RESUMEN
Remorins are a family of multigenic plasma membrane phosphoproteins involved in biotic and abiotic plant interaction mechanisms, partnering in molecular signaling cascades. Signaling activity of remorins depends on their phosphorylation states and subsequent clustering into nanosized membrane domains. The presence of a coiled-coil domain and a C-terminal domain is crucial to anchor remorins to negatively charged membrane domains; however, the exact role of the N-terminal intrinsically disordered domain (IDD) on protein clustering and lipid interactions is largely unknown. Here, we combine chemical biology and imaging approaches to study the partitioning of group 1 remorin into anionic model membranes mimicking the inner leaflet of the plant plasma membrane. Using reconstituted membranes containing a mix of saturated and unsaturated phosphatidylcholine, phosphatidylinositol phosphates, and sterol, we investigate the clustering of remorins to the membrane and monitor the formation of nanosized membrane domains. REM1.3 promoted membrane nanodomain organization on the exposed external leaflet of both spherical lipid vesicles and flat supported lipid bilayers. Our results reveal that REM1.3 drives a mechanism allowing lipid reorganization, leading to the formation of remorin-enriched nanodomains. Phosphorylation of the N-terminal IDD by the calcium protein kinase CPK3 influences this clustering and can lead to the formation of smaller and more disperse domains. Our work reveals the phosphate-dependent involvement of the N-terminal IDD in the remorin-membrane interaction process by driving structural rearrangements at lipid-water interfaces.
Asunto(s)
Proteínas Portadoras , Proteínas de Plantas , Proteínas Portadoras/metabolismo , Proteínas de Plantas/química , Membrana Celular/metabolismo , Plantas/metabolismo , Membrana Dobles de Lípidos/metabolismoRESUMEN
REMORINs (REMs) are a plant-specific protein family, proposed regulators of membrane-associated molecular assemblies and well-established markers of plasma membrane nanodomains. REMs play a diverse set of functions in plant interactions with pathogens and symbionts, responses to abiotic stresses, hormone signaling and cell-to-cell communication. In this review, we highlight the established and more putative roles of REMs throughout the literature. We discuss the physiological functions of REMs, the mechanisms underlying their nanodomain-organization and their putative role as regulators of nanodomain-associated molecular assemblies. Furthermore, we discuss how REM phosphorylation may regulate their functional versatility. Overall, through data-mining and comparative analysis of the literature, we suggest how to further study the molecular mechanisms underpinning the functions of REMs.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Membrana Celular/metabolismo , Microdominios de Membrana/metabolismo , Proteínas de Plantas/metabolismoRESUMEN
In eukaryotes, membrane contact sites (MCS) allow direct communication between organelles. Plants have evolved a unique type of MCS, inside intercellular pores, the plasmodesmata, where endoplasmic reticulum (ER)-plasma membrane (PM) contacts coincide with regulation of cell-to-cell signalling. The molecular mechanism and function of membrane tethering within plasmodesmata remain unknown. Here, we show that the multiple C2 domains and transmembrane region protein (MCTP) family, key regulators of cell-to-cell signalling in plants, act as ER-PM tethers specifically at plasmodesmata. We report that MCTPs are plasmodesmata proteins that insert into the ER via their transmembrane region while their C2 domains dock to the PM through interaction with anionic phospholipids. A Atmctp3/Atmctp4 loss of function mutant induces plant developmental defects, impaired plasmodesmata function and composition, while MCTP4 expression in a yeast Δtether mutant partially restores ER-PM tethering. Our data suggest that MCTPs are unique membrane tethers controlling both ER-PM contacts and cell-to-cell signalling.
Asunto(s)
Proteínas de Arabidopsis/genética , Arabidopsis/genética , Regulación de la Expresión Génica de las Plantas , Glicosiltransferasas/genética , Proteínas de la Membrana/genética , Plasmodesmos/genética , Arabidopsis/citología , Arabidopsis/crecimiento & desarrollo , Membrana Celular/metabolismo , Células Cultivadas , Retículo Endoplásmico/metabolismo , Perfilación de la Expresión Génica , Regulación del Desarrollo de la Expresión Génica , Genes Reporteros , Glicosiltransferasas/deficiencia , Proteínas Fluorescentes Verdes/genética , Proteínas Fluorescentes Verdes/metabolismo , Humanos , Proteínas Luminiscentes/genética , Proteínas Luminiscentes/metabolismo , Proteínas de la Membrana/deficiencia , Fosfolípidos/metabolismo , Células Vegetales , Plantas Modificadas Genéticamente , Plasmodesmos/metabolismo , Plasmodesmos/ultraestructura , Dominios Proteicos , Saccharomyces cerevisiae/genética , Saccharomyces cerevisiae/metabolismo , Transducción de Señal , Nicotiana/genética , Nicotiana/metabolismo , Proteína Fluorescente RojaRESUMEN
Plants respond to pathogens through dynamic regulation of plasma membrane-bound signaling pathways. To date, how the plant plasma membrane is involved in responses to viruses is mostly unknown. Here, we show that plant cells sense the Potato virus X (PVX) COAT PROTEIN and TRIPLE GENE BLOCK 1 proteins and subsequently trigger the activation of a membrane-bound calcium-dependent kinase. We show that the Arabidopsis thaliana CALCIUM-DEPENDENT PROTEIN KINASE 3-interacts with group 1 REMORINs in vivo, phosphorylates the intrinsically disordered N-terminal domain of the Group 1 REMORIN REM1.3, and restricts PVX cell-to-cell movement. REM1.3's phospho-status defines its plasma membrane nanodomain organization and is crucial for REM1.3-dependent restriction of PVX cell-to-cell movement by regulation of callose deposition at plasmodesmata. This study unveils plasma membrane nanodomain-associated molecular events underlying the plant immune response to viruses.
Asunto(s)
Proteínas Portadoras/metabolismo , Membrana Celular/inmunología , Fosfoproteínas/metabolismo , Proteínas de Plantas/metabolismo , Potexvirus/patogenicidad , Arabidopsis/genética , Arabidopsis/metabolismo , Proteínas de la Cápside/fisiología , Membrana Celular/metabolismo , Movimiento Celular , Enfermedades de las Plantas/virología , Hojas de la Planta/genética , Hojas de la Planta/inmunología , Plantas Modificadas Genéticamente/virología , Plasmodesmos/metabolismo , Proteínas Quinasas/metabolismoRESUMEN
REMORINs are nanodomain-organized proteins located in the plasma membrane and involved in cellular responses in plants. The dynamic assembly of the membrane nanodomains represents an essential tool of the versatile membrane barriers to control and modulate cellular functions. Nevertheless, the assembly mechanisms and protein organization strategies of nanodomains are poorly understood and many structural aspects are difficult to visualize. Using an ensemble of biophysical approaches, including solid-state nuclear magnetic resonance, cryo-electron microscopy and in vivo confocal imaging, we provide first insights on the role and the structural mechanisms of REMORIN trimerization. Our results suggest that the formation of REMORIN coiled-coil trimers is essential for membrane recruitment and promotes REMORIN assembly in vitro into long filaments by trimer-trimer interactions that might participate in nanoclustering into membrane domains in vivo.
Asunto(s)
Proteínas de Arabidopsis/química , Membrana Celular/metabolismo , Proteínas de Plantas/química , Multimerización de Proteína , Proteínas Recombinantes/química , Secuencia de Aminoácidos , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Microscopía por Crioelectrón , Espectroscopía de Resonancia Magnética , Microscopía Confocal , Microscopía Electrónica de Transmisión , Modelos Moleculares , Conformación Molecular , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Estructura Secundaria de Proteína , Proteínas Recombinantes/metabolismo , Proteínas Recombinantes/ultraestructura , Homología de Secuencia de AminoácidoRESUMEN
Plasmodesmata (PD) are nano-sized membrane-lined channels controlling intercellular communication in plants. Although progress has been made in identifying PD proteins, the role played by major membrane constituents, such as the lipids, in defining specialized membrane domains in PD remains unknown. Through a rigorous isolation of "native" PD membrane fractions and comparative mass spectrometry-based analysis, we demonstrate that lipids are laterally segregated along the plasma membrane (PM) at the PD cell-to-cell junction in Arabidopsis thaliana. Remarkably, our results show that PD membranes display enrichment in sterols and sphingolipids with very long chain saturated fatty acids when compared with the bulk of the PM. Intriguingly, this lipid profile is reminiscent of detergent-insoluble membrane microdomains, although our approach is valuably detergent-free. Modulation of the overall sterol composition of young dividing cells reversibly impaired the PD localization of the glycosylphosphatidylinositol-anchored proteins Plasmodesmata Callose Binding 1 and the ß-1,3-glucanase PdBG2 and altered callose-mediated PD permeability. Altogether, this study not only provides a comprehensive analysis of the lipid constituents of PD but also identifies a role for sterols in modulating cell-to-cell connectivity, possibly by establishing and maintaining the positional specificity of callose-modifying glycosylphosphatidylinositol proteins at PD. Our work emphasizes the importance of lipids in defining PD membranes.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Lípidos de la Membrana/metabolismo , Plasmodesmos/metabolismo , Microdominios de Membrana/metabolismoRESUMEN
The lipid composition of plasma membrane (PM) and the corresponding detergent-insoluble membrane (DIM) fraction were analyzed with a specific focus on highly polar sphingolipids, so-called glycosyl inositol phosphorylceramides (GIPCs). Using tobacco (Nicotiana tabacum) 'Bright Yellow 2' cell suspension and leaves, evidence is provided that GIPCs represent up to 40 mol % of the PM lipids. Comparative analysis of DIMs with the PM showed an enrichment of 2-hydroxylated very-long-chain fatty acid-containing GIPCs and polyglycosylated GIPCs in the DIMs. Purified antibodies raised against these GIPCs were further used for immunogold-electron microscopy strategy, revealing the distribution of polyglycosylated GIPCs in domains of 35 ± 7 nm in the plane of the PM. Biophysical studies also showed strong interactions between GIPCs and sterols and suggested a role for very-long-chain fatty acids in the interdigitation between the two PM-composing monolayers. The ins and outs of lipid asymmetry, raft formation, and interdigitation in plant membrane biology are finally discussed.
Asunto(s)
Membrana Celular/química , Lípidos de la Membrana/química , Nicotiana/química , Esfingolípidos/química , Técnicas de Cultivo de Célula/métodos , Membrana Celular/metabolismo , Ácidos Grasos/química , Ácidos Grasos/metabolismo , Glicoesfingolípidos/química , Lípidos de la Membrana/metabolismo , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Microscopía Confocal , Modelos Moleculares , Fitosteroles/química , Fitosteroles/metabolismo , Hojas de la Planta/química , Esfingolípidos/metabolismo , Nicotiana/citología , Nicotiana/metabolismoRESUMEN
The plant cuticle consists of cutin, a polyester of glycerol, hydroxyl, and epoxy fatty acids, covered and filled by waxes. While the biosynthesis of cutin building blocks is well documented, the mechanisms underlining their extracellular deposition remain unknown. Among the proteins extracted from dewaxed tomato (Solanum lycopersicum) peels, we identified GDSL1, a member of the GDSL esterase/acylhydrolase family of plant proteins. GDSL1 is strongly expressed in the epidermis of growing fruit. In GDSL1-silenced tomato lines, we observed a significant reduction in fruit cuticle thickness and a decrease in cutin monomer content proportional to the level of GDSL1 silencing. A significant decrease of wax load was observed only for cuticles of the severely silenced transgenic line. Fourier transform infrared (FTIR) analysis of isolated cutins revealed a reduction in cutin density in silenced lines. Indeed, FTIR-attenuated total reflectance spectroscopy and atomic force microscopy imaging showed that drastic GDSL1 silencing leads to a reduction in ester bond cross-links and to the appearance of nanopores in tomato cutins. Furthermore, immunolabeling experiments attested that GDSL1 is essentially entrapped in the cuticle proper and cuticle layer. These results suggest that GDSL1 is specifically involved in the extracellular deposition of the cutin polyester in the tomato fruit cuticle.
Asunto(s)
Hidrolasas de Éster Carboxílico/metabolismo , Frutas/enzimología , Lípidos de la Membrana/metabolismo , Solanum lycopersicum/enzimología , Hidrolasas de Éster Carboxílico/genética , Hidrolasas de Éster Carboxílico/aislamiento & purificación , Regulación hacia Abajo/genética , Frutas/química , Frutas/genética , Frutas/ultraestructura , Regulación de la Expresión Génica de las Plantas/genética , Silenciador del Gen , Solanum lycopersicum/química , Solanum lycopersicum/genética , Solanum lycopersicum/ultraestructura , Lípidos de la Membrana/química , Microscopía de Fuerza Atómica , Epidermis de la Planta/química , Epidermis de la Planta/enzimología , Epidermis de la Planta/genética , Epidermis de la Planta/ultraestructura , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Plantas Modificadas Genéticamente , Proteómica , Interferencia de ARN , Ceras/química , Ceras/metabolismoRESUMEN
Biological membranes play a crucial role in actively hosting, modulating and coordinating a wide range of molecular events essential for cellular function. Membranes are organized into diverse domains giving rise to dynamic molecular patchworks. However, the very definition of membrane domains has been the subject of continuous debate. For example, in the plant field, membrane domains are often referred to as nanodomains, nanoclusters, microdomains, lipid rafts, membrane rafts, signalling platforms, foci or liquid-ordered membranes without any clear rationale. In the context of plant-microbe interactions, microdomains have sometimes been used to refer to the large area at the plant-microbe interface. Some of these terms have partially overlapping meanings at best, but they are often used interchangeably in the literature. This situation generates much confusion and limits conceptual progress. There is thus an urgent need for us as a scientific community to resolve these semantic and conceptual controversies by defining an unambiguous nomenclature of membrane domains. In this Review, experts in the field get together to provide explicit definitions of plasma membrane domains in plant systems and experimental guidelines for their study. We propose that plasma membrane domains should not be considered on the basis of their size alone but rather according to the biological system being considered, such as the local membrane environment or the entire cell.
RESUMEN
PTEN (phosphatase and tensin homologue deleted on chromosome ten) proteins are dual phosphatases with both protein and phosphoinositide phosphatase activity. They modulate signalling pathways controlling growth, metabolism and apoptosis in animals and are implied in several human diseases. In the present paper we describe a novel class of PTEN pro-teins in plants, termed PTEN2, which comprises the AtPTEN (Arabidopsis PTEN) 2a and AtPTEN2b proteins in Arabidopsis. Both display low in vitro tyrosine phosphatase activity. In addition, AtPTEN2a actively dephosphorylates in vitro the 3' phosphate group of PI3P (phosphatidylinositol 3-phosphate), PI(3,4)P2 (phosphatidylinositol 3,4-bisphosphate) and PI(3,5)P2 (phosphatidylinositol 3,5-bisphosphate). In contrast with animal PTENs, PI(3,4,5)P3 (phosphatidylinositol 3,4,5-trisphosphate) is a poor substrate. Site-directed mutagenesis of AtPTEN2a and molecular modelling of protein-phosphoinositide interactions indicated that substitutions at the PTEN2 core catalytic site of the Lys267 and Gly268 residues found in animals, which are critical for animal PTEN activity, by Met267 and Ala268 found in the eudicot PTEN2 are responsible for changes in substrate specificity. Remarkably, the AtPTEN2a protein also displays strong binding activity for PA (phosphatidic acid), a major lipid second messenger in plants. Promoter::GUS (ß-glucuronidase) fusion, transcript and protein analyses further showed the transcriptional regulation of the ubiquitously expressed AtPTEN2a and AtPTEN2b by salt and osmotic stress. The results of the present study suggest a function for this novel class of plant PTEN proteins as an effector of lipid signalling in plants.
Asunto(s)
Proteínas de Arabidopsis/metabolismo , Arabidopsis/metabolismo , Regulación de la Expresión Génica de las Plantas/fisiología , Fosfohidrolasa PTEN/metabolismo , Ácidos Fosfatidicos/metabolismo , Monoéster Fosfórico Hidrolasas/metabolismo , Arabidopsis/genética , Proteínas de Arabidopsis/genética , Escherichia coli/metabolismo , Modelos Moleculares , Fosfohidrolasa PTEN/genética , Monoéster Fosfórico Hidrolasas/genética , Filogenia , Unión Proteica , Conformación Proteica , Transducción de Señal , Especificidad por SustratoRESUMEN
REMORIN proteins belong to a plant-specific multigene family that localise in plasma membrane nanodomains and in plasmodesmata. We previously showed that in Nicotiana benthamiana, group 1 StREM1.3 limits the cell-to-cell spread of a potexvirus without affecting viral replication. This prompted us to check whether an effect on viral propagation could apply to potyvirus species Turnip mosaic virus (TuMV) and Potato virus A (PVA). Our results show that StREM1.3 transient or stable overexpression in transgenic lines increases potyvirus propagation, while it is slowed down in transgenic lines underexpressing endogenous NbREMs, without affecting viral replication. TuMV and PVA infection do not alter the membranous localisation of StREM1.3. Furthermore, StREM1.3-membrane anchoring is necessary for its agonist effect on potyvirus propagation. StREM1.3 phosphocode seems to lead to distinct plant responses against potexvirus and potyvirus. We also showed that StREM1.3 interacts in yeast and in planta with the key potyviral movement protein CI (cylindrical inclusion) at the level of the plasma membrane but only partially at plasmodesmata pit fields. TuMV infection also counteracts StREM1.3-induced plasmodesmata callose accumulation at plasmodesmata. Altogether, these results showed that StREM1.3 plays an agonistic role in potyvirus cell-to-cell movement in N. benthamiana.
Asunto(s)
Potexvirus , Potyvirus , Movimiento Celular , Enfermedades de las Plantas , Proteínas de Plantas , Potexvirus/genética , Potyvirus/fisiología , Nicotiana , Proteínas Virales/metabolismoRESUMEN
Membranes show a tremendous variety of lipids and proteins operating biochemistry, transport and signalling. The dynamics and the organization of membrane constituents are regulated in space and time to execute precise functions. Our understanding of the molecular mechanisms that shape and govern membrane subcompartmentalization and inter-organelle contact sites still remains limited. Here, we review some reported mechanisms implicated in regulating plant membrane domains including those of plasma membrane, plastids, mitochondria and endoplasmic reticulum. Finally, we discuss several state-of-the-art methods that allow nowadays researchers to decipher the architecture of these structures at the molecular and atomic level.
Asunto(s)
Retículo Endoplásmico , Membranas Mitocondriales , Transporte Biológico , Membrana Celular , Membranas Intracelulares , Mitocondrias , PlastidiosRESUMEN
The plasma membrane (PM) is the biological membrane that separates the interior of all cells from the outside. The PM is constituted of a huge diversity of proteins and lipids. In this review, we will update the diversity of molecular species of lipids found in plant PM. We will further discuss how lipids govern global properties of the plant PM, explaining that plant lipids are unevenly distributed and are able to organize PM in domains. From that observation, it emerges a complex picture showing a spatial and multiscale segregation of PM components. Finally, we will discuss how lipids are key players in the function of PM in plants, with a particular focus on plant-microbe interaction, transport and hormone signaling, abiotic stress responses, plasmodesmata function. The last chapter is dedicated to the methods that the plant membrane biology community needs to develop to get a comprehensive understanding of membrane organization in plants.
Asunto(s)
Membrana Celular/química , Membrana Celular/metabolismo , Fosfolípidos/química , Fitosteroles/química , Esfingolípidos/química , Interacciones Microbiota-Huesped/fisiología , Microdominios de Membrana/química , Microdominios de Membrana/metabolismo , Proteínas de la Membrana/química , Proteínas de la Membrana/metabolismo , Fosfolípidos/metabolismo , Fitosteroles/metabolismo , Células Vegetales/química , Células Vegetales/ultraestructura , Plasmodesmos/química , Plasmodesmos/metabolismo , Esfingolípidos/metabolismo , Estrés Fisiológico/fisiologíaRESUMEN
Since the publication of the fluid mosaic as a relevant model for biological membranes, accumulating evidence has revealed the outstanding complexity of the composition and organization of the plant plasma membrane (PM). Powerful new methodologies have uncovered the remarkable multiscale and multicomponent heterogeneity of PM subcompartmentalization, and this is emerging as a general trait with different features and properties. It is now evident that the dynamics of such a complex organization are intrinsically related to signaling pathways that regulate key physiological processes. Listing and linking recent progress in precisely qualifying these heterogeneities will help to draw an integrated picture of the plant PM. Understanding the key principles governing such a complex dynamic organization will contribute to deciphering the crucial role of the PM in cell physiology.
Asunto(s)
Membrana Celular/fisiología , Fenómenos Fisiológicos de las Plantas , Transducción de SeñalRESUMEN
Crystal structures of peroxisomal Arabidopsis thaliana 3-ketoacyl-CoA thiolase (AtKAT), an enzyme of fatty acid beta-oxidation, are reported. The subunit, a typical thiolase, is a combination of two similar alpha/beta domains capped with a loop domain. The comparison of AtKAT with the Saccharomyces cerevisiae homologue (ScKAT) structure reveals a different placement of subunits within the functional dimers and that a polypeptide segment forming an extended loop around the open catalytic pocket of ScKAT converts to alpha-helix in AtKAT, and occludes the active site. A disulfide is formed between Cys192, on this helix, and Cys138, a catalytic residue. Access to Cys138 is determined by the structure of this polypeptide segment. AtKAT represents an oxidized, previously unknown inactive form, whilst ScKAT is the reduced and active enzyme. A high level of sequence conservation is observed, including Cys192, in eukaryotic peroxisomal, but not mitochondrial or prokaryotic KAT sequences, for this labile loop/helix segment. This indicates that KAT activity in peroxisomes is influenced by a disulfide/dithiol change linking fatty acid beta-oxidation with redox regulation.
Asunto(s)
Acetil-CoA C-Aciltransferasa/química , Proteínas de Arabidopsis/química , Ácidos Grasos/química , Peroxisomas/metabolismo , Estructura Terciaria de Proteína , Subunidades de Proteína/química , Acetil-CoA C-Aciltransferasa/genética , Acetil-CoA C-Aciltransferasa/metabolismo , Secuencia de Aminoácidos , Animales , Arabidopsis/enzimología , Proteínas de Arabidopsis/genética , Proteínas de Arabidopsis/metabolismo , Sitios de Unión , Cristalografía por Rayos X , Dimerización , Humanos , Modelos Moleculares , Datos de Secuencia Molecular , Oxidación-Reducción , Subunidades de Proteína/genética , Subunidades de Proteína/metabolismo , Proteínas de Saccharomyces cerevisiae/química , Proteínas de Saccharomyces cerevisiae/genética , Proteínas de Saccharomyces cerevisiae/metabolismo , Alineación de SecuenciaRESUMEN
Plasma Membrane is the primary structure for adjusting to ever changing conditions. PM sub-compartmentalization in domains is thought to orchestrate signaling. Yet, mechanisms governing membrane organization are mostly uncharacterized. The plant-specific REMORINs are proteins regulating hormonal crosstalk and host invasion. REMs are the best-characterized nanodomain markers via an uncharacterized moiety called REMORIN C-terminal Anchor. By coupling biophysical methods, super-resolution microscopy and physiology, we decipher an original mechanism regulating the dynamic and organization of nanodomains. We showed that targeting of REMORIN is independent of the COP-II-dependent secretory pathway and mediated by PI4P and sterol. REM-CA is an unconventional lipid-binding motif that confers nanodomain organization. Analyses of REM-CA mutants by single particle tracking demonstrate that mobility and supramolecular organization are critical for immunity. This study provides a unique mechanistic insight into how the tight control of spatial segregation is critical in the definition of PM domain necessary to support biological function.
Asunto(s)
Membrana Celular/química , Nicotiana/química , Nicotiana/fisiología , Proteínas de Plantas/análisis , Fenómenos Biofísicos , MicroscopíaRESUMEN
What are the most abundant sphingolipids on earth? The answer is Glycosyl Inositol Phosphoryl Ceramides (GIPCs) present in fungi and the green lineage. In this review, we discuss the putative role of plant GIPCs in the lipid bilayer asymmetry, in the lateral organization of membrane rafts and in the very long chain fatty acid inter-leaflet coupling of lipids in the plant plasma membrane (PM). A special focus on the structural similarities -and putative functions- of GIPCs is discussed by comparison with animal gangliosides, structural homologs of plant GIPCs.
Asunto(s)
Ceramidas/metabolismo , Esfingolípidos/metabolismo , Animales , Ceramidas/química , Glicosilación , Microdominios de Membrana/metabolismo , Modelos Biológicos , Plantas/metabolismoRESUMEN
The Triple Gene Block 1 (TGBp1) protein encoded by the Potato virus X is a multifunctional protein that acts as a suppressor of RNA silencing or facilitates the passage of virus from cell to cell by promoting the plasmodesmata opening. We previously showed that the membrane raft protein StRemorin1.3 is able to impair PVX infection. Here, we show that overexpressed StRemorin1.3 does not impair the silencing suppressor activity of TGBp1, but affects its ability to increase plasmodesmata permeability. A similar effect on plasmodesmata permeability was observed with other movement proteins, suggesting that REM is a general regulator of plasmodesmal size exclusion limit. These results add to our knowledge of the mechanisms underlying the StREM1.3 role in virus infection.
Asunto(s)
Proteínas Portadoras/fisiología , Fosfoproteínas/fisiología , Proteínas de Plantas/fisiología , Plasmodesmos/metabolismo , Potexvirus/fisiología , Solanum tuberosum/virología , Proteínas Virales/fisiología , Agrobacterium/genética , Regulación Viral de la Expresión Génica , Interacciones Huésped-Patógeno , Microscopía Fluorescente , Permeabilidad , Plasmodesmos/virología , Isoformas de Proteínas/fisiología , Transporte de Proteínas , Interferencia de ARN , Proteínas Recombinantes de Fusión/metabolismo , Solanum tuberosum/metabolismo , Nicotiana/metabolismoRESUMEN
The SlPPC2 phosphoenolpyruvate carboxylase (PEPC; EC 4.1.1.31) gene from tomato (Solanum lycopersicum) is differentially and specifically expressed in expanding tissues of developing tomato fruit. We recently showed that a 1966 bp DNA fragment located upstream of the ATG codon of the SlPPC2 gene (GenBank AJ313434) confers appropriate fruit-specificity in transgenic tomato. In this study, we further investigated the regulation of the SlPPC2 promoter gene by analysing the SlPPC2 cis-regulating region fused to either the firefly luciferase (LUC) or the ß-glucuronidase (GUS) reporter gene, using stable genetic transformation and biolistic transient expression assays in the fruit. Biolistic analyses of 5' SlPPC2 promoter deletions fused to LUC in fruits at the 8(th) day after anthesis revealed that positive regulatory regions are mostly located in the distal region of the promoter. In addition, a 5' UTR leader intron present in the 1966 bp fragment contributes to the proper temporal regulation of LUC activity during fruit development. Interestingly, the SlPPC2 promoter responds to hormones (ethylene) and metabolites (sugars) regulating fruit growth and metabolism. When tested by transient expression assays, the chimeric promoter:LUC fusion constructs allowed gene expression in both fruit and leaf, suggesting that integration into the chromatin is required for fruit-specificity. These results clearly demonstrate that SlPPC2 gene is under tight transcriptional regulation in the developing fruit and that its promoter can be employed to drive transgene expression specifically during the cell expansion stage of tomato fruit. Taken together, the SlPPC2 promoter offers great potential as a candidate for driving transgene expression specifically in developing tomato fruit from various tomato cultivars.