Arabidopsis host proteins associated with tobacco rattle virus replication complexes.     

病毒与分子伴侣

分子伴侣与病毒生命活动密切相关,从病毒复制的起始、转录的进行、翻译的完成到病毒粒子的装配成熟,甚至病毒在宿主体内的转运都有分子伴侣的参与。随着病毒与分子伴侣相互关系研究的深入,产生了抗病毒的又一可能途径。     

Viruses Roll the Dice: The Stochastic Behavior of Viral Genome Molecules Accelerates Viral Adaptation at the Cell and Tissue Levels

Recent studies on evolutionarily distant viral groups have shown that the number of viral genomes that establish cell infection after cell-to-cell transmission is unexpectedly small (1–20 genomes). This aspect of viral infection appears to be important for the adaptation and survival of viruses. To clarify how the number of viral genomes that establish cell infection is determined, we developed a simulation model of cell infection for tomato mosaic virus (ToMV), a positive-strand RNA virus. The model showed that stochastic processes that govern the replication or degradation of individual genomes result in the infection by a small number of genomes, while a large number of infectious genomes are introduced in the cell. It also predicted two interesting characteristics regarding cell infection patterns: stochastic variation among cells in the number of viral genomes that establish infection and stochastic inequality in the accumulation of their progenies in each cell. Both characteristics were validated experimentally by inoculating tobacco cells with a library of nucleotide sequence–tagged ToMV and analyzing the viral genomes that accumulated in each cell using a high-throughput sequencer. An additional simulation model revealed that these two characteristics enhance selection during tissue infection. The cell infection model also predicted a mechanism that enhances selection at the cellular level: a small difference in the replication abilities of coinfected variants results in a large difference in individual accumulation via the multiple-round formation of the replication complex (i.e., the replication machinery). Importantly, this predicted effect was observed in vivo. The cell infection model was robust to changes in the parameter values, suggesting that other viruses could adopt similar adaptation mechanisms. Taken together, these data reveal a comprehensive picture of viral infection processes including replication, cell-to-cell transmission, and evolution, which are based on the stochastic behavior of the viral genome molecules in each cell.     

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.     

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.     

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.     

植物病毒基因沉默抑制子研究进展

RNAi普遍存在于真核生物中,是植物应对外来病毒入侵的一种防御机制。但是植物病毒能通过产生不同的抑制子蛋白来抑制寄主基因沉默的发生。病毒抑制子通过干扰基因沉默的起始、siRNA的积累或干扰系统性基因沉默等方式抑制寄主的基因沉默。有的病毒抑制子蛋白还能促进病毒的积累和胞间移动,加强侵染组织的病毒病症状表现。主要阐述了RNAi的机制、病毒抑制PTGS的作用方式、几种常见的沉默抑制子以及抑制子与病毒侵染的关系。     

Host factors against plant viruses

Plant virus genome replication and movement is dependent on host resources and factors. However, plants respond to virus infection through several mechanisms, such as autophagy, ubiquitination, mRNA decay and gene silencing, that target viral components. Viral factors work in synchrony with pro-viral host factors during the infection cycle and are targeted by antiviral responses. Accordingly, establishment of virus infection is genetically determined by the availability of the pro-viral factors necessary for genome replication and movement, and by the balance between plant defence and viral suppression of defence responses. Sequential requirement of pro-viral factors and the antagonistic activity of antiviral factors suggest a two-step model to explain plant–virus interactions. At each step of the infection process, host factors with antiviral activity have been identified. Here we review our current understanding of host factors with antiviral activity against plant viruses.