Cell-to-cell transport of plant viruses is mediated by virus-encoded movement proteins and occurs through plasmodesmata interconnecting neighboring cells in plant tissues. Three movement proteins coded by the “triple gene block” (TGB) and named TGBp1, TGBp2 and TGBp3 have distinct functions in viral transport. TGBp1 binds viral genomic RNAs to form ribonucleoprotein complexes representing the transport form of viral genome, while TGBp2 and TGBp3 are necessary for intracellular delivery of such complexes to plasmodesmata. Recently, it was revealed that overexpression of Potato virus X TGBp3 triggers the unfolded protein response mitigating the endoplasmic reticulum (ER) stress leading to cell death if this protein reaches high levels in the ER. Here we report microscopic studies of the influence of the Poa semilatent hordeivirus TGBp3 overexpressed in Nicotiana benthamiana epidermal cells by particle bombardment on cell endomembranes and demonstrate that the protein C-terminal transmembrane segment contains a determinant responsible for vesiculation and coalescence of the endoplasmic reticulum and Golgi presumably accompanying the ER stress that can be induced upon high-level TGBp3 expression.
Cell-to-cell movement of Potato mop-top virus (PMTV) is mediated by three virus-encoded ‘triple gene block’ (TGB) proteins termed TGBp1, TGBp2 and TGBp3. TGBp1 binds virus RNAs to form viral ribonucleoprotein complexes (vRNPs), the transport form of viral genome. TGBp2 and TGBp3 are necessary for intracellular delivery of TGBp1-containing vRNPs to plasmodesmata. To analyze subcellular localization and transport of TGBp1 we used a single binary vector for agrobacterium-mediated co-expression of PMTV TGBp1 fused to green fluorescent protein and TGBp2/TGBp3. At two days post infiltration (dpi) TGBp1 was found in the nucleus and in association with microtubules (MTs). Similar localization pattern was revealed in cells expressing GFP-TGBp1 alone after particle bombardment. At 3 dpi, in addition to the nucleus and MTs, TGBp1 was detected in numerous granular bodies located both along the MTs and at the cell wall. The latter structures co-localized with plasmodesmata-associated callose depositions. At 4 dpi, GFP-TGBp1 was detected in cell wall-associated bodies and also in residual MTs, the nucleoplasm and large perinuclear inclusions resembling aggresomes. Therefore GFP-TGBp1 association with MTs preceded to its localization to plasmodesmata. Disassembly of cell MTs by colchicine prevented GFP-TGBp1 targeting to plasmodesmata and the MT-dependent aggresome formation. Deletion analysis also revealed a correlation between TGBp1 microtubule association and plasmodesmata targeting. We propose that TGBp1 interaction with MTs may be important for the formation of vRNP bodies destined for the transport to plasmodesmata as well as degradation of the excessive TGBp1.
Movement protein; virus movement; plasmodesmata; microtubules; subcellular localization.
The triple-gene-block protein 3 (TGBp3) of Bamboo mosaic virus (BaMV) is an integral endoplasmic reticulum (ER) membrane protein which is assumed to form a membrane complex to deliver the virus intracellularly. However, the virus entity that is delivered to plasmodesmata (PD) and its association with TGBp3-based complexes are not known. Results from chemical extraction and partial proteolysis of TGBp3 in membrane vesicles revealed that TGBp3 has a right-side-out membrane topology; i.e., TGBp3 has its C-terminal tail exposed to the outer surface of ER. Analyses of the TGBp3-specific immunoprecipitate of Sarkosyl-extracted TGBp3-based complex revealed that TGBp1, TGBp2, TGBp3, capsid protein (CP), replicase and viral RNA are potential constituents of virus movement complex. Substantial co-fractionation of TGBp2, TGBp3 and CP, but not TGBp1, in the early eluted gel filtration fractions in which virions were detected after TGBp3-specific immunoprecipitation suggested that the TGBp2- and TGBp3-based complex is able to stably associate with the virion. This notion was confirmed by immunogold-labeling transmission electron microscopy (TEM) of the purified virions. In addition, mutational and confocal microscopy analyses revealed that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. Taken together, our results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-based membrane complex and recruitment of TGBp1 to the PD by this complex.
Plant viruses spread their infectious entities from cell to cell via plasmodesmata (PD) through the assistance of virus-encoded movement proteins and host factors. Some RNA viruses encode three functionally coordinated movement proteins organized into a triple gene block (TGB) to facilitate their cell-to-cell movement. TGBp2 and TGBp3 are known to associate with the endoplasmic reticulum (ER) membrane and ER-derived vesicles. The ER- or vesicle-associated TGBp2 and TGBp3 presumably form a membrane complex to deliver the viruses. However, the identity of the “viral RNA cargo” and whether the cargo is able to associate with the TGBp2- and TGBp3-containing membrane complex during intracellular transport remain unclear for potex-like viruses. Taking advantage of an HA-tagged and a His-tagged TGBp3 construct of Bamboo mosaic virus (BaMV), we have been able to determine the membrane topology of TGBp3, isolate the TGBp3-based complex and detect the existence of a stable TGBp2-TGBp3-virion complex. Moreover, we have clarified that TGBp3 plays a key role in virus cell-to-cell movement by enhancing the TGBp2- and TGBp3-dependent PD localization of TGBp1. These results suggested that the cell-to-cell movement of potexvirus requires stable association of the virion cargo with the TGBp2- and TGBp3-containing membrane complex and recruitment of TGBp1 to the PD by this complex.
A new study of Potato virus X (PVX) revealed that a viral movement protein, named TGBp3, triggers the unfolded protein response (UPR) which monitors accumulation of aberrant proteins the endoplasmic reticulum (ER) and targets them for degradation. The PVX TGBp3 resides in ER and activates bZIP60, a transcription factor involved in the UPR pathway. Knockdown of bZIP60 hampers virus infection in protoplasts and whole plants. Preliminary evidence indicates that UPR regulates cellular cytotoxicity that could otherwise lead to cell death if the PVX TGBp3 reaches high levels in the ER. SKP1 expression appears to be linked to bZIP60 and is a component of the SCF-complex mediating proteasomal degradation of cellular substrates. SKP1 expression is induced by PVX TGBp3 and plays a role in regulating PVX spread in whole plants. We propose that SKP1 might be linked to TGBp1-mediated degradation of AGO1.
potato virus X; unfolded protein response; bZIP60; SKP1; viral movement protein; triple gene block proteins; TGB3
Sorting signal-mediated oligomerization and localization of the viral protein TGBp3 to curved ER tubules is essential for viral movement between cells in plants.
Many plant RNA viruses use their nonstructural proteins to target and move through the cortical endoplasmic reticulum (ER) tubules within the plant intercellular junction for cell-to-cell spreading. Most of these proteins, including the triple-gene-block 3 protein (TGBp3) of Potexvirus, are ER membrane proteins. We previously showed that TGBp3 of the Bamboo mosaic potexvirus partitions into tubular subdomains of the ER in both yeast and plants, but the mechanism and physiological significance of this localization is unclear. Here, we demonstrate that a sorting signal present in TGBp3 is necessary and sufficient for its oligomerization and for targeting integral membrane proteins into puncta within curved ER tubules. Mutations in the TGBp3 sorting signal impair viral spread, and plants infected with viruses harboring these mutants were either asymptomatic or had reduced symptoms. Thus, we propose that Potexvirus use the sorting signal in TGBp3 to target infectious viral derivatives to cortical ER tubules for transmission through the intercellular junctions in plants.
Most RNA viruses remodel the endomembrane network to promote virus replication, maturation, or egress. Rearrangement of cellular membranes is a crucial component of viral pathogenesis. The PVX TGBp2 protein induces vesicles of the granular type to bud from the endoplasmic reticulum network. Green fluorescent protein (GFP) was fused to the PVX TGBp2 coding sequence and inserted into the viral genome and into pRTL2 plasmids to study protein subcellular targeting in the presence and absence of virus infection. Mutations were introduced into the central domain of TGBp2, which contains a stretch of conserved amino acids. Deletion of a 10-amino-acid segment (m2 mutation) overlapping the segment of conserved residues eliminated the granular vesicle and inhibited virus movement. GFP-TGBp2m2 proteins accumulated in enlarged vesicles. Substitution of individual conserved residues in the same region similarly inhibited virus movement and caused the mutant GFP-TGBp2 fusion proteins to accumulate in enlarged vesicles. These results identify a novel element in the PVX TGBp2 protein which determines vesicle morphology. In addition, the data indicate that vesicles of the granular type induced by TGBp2 are necessary for PVX plasmodesmata transport.
Functions of viral proteins can be regulated through phosphorylation by serine/threonine kinases in plants, but little is known about the involvement of tyrosine kinases in plant virus infection. In this study, TGBp3, one of the three movement proteins encoded by a triple gene block (TGB) of Potato mop-top virus (PMTV), was detected for the first time in PMTV-infected plants and found to be tyrosine phosphorylated. Phosphorylation sites (Tyr87–89 and Tyr120) were located in two amino acid motifs conserved in the TGB-containing, rod-shaped plant viruses. Substitution of these tyrosine residues in both motifs was needed to abolish tyrosine phosphorylation of TGBp3. Substitution of Tyr87–89 with alanine residues enhanced the interaction between TGBp3 and TGBp2 and inhibited cell-to-cell movement of PMTV. On the other hand, substitution of Tyr120 with alanine resulted in no alteration in the interaction of TGBp3 with TGBp2, but the mutant virus was not infectious. The results suggest that tyrosine phosphorylation is a mechanism regulating the functions of plant virus movement proteins.
The triple-gene-block protein 2 (TGBp2) of Bamboo mosaic virus (BaMV) is a transmembrane protein which was proposed to be involved in viral RNA binding during virus transport. Here, we report on the RNA-binding properties of TGBp2. Using tyrosine fluorescence spectroscopy and UV-crosslinking assays, the TGBp2 solubilized with Triton X-100 was found to interact with viral RNA in a non-specific manner. These results raise the possibility that TGBp2 facilitates intracellular delivery of viral RNA through non-specific protein-RNA interaction.
Plasmodesmata provide the cytoplasmic conduits for cell-to-cell communication throughout plant tissues and participate in a diverse set of non–cell-autonomous functions. Despite their central role in growth and development and defence, resolving their modus operandi remains a major challenge in plant biology. Features of protein sequences and/or structure that determine protein targeting to plasmodesmata were previously unknown. We identify here a novel family of plasmodesmata-located proteins (called PDLP1) whose members have the features of type I membrane receptor-like proteins. We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking. PDLP1a is targeted to plasmodesmata via the secretory pathway in a Brefeldin A–sensitive and COPII-dependent manner, and resides at plasmodesmata with its C-terminus in the cytoplasmic domain and its N-terminus in the apoplast. Using a deletion analysis, we show that the single transmembrane domain (TMD) of PDLP1a contains all the information necessary for intracellular targeting of this type I membrane protein to plasmodesmata, such that the TMD can be used to target heterologous proteins to this location. These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication. They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.
In plants, cylindrical, microscopic channels called plasmodesmata provide intracellular connections between cells for communication and material transport, and are important for many aspects of plant growth and defence. We identify a novel family of plasmodesmata-located proteins (called PDLP1) with features of type I membrane receptor-like proteins. In line with the potential for this protein to regulate molecular movement from cell to cell, we show that altered expression of the protein changes the efficiency of protein diffusion from plasmodesmata. We have also analysed the manner in which PDLP1 is transported to plasmodesmata. We show that the single transmembrane domain (TMD) of the protein contains all the information necessary for targeting to plasmodesmata and that proper targeting depends upon specific interactions with other factors within the membrane. Notably, a single amino acid close to the C-terminus of the TMD is critical for determining the intracellular destination. Further, by fusing the TMD to yellow fluorescent protein, we establish that the TMD can be used to target heterologous proteins to plasmodesmata.
Little is know about the structure and function of plant cell-to-cell connections, called plasmodesmata. This paper describes a new family of plasmodesmal proteins and the processes controlling their subcellular trafficking.
Viruses encode RNA silencing suppressors to counteract host antiviral silencing. In this study, we analyzed the suppressors encoded by potato virus M (PVM), a member of the genus Carlavirus. In the conventional green fluorescent protein transient coexpression assay, the cysteine-rich protein (CRP) of PVM inhibited both local and systemic silencing, whereas the triple gene block protein 1 (TGBp1) showed suppressor activity only on systemic silencing. Furthermore, to elucidate the roles of these two suppressors during an active viral infection, we performed PVX vector-based assays and viral movement complementation assays. CRP increased the accumulation of viral RNA at the single-cell level and also enhanced viral cell-to-cell movement by inhibiting RNA silencing. However, TGBp1 facilitated viral movement but did not affect viral accumulation in protoplasts. These data suggest that CRP inhibits RNA silencing primarily at the viral replication step, whereas TGBp1 is a suppressor that acts at the viral movement step. Thus, our findings demonstrate a sophisticated viral infection strategy that suppresses host antiviral silencing at two different steps via two mechanistically distinct suppressors. This study is also the first report of the RNA silencing suppressor in the genus Carlavirus.
Our previous work has demonstrated that the NSvc4 protein of Rice stripe virus (RSV) functions as a cell-to-cell movement protein. However, the mechanisms whereby RSV traffics through plasmodesmata (PD) are unknown. Here we provide evidence that the NSvc4 moves on the actin filament and endoplasmic reticulum network, but not microtubules, to reach cell wall PD. Disruption of cytoskeleton using different inhibitors altered NSvc4 localization to PD, thus impeding RSV infection of Nicotiana benthamiana. Sequence analyses and deletion mutagenesis experiment revealed that the N-terminal 125 amino acids (AAs) of the NSvc4 determine PD targeting and that a transmembrane domain spanning AAs 106–125 is critical for PD localization. We also found that the NSvc4 protein can localize to chloroplasts in infected cells. Analyses using deletion mutants revealed that the N-terminal 73 AAs are essential for chloroplast localization. Furthermore, expression of NSvc4 from a Potato virus X (PVX) vector resulted in more severe disease symptoms than PVX alone in systemically infected N. benthamiana leaves. Expression of NSvc4 in Spodoptera frugiperda 9 cells did not elicit tubule formation, but instead resulted in punctate foci at the plasma membrane. These findings shed new light on our understanding of the movement mechanisms whereby RSV infects host plants.
rice stripe virus; movement; chloroplast; tubules
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.
To successfully infect plants, viruses replicate in an initially infected cell and then move to neighboring cells through plasmodesmata (PDs). However, the nature of the viral entity that crosses over the cell barrier into non-infected ones is not clear. The membrane-associated 6K2 protein of turnip mosaic virus (TuMV) induces the formation of vesicles involved in the replication and intracellular movement of viral RNA. This study shows that 6K2-induced vesicles trafficked toward the plasma membrane and were associated with plasmodesmata (PD). We demonstrated also that 6K2 moved cell-to-cell into adjoining cells when plants were infected with TuMV. 6K2 was then fused to photo-activable GFP (6K2:PAGFP) to visualize how 6K2 moved intercellularly during TuMV infection. After activation, 6K2:PAGFP-tagged vesicles moved to the cell periphery and across the cell wall into adjacent cells. These vesicles were shown to contain the viral RNA-dependent RNA polymerase and viral RNA. Symplasmic movement of TuMV may thus be achieved in the form of a membrane-associated viral RNA complex induced by 6K2.
plant RNA virus; potyvirus; replication complex; membrane association; intercellular movement
Transgenic mice that overexpress the nuclear form of sterol regulatory element binding protein-1a (SREBP-1a) in liver (TgBP-1a mice) were shown previously to overproduce cholesterol and fatty acids and to accumulate massive amounts of cholesterol and triglycerides in hepatocytes. Despite the hepatic overproduction of lipids, the plasma levels of cholesterol (∼45 mg/dl) and triglycerides (∼55 mg/dl) were not elevated, perhaps owing to degradation of lipid-enriched particles by low-density lipoprotein (LDL) receptors. To test this hypothesis, in the current studies we bred TgBP-1a mice with LDL receptor knockout mice. As reported previously, LDLR–/– mice manifested a moderate elevation in plasma cholesterol (∼215 mg/dl) and triglycerides (∼155 mg/dl). In contrast, the doubly mutant TgBP-1a;LDLR–/– mice exhibited marked increases in plasma cholesterol (∼1,050 mg/dl) and triglycerides (∼900 mg/dl). These lipids were contained predominantly within large very-low-density lipoprotein (VLDL) particles that were relatively enriched in cholesterol and apolipoprotein E. Freshly isolated hepatocytes from TgBP-1a and TgBP-1a;LDLR–/– mice overproduced cholesterol and fatty acids and secreted increased amounts of these lipids into the medium. Electron micrographs of livers from TgBP-1a mice showed large amounts of enlarged lipoproteins within the secretory pathway. We conclude that the TgBP-1a mice produce large lipid-rich lipoproteins, but these particles do not accumulate in plasma because they are degraded through the action of LDL receptors.
J. Clin. Invest. 103:1067–1076 (1999).
Intercellular transport of viruses through cytoplasmic connections, termed plasmodesmata (PD), is essential for systemic infection in plants by viruses. Previous genetic and ultrastructural data revealed that the potyvirus cyclindrical inclusion (CI) protein is directly involved in cell-to-cell movement, likely through the formation of conical structures anchored to and extended through PD. In this study, we demonstrate that plasmodesmatal localization of CI in N. benthamiana leaf cells is modulated by the recently discovered potyviral protein, P3N-PIPO, in a CI:P3N-PIPO ratio-dependent manner. We show that P3N-PIPO is a PD-located protein that physically interacts with CI in planta. The early secretory pathway, rather than the actomyosin motility system, is required for the delivery of P3N-PIPO and CI to PD. Moreover, CI mutations that disrupt virus cell-to-cell movement compromise PD-localization capacity. These data suggest that the CI and P3N-PIPO complex coordinates the formation of PD-associated structures that facilitate the intercellular movement of potyviruses in infected plants.
Plant viral pathogens cause an estimated US$60 billion loss in crop yields worldwide each year. Potyviruses, accounting for ∼30% of known plant viruses, include many agriculturally important viruses. Despite their importance, the cell-to-cell spread of potyviruses remains poorly understood. Previous studies have shown that at early time points of infection, the virus-encoded CI protein, one of 11 known potyviral proteins, is associated with cone-shaped structures at plasmodesmata (PD) and is involved in viral cell-to-cell movement. In this paper, we show that a newly identified potyviral protein, P3N-PIPO, is a PD-located protein and directs the CI protein to PD, facilitating the deposition of the cone-shaped structures of CI at PD by interacting with CI protein. We demonstrate that the mutant of CI, which impairs potyviral cell-to-cell movement, loses its ability to accumulate at PD. We further reveal that P3N-PIPO utilizes the secretory pathway rather than the actomyosin motility system for trafficking to PD. Taken together, the data presented in this study suggest that CI and P3N-PIPO coordinates the formation of conical structure at PD for potyviral cell-to-cell spread.
The interaction between tobacco mosaic virus and its host plant cells has been intensively studied as a model for macromolecular trafficking. The observation that GFP-labelled TMV movement protein localises to microtubules led to the suggestion that microtubules are required for the cell to cell movement of the virus. In a recent paper we have demonstrated that the targeting of TMV movement protein to plasmodesmata requires the actin and ER networks, which supports previous evidence from our laboratory that showed that disruption of microtubules did not prevent cell to cell movement of TMV virus, and that a mutated movement protein, which did not localise to micro-tubules, showed enhanced viral movement. In this addendum we speculate where the TMV movement protein accumulates within plasmodesmata, and the relationship of this accumulation to the cell to cell movement of the virus.
actin; endoplasmic reticulum; microtubule; plasmodesmata; targeting; tobacco mosaic virus
Plant viruses spread cell-to-cell in infected plants by exploiting plasmodesmata (PD), gatable channels in the cell wall that provide cytoplasmic passageways for the trafficking of informational macromolecules. Since it became known that the intercellular spread of Tobacco mosaic virus (TMV) depends on virus-encoded movement protein (MP), the mechanism by which this protein mediates in the targeting of this virus to PD is subject to intense studies. TMV movement occurs in a non-encapsidated form and thus promises to reveal important host functions involved in the intra-and intercellular trafficking of RNA molecules. We have recently presented new evidence that the cell-to-cell trafficking of TMV RNA (vRNA) involves the formation and intracellular trafficking of distinct MP particles. Upon assembly, these particles detach from cortical microtubule (MT) sites and then move with the flow of ER through the cell. During passage the particles continue to undergo transient interactions with MT which may guide the particles to their destination. The comprehensive analysis of particle composition may lead to important insights into the regulation of RNA transport in plants and may also reveal potential similarities to RNA transport mechanisms in animals and humans.
Tobacco mosaic virus; movement protein; RNA transport; plasmodesmata; microtubules; endoplasmic reticulum
Cell-to-cell movement of tobacco mosaic virus (TMV) is used to illustrate macromolecular traffic through plant intercellular connections, the plasmodesmata. This transport process is mediated by a specialized viral movement protein, P30. In the initially infected cell, P30 is produced by transcription of a subgenomic RNA derived from the invading virus. Presumably, P30 then associates with a certain proportion of the viral RNA molecules, sequestering them from replication and mediating their transport into neighbouring uninfected host cells. This nucleoprotem complex is targeted to plasmodesmata, possibly via interaction with the host cell's cytoskeleton. Prior to passage through a plasmodesma, the plasmodesmatal channel is dilated by the movement protein. It is proposed that targeting of P30-TMV RNA complexes to plasmodesmatal involves binding to a specific cell-wall-associated receptor molecule. This protein, designated p38, also functions as a protein kinase, phosphorylating P30 at its carboxy-terminus and minimizing P30-induced interference with plasmodesmatal permeability during viral infection.
Plasmodesmata (PD) are the communication channels which allow the trafficking of macromolecules between neighboring cells. Such cell-to-cell movement of macromolecules is regulated during plant growth and development; however, little is known about the regulation mechanism of PD size exclusion limit (SEL). Plant viral movement proteins (MPs) enhance the invasion of viruses from cell to cell by increasing the SEL of the PD and are therefore a powerful means for the study of the plasmodesmal regulation mechanisms. In a recent study, we reported that the actin cytoskeleton is involved in the increase of the PD SEL induced by MPs. Microinjection experiments demonstrated that actin depolymerization was required for the Cucumber mosaic virus (CMV) MP-induced increase in the PD SEL. In vitro experiments showed that CMV MP severs actin filaments (F-actin). Furthermore, through the analyses of two CMV MP mutants, we demonstrated that the F-actin severing ability of CMV MP was required to increase the PD SEL. These results are similar to what has been found in Tobacco mosaic virus MP. Thus, our data suggest that actin dynamics may participate in the regulations of the PD SEL.
plasmodesmata; size exclusion limit; movement protein; actin filaments; F-actin severing
Cell-to-cell signal transduction is vital for orchestrating the whole-body physiology of multi-cellular organisms, and many endogenous macromolecules, proteins, and nucleic acids function as such transported signals. In plants, many of these molecules are transported through plasmodesmata (Pd), the cell wall-spanning channel structures that interconnect plant cells. Furthermore, Pd also act as conduits for cell-to-cell movement of most plant viruses that have evolved to pirate these channels to spread the infection. Pd transport is presumed to be highly selective, and only a limited repertoire of molecules is transported through these channels. Recent studies have begun to unravel mechanisms that actively regulate the opening of the Pd channel to allow traffic. This macromolecular transport between cells comprises two consecutive steps: intracellular targeting to Pd and translocation through the channel to the adjacent cell. Here, we review the current knowledge of molecular species that are transported though Pd and the mechanisms that control this traffic. Generally, Pd traffic can occur by passive diffusion through the trans-Pd cytoplasm or through the membrane/lumen of the trans-Pd ER, or by active transport that includes protein–protein interactions. It is this latter mode of Pd transport that is involved in intercellular traffic of most signal molecules and is regulated by distinct and sometimes interdependent mechanisms, which represent the focus of this article.
Plasmodesmata; cell-to-cell transport; plant viruses; transcription factors
RNA silencing is a natural defense mechanism against genetic stress factors, including viruses. A mutant hordeivirus (Barley stripe mosaic virus [BSMV]) lacking the γb gene was confined to inoculated leaves in Nicotiana benthamiana, but systemic infection was observed in transgenic N. benthamiana expressing the potyviral silencing suppressor protein HCpro, suggesting that the γb protein may be a long-distance movement factor and have antisilencing activity. This was shown for γb proteins of both BSMV and Poa semilatent virus (PSLV), a related hordeivirus. Besides the functions in RNA silencing suppression, γb and HCpro had analogous effects on symptoms induced by the hordeiviruses. Severe BSMV-induced symptoms were correlated with high HCpro concentrations in the HCpro-transgenic plants, and substitution of the γb cistron of BSMV with that of PSLV led to greatly increased symptom severity and an altered pattern of viral gene expression. The efficient systemic infection with the chimera was followed by the development of dark green islands (localized recovery from infection) in leaves and exemption of new developing leaves from infection. Recovery and the accumulation of short RNAs diagnostic of RNA silencing in the recovered tissues in wild-type N. benthamiana were suppressed in HCpro-transgenic plants. These results provide evidence that potyviral HCpro and hordeivirus γb proteins contribute to systemic viral infection, symptom severity, and RNA silencing suppression. HCpro's ability to suppress the recovery of plants from viral infection emphasizes recovery as a manifestation of RNA silencing.
Plasmodesmata—intercellular channels that communicate adjacent cells—possess complex membranous structures. Recent evidences indicate that plasmodesmata contain membrane microdomains. In order to understand how these submembrane regions collaborate to plasmodesmata function, it is necessary to characterize their size, composition and dynamics. An approach that can shed light on these microdomain features is based on the use of Arabidopsis mutants in sphingolipid synthesis. Sphingolipids are canonical components of microdomains together with sterols and some glycerolipids. Moreover, sphingolipids are transducers in pathways that display programmed cell death as a defense mechanism against pathogens. The study of Arabidopsis mutants would allow determining which structural features of the sphingolipids are important for the formation and stability of microdomains, and if defense signaling networks using sphingoid bases as second messengers are associated to plasmodesmata operation. Such studies need to be complemented by analysis of the ultrastructure and the use of protein probes for plasmodesmata microdomains and may constitute a very valuable source of information to analyze these membrane structures.
sphingolipid Arabidopsis mutants; sphingolipids and microdomains; long chain bases; sphingoid bases; microdomains and plasmodesmata
Many plant viruses encode for specialized movement proteins (MP) to facilitate passage of viral material to and through plasmodesmata (PD). To analyze intracellular trafficking of potato leaf roll virus (PLRV) movement protein (MP17) we performed GFP fusion experiments with distinct deletion variants of MP17. These studies revealed that the C-terminus of MP17 is essential but not sufficient for PD targeting. Interestingly, fusion of GFP to three C-terminal MP17 deletion variants resulted in the accumulation of GFP in chloroplasts. This indicates that MP17 harbors hidden plastid targeting sequences. Previous studies showed that posttranslational protein phosphorylation influences PD targeting of MP and virus spread. Analysis of MP17-derived phospho-peptides by mass spectrometry revealed four phosphorylated serine residues (S71, S79, S137, and S140). Site-directed mutagenesis of S71/S79 and S137/S140 showed that the C-terminal serine residues S137/S140 are dispensable for PD targeting. However, exchange of S71/S79 to A71/A79 abolished PD targeting of the mutated MP17 protein. To mimic phosphorylation of S71/S79 both amino acids were substituted by aspartic acid. The resulting D71/D79 variant of MP17 was efficiently targeted to PD. Further deletion analysis showed that PD targeting of MP17 is dependent on the C-terminus, phosphorylation of S71 and/or S79 and a N-terminal domain.
MP17; PLRV; phosphorylation; posttranslational modification; intracellular trafficking; plasmodesmata
Plant virus movement proteins compartmentalize replication complexes at plasmodesmata for localized RNA synthesis and directional trafficking of the virus between cells.
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.
In plant development, cell-to-cell signaling is mediated by mobile signals, including transcription factors and small RNA molecules. This communication is essential for growth and patterning. Short-range movement of signals occurs in the extracellular space via the apoplastic pathway or directly from cell-to-cell via the symplastic pathway. Symplastic transport is mediated by plant specific structures called plasmodesmata, which are plasma membrane-lined pores that traverse the cell walls of adjacent cells thus connecting their cytoplasms. However, a thorough understanding of molecules moving via plasmodesmata and regulatory networks relying on symplastic signaling is lacking. Traffic via plasmodesmata is highly regulated, and callose turnover is known to be one mechanism. In Arabidopsis, plasmodesmata apertures can be regulated in a spatially and temporally specific manner with the icals3m, an inducible vector system expressing the mutated CalS3 gene encoding a plasmodesmata localized callose synthase that increases callose deposition at plasmodesmata. We discuss strategies to use the icals3m system for global analyses on symplastic signaling in plants.
symplastic communication; mobile signal; Arabidopsis; root development; plasmodesmata; callose