Plasma Membrane Localization of Solanum tuberosum Remorin from Group 1, Homolog 3 Is Mediated by Conformational Changes in a Novel C-Terminal Anchor and Required for the Restriction of Potato Virus X Movement]

The formation of plasma membrane (PM) microdomains plays a crucial role in the regulation of membrane signaling and trafficking. Remorins are a plant-specific family of proteins organized in six phylogenetic groups, and Remorins of group 1 are among the few plant proteins known to specifically associate with membrane rafts. As such, they are valuable to understand the molecular bases for PM lateral organization in plants. However, little is known about the structural determinants underlying the specific association of group 1 Remorins with membrane rafts. We used a structure-function approach to identify a short C-terminal anchor (RemCA) indispensable and sufficient for tight direct binding of potato (Solanum tuberosum) REMORIN 1.3 (StREM1.3) to the PM. RemCA switches from unordered to α-helical structure in a nonpolar environment. Protein structure modeling indicates that RemCA folds into a tight hairpin of amphipathic helices. Consistently, mutations reducing RemCA amphipathy abolished StREM1.3 PM localization. Furthermore, RemCA directly binds to biological membranes in vitro, shows higher affinity for Detergent-Insoluble Membranes lipids, and targets yellow fluorescent protein to Detergent-Insoluble Membranes in vivo. Mutations in RemCA resulting in cytoplasmic StREM1.3 localization abolish StREM1.3 function in restricting potato virus X movement. The mechanisms described here provide new insights on the control and function of lateral segregation of plant PM.Protein-lipid interactions are increasingly recognized as key regulatory processes for signal perception and cellular signaling cascades (Cho and Stahelin, 2005). During signal transduction and trafficking, a number of soluble proteins dynamically associate with plasma membranes (PMs) to deliver their cargo and to recruit pathway components to the sites of action (Seong et al., 2011). For such proteins, membrane association can be critical for function (Porter and Koelle, 2010).PM targeting of peripheral proteins is achieved through (1) binding to integral membrane proteins, (2) posttranslational modifications, or (3) directly by intrinsic membrane anchor domains. Posttranslational modifications function as auxiliary modifications for transient or weak association of soluble proteins to the intracellular face of the PM. In plants, these include N-myristoylation, S-palmitoylation, prenylation by farnesyl or geranylgeranyl moieties, or attachment of glycosylphosphatidylinositol (GPI) anchors (Thompson and Okuyama, 2000). GPI anchors, for example, tightly associate proteins to the extracellular face of PMs by interaction of the inositol head group of the membrane lipid phosphatidylinositol with a glucosamine residue linked to the C-terminal amino acid of the protein (Paulick and Bertozzi, 2008). As an alternative mechanism, globular structures either recognize phospholipids in a stereospecific manner or associate with membranes by their biophysical properties (for review, see Lemmon, 2008). Other proteins expose unstructured clusters of basic and hydrophobic residues to mediate PM binding (McLaughlin et al., 2002; McLaughlin and Murray, 2005).Selective recognition of membrane compartments or domains by protein anchors can be critical in triggering the appropriate downstream trafficking and signaling events (for review, see Gruenberg, 2003; De Matteis and Godi, 2004). Membrane domain selectivity can be specified by the anchoring posttranslational modification or by a protein anchor domain. For instance, proteins carrying GPI anchors are overrepresented in membrane rafts, indicating that addition of this lipid anchor directs proteins to these microdomains (Cordy et al., 2003; Kierszniowska et al., 2009). Membrane rafts are enriched in highly saturated long-chain sphingolipids, sterols, and saturated phospholipids, creating tightly packed domains, designated as “liquid ordered.” These lipids display a stronger affinity to saturated acyl chains as found in GPI-anchored and acylated proteins (Brown, 2006). The composition of membrane rafts also prevents solubilization by detergent at low temperature with nonionic detergent and allows the partial purification of rafts in so-called Detergent-Insoluble Membrane (DIM) fractions, which are supposedly biochemical counterparts of membrane rafts. Many signaling proteins are found in membrane rafts, supporting the hypothesis that they serve as key platforms for cellular signal transduction and cell-to-cell communication (Lingwood and Simons, 2010; Simon-Plas et al., 2011). For example, in human (Homo sapiens) cells, key soluble signaling components such as the Ser/Thr kinase Akt (protein kinase B) are recruited to membrane rafts where they activate signal transduction cascades (Lasserre et al., 2008). Nevertheless, few protein motifs were described to contribute to raft targeting (Rossin et al., 2010), although the 6-amino-acid-long raft target signal from human Tyr phosphatase Src homology 2-containing phosphatase1 is the only known motif sufficient to anchor soluble proteins specifically to domains of the intracellular face of the PM (Sankarshanan et al., 2007). However, the anchoring mechanism itself remains to be unraveled in plants even though DIMs also exist (Mongrand et al., 2010) and functional PM domains have been reported (Bhat et al., 2005). The molecular basis for specific targeting and binding of proteins to membrane rafts has never been described.Remorins form a diverse family of plant-specific proteins organized in six distinct phylogenetic groups (Raffaele et al., 2007). Remorins from group 1 have been reported to localize to the PM despite their overall hydrophilic nature (Reymond et al., 1996; Raffaele et al., 2007). Moreover, group 1 Remorins almost exclusively associate to DIMs and localize to membrane microdomains in a sterol-dependent manner (Lefebvre et al., 2007; Kierszniowska et al., 2009; Raffaele et al., 2009). The function of Remorins is mostly unknown, but we showed in a previous study that StREM1.3 (for potato (Solanum tuberosum) Remorin from group 1, homolog 3; initially described in Reymond et al., 1996) regulates cell-to-cell propagation of the potato virus X (PVX), likely by directly interacting with the viral movement protein Triple Gene Block protein1 (TGBp1; Raffaele et al., 2009). StREM1.3 localizes to the inner leaflet of PMs and along plasmodesmata, bridges connecting neighbor cells essential for cell-to-cell communication in plants (Maule, 2008). Other members of the Remorin family group 1 are likely involved in innate immune responses (Liu et al., 2009; Widjaja et al., 2009; Keinath et al., 2010). Remorins from group 2 are involved in the control of infection by symbiotic bacteria at nodular infection threads and the peribacteroid membrane (Lefebvre et al., 2010) These data suggest general roles for Remorins in regulating signaling in plant-microbe interactions (Jarsch and Ott, 2011).Elucidating the mechanisms driving StREM1.3 association with PM microdomains therefore provides a unique opportunity for understanding the regulation and function of membrane lateral segregation in plants. StREM1.3 does not contain predictable transmembrane or membrane-associated domains. The bases for its association to PMs and selective targeting to DIMs are unknown. Here we identified a novel membrane anchor domain required for StREM1.3 tight and direct association with the detergent-insoluble fraction of the PM. We combined biophysics, in silico analysis, and directed mutagenesis to unravel the molecular bases of StREM1.3 membrane binding and its biological significance in the control of PVX propagation.     

Potato virus X: structure, disassembly and reconstitution

This paper summarizes some structural characteristics of Potato virus X (PVX), the flexuous filamentous plant potexvirus. A model of PVX coat protein (CP) tertiary structure in the virion proposed on the basis of tritium planigraphy combined with predictions of the protein tertiary structure is described. A possible role of glycosylation and phosphorylation in the CP structure and function is discussed. Two forms of PVX virion disassembly are discussed: (i) the virion co-translational disassembly after PVX CP in situ phosphorylation and (ii) disassembly of PVX triggered by different factors after linear destabilization of the virion by binding of the PVX-coded movement protein (TGBp1) to one end of the polar CP-helix. Special emphasis was placed on a translational activation of encapsidated PVX RNA and rapid disassembly of TGBp1-PVX complexes into free RNA and CP. The results of experiments on the PVX CP repolymerization and PVX reconstitution are considered. In particular, the products assembled from PVX RNA, CP and TGBp1 were examined. Single-tailed particles were found with a helical, head-like structure consisting of helically arranged CP subunits located at the 5'-tail of RNA; the TGBp1 was bound to the end of the head. Translatable 'RNA-CP-TGBp1' complexes may represent the transport form of the PVX infection.     

Virus Entry, Assembly, Budding, and Membrane Rafts

As intracellular parasites, viruses rely heavily on the use of numerous cellular machineries for completion of their replication cycle. The recent discovery of the heterogeneous distribution of the various lipids within cell membranes has led to the proposal that sphingolipids and cholesterol tend to segregate in microdomains called membrane rafts. The involvement of membrane rafts in biosynthetic traffic, signal transduction, and endocytosis has suggested that viruses may also take advantage of rafts for completion of some steps of their replication cycle, such as entry into their cell host, assembly, and budding. In this review, we have attempted to delineate all the reliable data sustaining this hypothesis and to build some models of how rafts are used as platforms for assembly of some viruses. Indeed, if in many cases a formal proof of raft involvement in a virus replication cycle is still lacking, one can reasonably suggest that, owing to their ability to specifically attract some proteins, lipid microdomains provide a particular milieu suitable for increasing the efficiency of many protein-protein interactions which are crucial for virus infection and growth.     

Potato virus X as a vector for gene expression in plants

The suitability of potato virus X (PVX) as a gene vector in plants was tested by analysis of two viral constructs. In the first, the GUS gene of Escherichia coli was substituted for the viral coat protein gene. In the second, GUS was added into the viral genome coupled to a duplicated copy of the viral promoter for the coat protein mRNA. The viral construct with the substituted coat protein gene accumulated poorly in inoculated protoplasts and failed to spread from the site of infection in plants. These results suggest a role for the viral coat protein in key stages of the viral infection cycle and show that gene replacement constructs are not suitable for the production of PVX-based gene vector. The construct with GUS coupled to the duplicated promoter for coat protein mRNA also accumulated less well in protoplasts than the unmodified PVX, but did infect systemically and directed high level synthesis of GUS in inoculated and systemically infected tissue. Although there was some genome instability in the PVX construct, much of the viral RNA in the systemically infected tissue had retained the foreign gene insertion, especially in infected Nicotiana clevelandii plants. These data point to a general utility of PVX as a vector for unregulated gene expression in plants.     

Purification of Potato Leaf Plasma Membrane Protein pp34, a Protein Phosphorylated in Response to Oligogalacturonide Signals for Defense and Development

A potato (Solanum tuberosum L.) plasma membrane protein called pp34, the only known example of a plasma membrane protein that is phosphorylated specifically in response to defined Oligogalacturonide signals in plants, has been purified to apparent homogeneity. Polyclonal antibodies raised in rabbits against the purified pp34 protein immunoprecipitated a single thiophosphorylated protein species from potato plasma membranes, as analyzed by two-dimensional denaturing electrophoresis and fluorography. The pp34 antibodies also recognized a single protein in tomato (Lycopersicon esculentum L.) membranes that is thiophosphorylated in response to Oligogalacturonide elicitors, as demonstrated by western blotting and specific immunoprecipitation. These experiments confirm the identity of the tomato membrane protein as a pp34 homolog and establish the high monospecificity of the pp34 antibodies. This will permit further investigation of the role of protein phosphorylation in oligouronide signaling for defensive genes in potato and tomato plants.     

Polyphosphoinositides Are Enriched in Plant Membrane Rafts and Form Microdomains in the Plasma Membrane

In this article, we analyzed the lipid composition of detergent-insoluble membranes (DIMs) purified from tobacco (Nicotiana tabacum) plasma membrane (PM), focusing on polyphosphoinositides, lipids known to be involved in various signal transduction events. Polyphosphoinositides were enriched in DIMs compared with whole PM, whereas all structural phospholipids were largely depleted from this fraction. Fatty acid composition analyses suggest that enrichment of polyphosphoinositides in DIMs is accompanied by their association with more saturated fatty acids. Using an immunogold-electron microscopy strategy, we were able to visualize domains of phosphatidylinositol 4,5-bisphosphate in the plane of the PM, with 60% of the epitope found in clusters of approximately 25 nm in diameter and 40% randomly distributed at the surface of the PM. Interestingly, the phosphatidylinositol 4,5-bisphosphate cluster formation was not significantly sensitive to sterol depletion induced by methyl-β-cyclodextrin. Finally, we measured the activities of various enzymes of polyphosphoinositide metabolism in DIMs and PM and showed that these activities are present in the DIM fraction but not enriched. The putative role of plant membrane rafts as signaling membrane domains or membrane-docking platforms is discussed.Polyphosphoinositides are phosphorylated derivatives of phosphatidylinositol (PtdIns) implicated in many aspects of cell function. They control a surprisingly large number of processes in animal, yeast, and plant cells, including exocytosis, endocytosis, cytoskeletal adhesion, and signal transduction not only as second-messenger precursors but also as signaling molecules on their own by interacting with protein partners, allowing spatially selective regulation at the cytoplasm-membrane interface (for review, see Di Paolo and De Camilli, 2006). Polyphosphoinositides also control the activity of ion transporters and channels during biosynthesis or vesicle trafficking (Liu et al., 2005; Monteiro et al., 2005b). In plants, phosphatidylinositol 4,5-bisphosphate [PtdIns(4,5)P₂] is present in very small quantities (for review, see Stevenson et al., 2000; Meijer and Munnik, 2003) and was visualized in vivo by expressing a fluorescent protein (GFP or yellow fluorescent protein) fused to the pleckstrin homology (PH) domain of the human phospholipase C δ1 (PLCδ1) that specifically binds PtdIns(4,5)P₂. The fused protein yellow fluorescent protein-PHPLCδ1 was present in the cytoplasm but concentrated at the plant plasma membrane (PM) in response to salt stress or upon treatment with the PLC inhibitor {"type":"entrez-nucleotide","attrs":{"text":"U73122","term_id":"4098075","term_text":"U73122"}}U73122 (van Leeuwen et al., 2007). In pollen tubes and root hairs, where spatially focused cell expansion occurs, highly localized PtdIns(4,5)P₂ has been evidenced at the membrane tip (Braun et al., 1999; Kost et al., 1999). PtdIns(4,5)P₂ likely functions as an effector of small G proteins at the apex of cells influencing membrane fusion events (Monteiro et al., 2005a). In guard cells, the level of PtdIns(4,5)P₂ increases at the PM upon illumination (Lee et al., 2007). Combining imaging, patch clamp, and genetic evidence, Lee et al. (2007) further proposed that PtdIns(4,5)P₂ is important for stomatal opening. Stomatal guard cells have also been reported to contain phosphatidylinositol 3-phosphate (PtdIns3P) and phosphatidylinositol 4-phosphate (PtdIns4P), the products of PtdIns 3-kinase and PtdIns 4-kinase activities, respectively. Jung et al. (2002) demonstrated that PtdIns3P and PtdIns4P play an important role in the modulation of stomatal closing and that reductions in the levels of functional PtdIns3P and PtdIns4P enhance stomatal opening. Recently, the hyperosmotic stress response was studied in Arabidopsis (Arabidopsis thaliana). Several groups (Pical et al., 1999; DeWald et al., 2001; Konig et al., 2007, 2008b) have shown that plants exhibit a transient increase in polyphosphoinositides after hyperosmotic stress, providing a model for comparing constitutive and stress-inducible polyphosphoinositide pools. Under nonstress conditions, structural phospholipids and PtdIns contained 50 to 70 mol % polyunsaturated fatty acids (PUFA), whereas polyphosphoinositides were more saturated (10–20 mol % PUFA; Konig et al., 2007). Upon hyperosmotic stress, polyphosphoinositides with up to 70 mol % PUFA were formed that differed from constitutive species and coincided with a transient loss in unsaturated PtdIns. These patterns indicate the inducible turnover of an unsaturated PtdIns pool and the presence of distinct polyphosphoinositide pools in plant membranes (Konig et al., 2007).Since these biological phenomena are likely to occur in distinct regions of the PM, it has been our working hypothesis that in plant cells polyphosphoinositides are localized in various microdomains to participate in different cellular functions. Two decades ago, Metcalf et al. (1986) already suggested that the plant PM contains stable immiscible domains of fluid and gel-like lipids using fluorescent lipid and phospholipid probes incorporated into soybean (Glycine max) protoplasts prepared from cultured soybean cells. To this day, it has been generally accepted that lipids and proteins of the PM are not homogeneously distributed within membranes but rather form various domains of localized enrichment. The best-characterized membrane domains are membrane rafts (MRs; Pike, 2006). MRs are liquid-ordered subdomains within eukaryotic membranes that are hypothesized to play important roles in a variety of biological functions by coordinating and compartmentalizing diverse sets of proteins to facilitate signal transduction mechanisms, focal regulation of cytoskeleton, and membrane trafficking (for review, see Rajendran and Simons, 2005; Brown, 2006). Both evidenced in plants and animals, MRs are enriched in sphingolipids and sterols and largely deprived in phospholipids (for review, see Brown and London, 2000; Bhat and Panstruga, 2005). Sterols interact preferentially, although not exclusively, with sphingolipids due to their structure and the saturation of their hydrocarbon chains. Because of the rigid nature of the sterol group, sterols have the ability to pack in between the lipids in rafts, serving as molecular spacers and filling voids between associated sphingolipids (Binder et al., 2003). Acyl chains of MR lipids tend to be more rigid and in a less fluid state (Roche et al., 2008). In agreement, the hydrophobic chains of the phospholipids within the raft are more saturated and tightly packed than those of lipids in the surrounding bilayer (Mongrand et al., 2004). MRs can be isolated from PM by extraction with nonionic detergents such as Triton X-100 (TX100) or Brij-98 at low temperatures. Fluid nonraft domains will solubilize while the MRs remain intact and can be enriched after centrifugation, floating in a Suc density gradient. Floating purified fractions, therefore, are called detergent-insoluble membranes (DIMs) or detergent-resistant membranes and are thought to be the biochemical counterpart of in vivo MRs.In plants, a few results suggest the role in vivo of dynamic clustering of PM proteins, and they refer to plant-pathogen interaction. A cell biology study reported the pathogen-triggered focal accumulation of components of the plant defense pathway in the PM, a process reminiscent of MRs (Bhat et al., 2005). The proteomic analysis of tobacco (Nicotiana tabacum) DIMs led to the identification of 145 proteins, among which a high proportion were linked to signaling in response to biotic stress, cellular trafficking, and cell wall metabolism (Morel et al., 2006). Therefore, these domains are likely to constitute, as in animal cells, signaling membrane platforms concentrating lipids and proteins necessary for the generation of signaling molecules of physiological relevance. This hypothesis was confirmed by a quantitative proteomic study describing the dynamic association of proteins with DIMs upon challenge of tobacco cells with an elicitor of defense reaction (Stanislas et al., 2009). Recently, Raffaele et al. (2009) showed that a group of proteins specific to vascular plants, called remorins (REMs), share the biochemical properties of other MR proteins and are clustered into microdomains of approximately 70 nm in diameter in the PM and plasmodesmata in tobacco, providing a link between biochemistry (DIM purification) and imaging (membrane microdomain observation).Several investigators have previously suggested that PtdIns(4,5)P₂-rich raft assemblies exist in animal cell membranes to provide powerful organizational principles for tight spatial and temporal control of signaling in motility. Laux et al. (2000) demonstrated that PtdIns(4,5)P₂ formed microdomains in the PM of animal cells, and at least part of these microdomains was colocalized with the myristoylated Ala-rich type C kinase substrate, a protein enriched in MRs, and involved in the regulation of the actin cytoskeleton. The relationship between the spatial organization of PtdIns(4,5)P₂ microdomains and exocytotic machineries has been evidenced in rat. Both PtdIns(4,5)P₂ and syntaxin, a protein essential for exocytosis, exhibited punctate clusters in isolated PM. PtdIns(4,5)P₂ also accumulated at sites of cell surface motility together with a Rho-type GTPase. Therefore, PtdIns(4,5)P₂ may coordinate membrane dynamics and actin organization as well as integrate signaling (Aoyagi et al., 2005). These results provide evidence of compartmentalization of PtdIns(4,5)P₂-dependent signaling in cell membranes.Little is known in plants about whether and how separate pools of polyphosphoinositides come about and how they are regulated. In this article, we have analyzed the lipid composition of DIMs enriched from tobacco PM, with a particular focus on phospholipids involved in signaling events, such as polyphosphoinositides. We showed that polyphosphoinositides were enriched in DIMs, whereas structural phospholipids were largely excluded. We were able to calculate that almost half of the PtdInsP and PtdIns(4,5)P₂ were present in MR domains. Fatty acid composition analyses demonstrate that this enrichment is accompanied by the presence of more saturated fatty acids in polyphosphoinositides. Consistently, using an electron microscopy approach with immunogold labeling and a pattern-identifying statistical analysis, we showed that more than half of the PtdIns(4,5)P₂ labeling is clustered into microdomains of approximately 25 nm in diameter in the PM. Finally, we measured the activities of lipid-using enzymes present in DIMs/PM and showed that activities responsible for polyphosphoinositide metabolism are present in the DIM fraction.     

Association of the P6 Protein of Cauliflower mosaic virus with Plasmodesmata and Plasmodesmal Proteins

The P6 protein of Cauliflower mosaic virus (CaMV) is responsible for the formation of inclusion bodies (IBs), which are the sites for viral gene expression, replication, and virion assembly. Moreover, recent evidence indicates that ectopically expressed P6 inclusion-like bodies (I-LBs) move in association with actin microfilaments. Because CaMV virions accumulate preferentially in P6 IBs, we hypothesized that P6 IBs have a role in delivering CaMV virions to the plasmodesmata. We have determined that the P6 protein interacts with a C2 calcium-dependent membrane-targeting protein (designated Arabidopsis [Arabidopsis thaliana] Soybean Response to Cold [AtSRC2.2]) in a yeast (Saccharomyces cerevisiae) two-hybrid screen and have confirmed this interaction through coimmunoprecipitation and colocalization assays in the CaMV host Nicotiana benthamiana. An AtSRC2.2 protein fused to red fluorescent protein (RFP) was localized to the plasma membrane and specifically associated with plasmodesmata. The AtSRC2.2-RFP fusion also colocalized with two proteins previously shown to associate with plasmodesmata: the host protein Plasmodesmata-Localized Protein1 (PDLP1) and the CaMV movement protein (MP). Because P6 I-LBs colocalized with AtSRC2.2 and the P6 protein had previously been shown to interact with CaMV MP, we investigated whether P6 I-LBs might also be associated with plasmodesmata. We examined the colocalization of P6-RFP I-LBs with PDLP1-green fluorescent protein (GFP) and aniline blue (a stain for callose normally observed at plasmodesmata) and found that P6-RFP I-LBs were associated with each of these markers. Furthermore, P6-RFP coimmunoprecipitated with PDLP1-GFP. Our evidence that a portion of P6-GFP I-LBs associate with AtSRC2.2 and PDLP1 at plasmodesmata supports a model in which P6 IBs function to transfer CaMV virions directly to MP at the plasmodesmata.Through the years, numerous studies have focused on the characterization of viral replication sites within the cell, as well as how plant virus movement proteins (MPs) modify the plasmodesmata to facilitate cell-to-cell movement (for review, see Benitez-Alfonso et al., 2010; Laliberté and Sanfaçon, 2010; Niehl and Heinlein, 2011; Ueki and Citovsky, 2011; Verchot, 2012). It is accepted that plant virus replication is associated with host membranes, and at some point, the viral genomic nucleic acid must be transferred from the site of replication in the cell to the plasmodesmata. This step could involve transport from a distant site within the cell, or alternatively, it may be that replication is coupled with transport at the entrance of the plasmodesmata (Tilsner et al., 2013). However, even with the latter model, there is ample evidence that the viral proteins necessary for replication or cell-to-cell movement utilize intracellular trafficking pathways within the cell to become positioned at the plasmodesma. These pathways may involve microfilaments, microtubules, or specific endomembranes that participate in macromolecular transport pathways, or combinations of these elements (Harries et al., 2010; Schoelz et al., 2011; Patarroyo et al., 2012; Peña and Heinlein, 2012; Tilsner and Oparka 2012; Liu and Nelson, 2013).The P6 protein of Cauliflower mosaic virus (CaMV) is one viral protein that had not been considered to play a role in viral movement until recently. P6 is the most abundant protein component of the amorphous, electron-dense inclusion bodies (IBs) present during virus infection (Odell and Howell, 1980; Shockey et al., 1980). Ectopic expression of P6 in Nicotiana benthamiana leaves resulted in the formation of inclusion-like bodies (I-LBs) that were capable of intracellular movement along actin microfilaments. Furthermore, treatment of Nicotiana edwardsonii leaves with latrunculin B abolished the formation of CaMV local lesions, suggesting that intact microfilaments are required for CaMV infection (Harries et al., 2009a). A subsequent paper showed that P6 physically interacts with Chloroplast Unusual Positioning1 (CHUP1), a plant protein localized to the chloroplast outer membrane that contributes to movement of chloroplasts on microfilaments in response to changes in light intensity (Oikawa et al., 2003, 2008; Angel et al., 2013). The implication was that P6 might hijack CHUP1 to facilitate movement of the P6 IBs on microfilaments. Silencing of CHUP1 in N. edwardsonii, a host for CaMV, slowed the rate of local lesion formation, suggesting that CHUP1 contributes to intracellular movement of CaMV (Angel et al., 2013).In addition to its role in intracellular trafficking, the P6 protein has been shown to have at least four other distinct functions in the viral infection cycle. P6-containing IBs induced during virus infection are likely virion factories, as they are the primary site for CaMV protein synthesis, genome replication, and assembly of virions (Hohn and Fütterer, 1997). Second, P6 interacts with host ribosomes to facilitate reinitiation of translation of genes on the polycistronic 35S viral RNA, a process called translational transactivation (Bonneville et al., 1989; Park et al., 2001; Ryabova et al., 2002). The translational transactivator region of P6 (Fig. 1) defines the essential sequences required for translational transactivation (DeTapia et al., 1993). Third, P6 is an important pathogenicity determinant. P6 functions as an avirulence determinant in some solanaceous and cruciferous species (Daubert et al., 1984; Schoelz et al., 1986; Hapiak et al., 2008) and is a chlorosis symptom determinant in susceptible hosts (Daubert et al., 1984; Baughman et al., 1988; Goldberg et al., 1991; Cecchini et al., 1997). Finally, P6 has the capacity to compromise host defenses, as it is a suppressor of RNA silencing and cell death (Love et al., 2007; Haas et al., 2008), and it modulates signaling by salicylic acid, jasmonic acid, ethylene, and auxin (Geri et al., 2004; Love et al., 2012; Laird et al., 2013). Domain D1 of P6 has been shown to be necessary but not sufficient for suppression of silencing and salicylic acid-mediated defenses (Laird et al., 2013).Open in a separate windowFigure 1.CaMV and host constructs used for confocal microscopy or coimmunoprecipitation (co-IP). A, Structure of CaMV P6 and Arabidopsis (Arabidopsis thaliana) Soybean Response to Cold (AtSRC2.2) proteins. The functions of P6 domains D1 to D4 tested for interaction with AtSRC2.2 are indicated by the shaded boxes. The Mini TAV is the minimal region for the translational transactivation function. The NLSa sequence corresponds to the nuclear localization signal of influenza virus. The NLS sequence corresponds to the nuclear localization signal of human ribosomal protein L22. B, Structure of P6 (Angel et al., 2013), AtSRC2.2, PDLP (Thomas et al., 2008), and CaMV MP fusions developed for confocal microscopy and/or co-IP. aa, Amino acid.Because P6-containing IBs are the site for virion accumulation and they are capable of movement, they may be responsible for delivering virions to the CaMV MP located at the plasmodesmata (for review, see Schoelz et al., 2011). The vast majority of CaMV virions accumulate in association with P6-containing IBs. Furthermore, P6 physically interacts with the CaMV capsid and MP, as well as the two proteins necessary for aphid transmission, P2 and P3 (Himmelbach et al., 1996; Ryabova et al., 2002; Hapiak et al., 2008; Lutz et al., 2012). Recent studies have indicated that P6 IBs serve as a reservoir for virions, in which the virions may be rapidly transferred to P2 electron-lucent IBs for acquisition by aphids (Bak et al., 2013). It stands to reason that P6 IBs may also serve as a reservoir for CaMV virions to be transferred to the CaMV MP in the plasmodesmata.CaMV virions move from cell to cell through plasmodesmata modified into tubules through the function of its MP (Perbal et al., 1993; Kasteel et al., 1996). However, studies have suggested that CaMV virions do not appear to directly interact with the MP. Instead, the MP interacts with the CaMV P3 protein (also known as the virion-associated protein [VAP]), which forms a trimeric structure that is anchored into the virions (Leclerc et al., 1998; Leclerc et al., 2001). Electron microscopy studies have indicated that MP and VAP colocalize with virions only at the entrance to or within the plasmodesmata, and it has been suggested that the VAP/virion complex travels to the plasmodesmata independently from the MP (Stavolone et al., 2005). Consequently, there is a need for a second CaMV protein such as P6 to fulfill the role of delivery of virions to the plasmodesmata (Schoelz et al., 2011).Additional studies have shown that the CaMV MP is incorporated into vesicles and is trafficked on the endomembrane system to reach the plasmodesma (Carluccio et al., 2014). These authors suggest that the CaMV MP is recycled in a vesicular transport pathway between plasmodesmata and early endosome compartments. The CaMV MP interacts with µA-Adaptin (Carluccio et al., 2014) and Movement Protein-Interacting7 (Huang et al., 2001), two proteins shown to have a role in vesicular trafficking. Once the MP arrives at plasmodesmata, it interacts with the Plasmodesmata-Localized Protein (PDLP) proteins, which comprise a family of eight proteins associated with plasmodesmata (Amari et al., 2010). In addition to its interaction with CaMV MP, PDLP1 interacts with the 2B protein of Grapevine fan leaf virus (GFLV) at the base of tubules formed by the 2B protein. Furthermore, an Arabidopsis transfer DNA (T-DNA) mutant line in which three PDLP genes had been knocked out (pdlp1-pdlp2-pdlp3) responded to GFLV and CaMV inoculation with a delayed infection (Amari et al., 2010). This has led to the suggestion that the PDLPs might act as receptors for the MPs of the tubule-forming viruses such as GFLV and CaMV (Amari et al., 2010, 2011).To better understand the function of the P6 protein during CaMV intracellular movement, we have utilized a yeast (Saccharomyces cerevisiae) two-hybrid assay to identify host proteins that interact with CaMV P6. We show that P6 physically interacts with a C2-calcium-dependent protein (designated AtSRC2.2). AtSRC2.2 is a membrane-bound protein that is capable of forming punctate spots associated with plasmodesmata. The localization of AtSRC2.2 with plasmodesmata led to an analysis of interactions between P6 I-LBs, AtSRC2.2, PDLP1, and the CaMV MP and also revealed that a portion of P6 I-LBs are found adjacent to plasmodesmata. These results provide further evidence for a model in which P6 IBs are capable of delivery of virions to plasmodesmata for their transit to other host cells.     

马铃薯X病毒湖南分离物的鉴定与分组研究

从湖南石门采集表现重花叶症状的马铃薯叶片中分离纯化到一株线状病毒HN021.经双链RNA(ds-RNA)抽提、寄主反应测定、病毒粒子和内含体的形状观察, 初步确定该病毒为马铃薯X病毒 (Potato virus X).以ds-RNA作为模板,用相应引物对HN021分离物的ORF4-UTR-ORF5片段进行RT-PCR,得到1kb左右的双链cDNA片段.对该片段进行克隆和测序,并将测序所得的核苷酸序列与Genbank登录的11株不同分离物的相应片段的核苷酸序列进行同源性比较和分析.结果表明,HN021与分离自南美洲的三株分离物(COAT,KPA和HB)的同源性为78.4%~79.4%,与其它8株(分离自亚洲、欧洲、大洋州和北美洲)分离物的同源性为96.4%-97.8%.从氨基酸水平比较,HN021与COAT,KPA和HB三者CP和8kDa蛋白氨基酸序列同源性分别为86.5%~89.0%和74.3%~75.7%,相应地与其它8株分离物的同源性分别为97.1%-98.7% 和97.1%-100%.序列分析的结果证实了HN021分离物为马铃薯X病毒,同时表明PVX明显存在两个组(组Ⅰ和组Ⅱ),HN021和其它来自亚洲、欧洲、大洋州、北美洲分离物的组II,3个南美洲分离物属于组I.     

马铃薯X病毒湖南分离物的鉴定与分组研究

从湖南石门采集表现重花叶症状的马铃薯叶片中分离纯化到一株线状病毒HN021。经双链RNA(ds—RNA)抽提、寄主反应测定、病毒粒子和内含体的形状观察,初步确定该病毒为马铃薯X病毒(Potato virus X)。以ds—RNA作为模板,用相应引物对HN021分离物的ORF4-UTR-ORF5片段进行RT—PCRP得到1kb左右的双链cDNA片段。对该片段进行克隆和测序,并将测序所得的核苷酸序列与Genbank(登录的11株不同分离物的相应片段的核苷酸序列进行同源性比较和分析。结果表明,HN021与分离自南美洲的三株分离物(COAT,KPA和HB)的同源性为78．4％—79．4％,与其它8株(分离自亚洲、欧洲、大洋洲和北美洲)分离物的同源性为96．4％—97．8％。从氨基酸水平比较,HN021与COAT,KPA和HB三者CP和8kDa蛋白氨基酸序列同源性分别为86．5％—89．0％和74．3％—75．7％,相应地与其它8株分离物的同源性分别为97．1％—98．7％和97．1％—100％。序列分析的结果证实了HN021分离物为马铃薯X病毒,同时表明PVX明显存在两个组(组Ⅰ和组Ⅱ),HN021和其它来自亚洲、欧洲、大洋洲、北美洲分离物的组Ⅱ,3个南美洲分离物属于组Ⅰ。     

Grain setting defect1, Encoding a Remorin Protein,Affects the Grain Setting in Rice through Regulating Plasmodesmatal Conductance

Effective grain filling is one of the key determinants of grain setting in rice (Oryza sativa). Grain setting defect1 (GSD1), which encodes a putative remorin protein, was found to affect grain setting in rice. Investigation of the phenotype of a transfer DNA insertion mutant (gsd1-Dominant) with enhanced GSD1 expression revealed abnormalities including a reduced grain setting rate, accumulation of carbohydrates in leaves, and lower soluble sugar content in the phloem exudates. GSD1 was found to be specifically expressed in the plasma membrane and plasmodesmata (PD) of phloem companion cells. Experimental evidence suggests that the phenotype of the gsd1-Dominant mutant is caused by defects in the grain-filling process as a result of the impaired transport of carbohydrates from the photosynthetic site to the phloem. GSD1 functioned in affecting PD conductance by interacting with rice ACTIN1 in association with the PD callose binding protein1. Together, our results suggest that GSD1 may play a role in regulating photoassimilate translocation through the symplastic pathway to impact grain setting in rice.Grain filling, a key determinant of grain yield in rice (Oryza sativa), hinges on the successful translocation of photoassimilates from the leaves to the fertilized reproductive organs through the phloem transport system. Symplastic phloem loading, which is one of the main pathways responsible for the transport of photoassimilates in rice, is mediated by plasmodesmata (PD) that connect phloem companion cells with sieve elements and surrounding parenchyma cells (Kaneko et al., 1980; Chonan et al., 1981; Eom et al., 2012). PD are transverse cell wall channels structured with the cytoplasmic sleeve and the modified endoplasmic reticulum desmotubule between neighboring cells (Maule, 2008). A number of proteins affect the structure and functional performance of the PD, which in turn impacts the cell-to-cell transport of small and large molecules through the PD during plant growth, development, and defense (Cilia and Jackson, 2004; Sagi et al., 2005; Lucas et al., 2009; Simpson et al., 2009; Stonebloom et al., 2009). For example, actin and myosin, which link the desmotubule to the plasma membrane (PM) at the neck region of PD, are believed to play a role in regulating PD permeability by controlling PD aperture (White et al., 1994; Ding et al., 1996; Reichelt et al., 1999). Callose deposition can also impact the size of the PD aperture at the neck region (Radford et al., 1998; Levy et al., 2007) and callose synthase genes such as Glucan Synthase-Like7 (GSL7, also named CalS7), GSL8, and GSL12 have been shown to play a role in regulating symplastic trafficking (Guseman et al., 2010; Barratt et al., 2011; Vatén et al., 2011; Xie et al., 2011). Other proteins that have been shown to impact the structure and function of the PD include glycosylphosphatidylinositol (GPI)-anchored proteins, PD callose binding protein1 (PDCB1), which is also associated with callose deposition (Simpson et al., 2009), and LYSIN MOTIF DOMAIN-CONTAINING GLYCOSYLPHOSPHATIDYLINOSITOL-ANCHORED PROTEIN2, which limits the molecular flux through the PD by chitin perception (Faulkner et al., 2013). Changes in PD permeability can have major consequences for the translocation of photoassimilates needed for grain filling in rice. However, the genes and molecular mechanisms underlying the symplastic transport of photoassimilates remain poorly characterized.Remorins are a diverse family of plant-specific proteins with conserved C-terminal sequences and highly variable N-terminal sequences. Remorins can be classified into six distinct phylogenetic groups (Raffaele et al., 2007). The functions of most remorins are unknown, but some members of the family have been shown to be involved in immune response through controlling the cell-to-cell spread of microbes. StREM1.3, a remorin that is located in PM rafts and the PD, was shown to impair the cell-to-cell movement of a plant virus X by binding to Triple Gene Block protein1 (Raffaele et al., 2009). Medicago truncatula symbiotic remorin1 (MtSYMREM1), a remorin located at the PM in Medicago truncatula, was shown to facilitate infection and the release of rhizobial bacteria into the host cytoplasm (Lefebvre et al., 2010). Overexpression of LjSYMREM1, the ortholog of MtSYMREM1 in Lotus japonicus, resulted in increased root nodulation (Lefebvre et al., 2010; Tóth et al., 2012). Although a potential association between remorins and PD permeability has been proposed (Raffaele et al., 2009), the diversity observed across remorins, plus the fact that remorin mutants generated through different approaches fail to show obvious phenotypes (Reymond et al., 1996; Bariola et al., 2004), have made it challenging to characterize the function of remorins in cell-to-cell transport.In this study, we identified a rice transfer DNA (T-DNA) insertion mutant (grain setting defect1-Dominant [gsd1-D]), with a grain setting-deficient phenotype caused by overexpression of GSD1, a remorin gene with unknown function. GSD1 is expressed specifically in phloem companion cells and is localized in the PD and PM. We provide evidence to show that overexpression of GSD1 leads to deficient grain setting in rice, likely as a consequence of reduced sugar transport resulting from decreased PD permeability in phloem companion cells.     

Rafts promote assembly and atypical targeting of a nonenveloped virus, rotavirus, in Caco-2 cells

Rotavirus follows an atypical pathway to the apical membrane of intestinal cells that bypasses the Golgi. The involvement of rafts in this process was explored here. VP4 is the most peripheral protein of the triple-layered structure of this nonenveloped virus. High proportions of VP4 associated with rafts within the cell as early as 3 h postinfection. In the meantime a significant part of VP4 was targeted to the Triton X-100-resistant microdomains of the apical membrane, suggesting that this protein possesses an autonomous signal for its targeting. At a later stage the other structural rotavirus proteins were also found in rafts within the cells together with NSP4, a nonstructural protein required for the final stage of virus assembly. Rafts purified from infected cells were shown to contain infectious particles. Finally purified VP4 and mature virus were shown to interact with cholesterol- and sphingolipid-enriched model lipid membranes that changed their phase preference from inverted hexagonal to lamellar structures. Together these results indicate that a direct interaction of VP4 with rafts promotes assembly and atypical targeting of rotavirus in intestinal cells.     

TIP,a novel host factor linking callose degradation with the cell-to-cell movement of Potato virus X

The cell-to-cell movement of Potato virus X (PVX) requires four virus-encoded proteins, the triple gene block (TGB) proteins (TGB25K, TGB12K, and TGB8K) and the coat protein. TGB12K increases the plasmodesmal size exclusion limit (SEL) and may, therefore, interact directly with components of the cell wall or with plant proteins associated with bringing about this change. A yeast two-hybrid screen using TGB12K as bait identified three TGB12K-interacting proteins (TIP1, TIP2, and TIP3). All three TIPs interacted specifically with TGB12K but not with TGB25K or TGB8K. Similarly, all three TIPs interacted with beta-1,3-glucanase, the enzyme that may regulate plasmodesmal SEL through callose degradation. Sequence analyses revealed that the TIPs encode very similar proteins and that TIP1 corresponds to the tobacco ankyrin repeat-containing protein HBP1. A TIP1::GFP fusion protein localized to the cytoplasm. Coexpression of this fusion protein with TGB12K induced cellular changes manifested as deposits of additional cytoplasm at the cell periphery. This work reports a direct link between a viral movement protein required to increase plasmodesmal SEL and a host factor that has been implicated as a key regulator of plasmodesmal SEL. We propose that the TIPs are susceptibility factors that modulate the plasmodesmal SEL.     

The Mammalian Calcium-binding Protein, Nucleobindin (CALNUC), Is a Golgi Resident Protein

We have identified CALNUC, an EF-hand, Ca²⁺-binding protein, as a Golgi resident protein. CALNUC corresponds to a previously identified EF-hand/calcium-binding protein known as nucleobindin. CALNUC interacts with Gα_i3 subunits in the yeast two-hybrid system and in GST-CALNUC pull-down assays. Analysis of deletion mutants demonstrated that the EF-hand and intervening acidic regions are the site of CALNUC's interaction with Gα_i3. CALNUC is found in both cytosolic and membrane fractions. The membrane pool is tightly associated with the luminal surface of Golgi membranes. CALNUC is widely expressed, as it is detected by immunofluorescence in the Golgi region of all tissues and cell lines examined. By immunoelectron microscopy, CALNUC is localized to cis-Golgi cisternae and the cis-Golgi network (CGN). CALNUC is the major Ca²⁺-binding protein detected by ⁴⁵Ca²⁺-binding assay on Golgi fractions. The properties of CALNUC and its high homology to calreticulin suggest that it may play a key role in calcium homeostasis in the CGN and cis-Golgi cisternae.     

A chimeric Potato virus X encoding a heterologous peptide affects Nicotiana benthamiana chloroplast structure

Abstract

The cytopathology of a Potato virus X (PVX) recombinant variant (encoding as fusion of an epitope of immunological interest with the N‐terminus of the coat protein, PVXSmaP18DD) has been compared with that induced by the wild‐type virus (PVX wt) in Nicotiana benthamiana plants. Both PVX wt and PVXSmaP18DD caused similar ultrastructural alterations, characterized by the presence of laminated inclusion components and bulk virus accumulations in mesophyll cells. However, some striking differences were observed not only in the morphology of these accumulations (typically ordered in PVX wt infection and disordered in PVXSmaP18DD infection) but also because the chimeric virus caused peculiar alterations in chloroplasts structure.

Abbreviations: CP, coat protein; d.p.i., days post inoculation; LIC, laminated inclusion components; PVX, Potato virus X