In Vivo Aniline Blue Staining and Semiautomated Quantification of Callose Deposition at Plasmodesmata.

The deposition and turnover of callose (beta-1,3 glucan polymer) in the cell wall surrounding the neck regions of plasmodesmata (PD) controls the cell-to-cell diffusion rate of molecules and, therefore, plays an important role in the regulation of intercellular communication in plants.Here we describe a simple and fast in vivo staining procedure for the imaging and quantification of callose at PD. We also introduce calloseQuant, a plug-in for semiautomated image analysis and non-biased quantification of callose levels at PD using ImageJ.

Intercellular trafficking via plasmodesmata: molecular layers of complexity.

Plasmodesmata are intercellular pores connecting together most plant cells. These structures consist of a central constricted form of the endoplasmic reticulum, encircled by some cytoplasmic space, in turn delimited by the plasma membrane, itself ultimately surrounded by the cell wall. The presence and structure of plasmodesmata create multiple routes for intercellular trafficking of a large spectrum of molecules (encompassing RNAs, proteins, hormones and metabolites) and also enable local signalling events. Movement across plasmodesmata is finely controlled in order to balance processes requiring communication with those necessitating symplastic isolation. Here, we describe the identities and roles of the molecular components (specific sets of lipids, proteins and wall polysaccharides) that shape and define plasmodesmata structural and functional domains. We highlight the extensive and dynamic interactions that exist between the plasma/endoplasmic reticulum membranes, cytoplasm and cell wall domains, binding them together to effectively define plasmodesmata shapes and purposes.

Identification of two additional plasmodesmata localization domains in the tobacco mosaic virus cell-to-cell-movement protein.

Despite decades of intensive studies, the failure to identify plasmodesmata (PD) localization sequences has constrained our understanding of Tobacco mosaic virus (TMV) movement. Recently, we identified the first PD localization signal (major PLS) in the TMV movement protein (MP), which encompasses the first 50 amino acid residues of the MP. Although the major PLS is sufficient for PD targeting, the efficiency is lower than the full-length TMV MP. To address this efficiency gap, we identified two additional PLS domains encompassing amino acid residues 61 to 80, and 147 to 170 of the MP and showed that these two domains target to PD, but do not transit to adjacent cells. We also demonstrated that the MP61-80 fragment interacts with Arabidopsis synaptotagmin A, which was also shown to interact with the major TMV MP PLS. Therefore, our findings have provided new insights to more fully understand the mechanism underlying plasmodesmal targeting of TMV MP.

Plant lipids: Key players of plasma membrane organization and function.

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.

Stereochemical identification of glucans by oligothiophenes enables cellulose anatomical mapping in plant tissues.

Efficient use of plant-derived materials requires enabling technologies for non-disruptive composition analysis. The ability to identify and spatially locate polysaccharides in native plant tissues is difficult but essential. Here, we develop an optical method for cellulose identification using the structure-responsive, heptameric oligothiophene h-FTAA as molecular fluorophore. Spectrophotometric analysis of h-FTAA interacting with closely related glucans revealed an exceptional specificity for ß-linked glucans. This optical, non-disruptive method for stereochemical differentiation of glycosidic linkages was next used for in situ composition analysis in plants. Multi-laser/multi-detector analysis developed herein revealed spatial localization of cellulose and structural cell wall features such as plasmodesmata and perforated sieve plates of the phloem. Simultaneous imaging of intrinsically fluorescent components revealed the spatial relationship between cell walls and other organelles, such as chloroplasts and lignified annular thickenings of the trachea, with precision at the sub-cellular scale. Our non-destructive method for cellulose identification lays the foundation for the emergence of anatomical maps of the chemical constituents in plant tissues. This rapid and versatile method will likely benefit the plant science research fields and may serve the biorefinery industry as reporter for feedstock optimization as well as in-line monitoring of cellulose reactions during standard operations.

Isolation of Plasmodesmata.

Plasmodesmata (PD) are plasma membrane lined pores that cross the plant cell wall and connect adjacent cells. Plasmodesmata are composed of elements of the endoplasmic reticulum, plasma membrane, cytosol, and cell wall and thus, as multicomposite structures that are embedded in the cell wall, they are notoriously difficult to isolate from whole plant tissue. However, understanding PD structure, function, and regulation necessitates identification of their molecular components and therefore proteomic and lipidomic analyses of PD fractions are an essential strategy for plasmodesmal biology. Here we outline a simple two-step purification procedure that allows isolation of PD-derived membranes from Arabidopsis suspension cells. The method involves isolation of purified cell wall fragments containing intact PD which is followed by enzymatic degradation of the cell wall to release the PD. This membrane-rich fraction can be subjected to protein and lipid extraction for molecular characterization of PD components. The first step of this procedure involves the isolation of cell wall fragments containing intact PD, free from contamination from other cellular compartments. Purified PD membranes are then released from the cell wall matrix by enzymatic degradation. Isolated PD membranes provide a suitable starting material for the analysis of PD-associated proteins and lipids.

Plasmodesmata: a signaling hub at the cellular boundary.

Effective intercellular communication is crucial for the survival of plants. Because plant cells are encased in rigid cell walls, direct cell-to-cell exchange of cytoplasmic content is only possible through plasmodesmata (PD), membrane-lined nanotubes that connect the cytoplasm of adjacent cells. PD are highly dynamic communication channels that can undergo various structural and functional modifications. Recent findings in the field suggest that defense signaling pathways are tightly linked to the regulation of PD, and the restriction of PD-mediated cell-to-cell communication is an essential innate immune response to microbial pathogens. Moreover, several plasma membrane-bound signaling components, including receptor-like kinases that are known to have non-cell autonomous function or pathogen perception at the cell periphery, are found to also partition to PD. These findings hint at the novel role of PD as a signaling hub for both symplasmic and cross-membrane pathways.

Immunofluorescence detection of callose deposition around plasmodesmata sites.

Accumulation of callose (ß-1,3 glucans) at the plasmodesmata (PD) neck region dynamically regulates symplastic intercellular transport. Here we describe a 2-3-day immuno-labelling protocol to determine callose levels in the cell wall region at PD. The method relies on exposure of internal cell walls by hand-sectioning of the sample and digestion of the cell wall with enzymes in order to improve antibody penetration to deep tissue layers. By using this protocol, combined with high-resolution confocal imaging, we successfully detected PD-associated callose in Arabidopsis root apical meristem, vascular tissue, and developing lateral root primordia.

Imaging callose at plasmodesmata using aniline blue: quantitative confocal microscopy.

Callose (ß-1,3-glucan) is both structural and functional component of plasmodesmata (Pd). The turnover of callose at Pd controls the cell-to-cell diffusion rate of molecules through Pd. An accurate assessment of changes in levels of Pd-associated callose has become a first-choice experimental approach in the research of intercellular communication in plants.Here we describe a detailed and easy-to-perform procedure for imaging and quantification of Pd-associated callose using fixed plant tissue stained with aniline blue. We also introduce an automated image analysis protocol for non-biased quantification of callose levels at Pd from fluorescence images using ImageJ. Two experimental examples of Pd-callose quantification using the automated method are provided as well.

Ophioviruses CPsV and MiLBVV movement protein is encoded in RNA 2 and interacts with the coat protein.

Citrus psorosis virus (CPsV) and Mirafiori lettuce big-vein virus (MiLBVV), members of the Ophioviridae family, have segmented negative-sense single-stranded RNA genomes. To date no reports have described how ophioviruses spread within host plants and/or the proteins involved in this process. Here we show that the 54K protein of CPsV is encoded by RNA 2 and describe its subcellular distribution. Upon transient expression in Nicotiana benthamiana epidermal cells the 54K protein, and also its 54K counterpart protein of MiLBVV, localize to plasmodesmata and enhance GFP cell-to-cell diffusion between cells. Both proteins, but not the coat proteins (CP) of the respective viruses, functionally trans-complement cell-to-cell movement-defective Potato virus X (PVX) and Tobacco mosaic virus (TMV) mutants. The 54K and 54K proteins interact with the virus-specific CP in the cytoplasm, suggesting a potential role of CP in ophiovirus movement. This is the first study characterizing the movement proteins (MP) of ophioviruses.

The secretory pathway and the actomyosin motility system are required for plasmodesmatal localization of the P7-1 of rice black-streaked dwarf virus.

Rice black-streaked dwarf virus (RBSDV), a plant-infecting reovirus (genus Fijivirus), generally induces virus-containing tubules in infected cells. The nonstructural protein P7-1, encoded by the first open reading frame of segment 7, is involved in forming the structural matrix of these tubules. In experiments to investigate the subcellular localization of P7-1 in Nicotiana benthamiana epidermal cells, fluorescence of P7-1:eGFP was observed in the nucleus, cytoplasm and cell periphery, and in punctate points along the cell wall of plasmolyzed cells. Co-localization with plasmodesmata-located protein 1 showed that P7-1 formed the punctate points at plasmodesmata. Mutational analysis demonstrated that transmembrane domain 1 and adjacent residues were necessary and sufficient for P7-1 to form punctate structures at the cell wall in the plasmolyzed cells. Chemical drug and protein inhibitor treatments indicated that P7-1 utilized the ER-to-Golgi secretory pathway and the actomyosin motility system for its intracellular transport. The plasmodesmatal localization of RBSDV P7-1 is therefore dependent on the secretory pathway and the actomyosin motility system.

The ORF3-encoded proteins of vitiviruses GVA and GVB induce tubule-like and punctate structures during virus infection and localize to the plasmodesmata.

The genomic RNA of vitiviruses contains 5 open reading frames (ORF). ORF3 encodes a protein to which the function of a movement protein (MP) was assigned, based on sequence homology with other viral proteins. The aim of the research described in this paper was to gain further insight in distribution profile of the ORF3 product encoded by the vitiviruses Grapevine virus A (GVA) and Grapevine virus B (GVB). Expression of the GVA MP-GFP fusion protein via the virus genome in Nicotiana benthamiana leaves resulted in the formation of irregular spots and fibrous network structures on the outermost periphery of epidermal cells. Expression of GVA MP-GFP and GVB MP-GFP was involved in the formation of the tubule-like and punctate structures on the periphery of N. benthamiana and Vitis vinifera protoplasts. Co-expression of the GVA MP-GFP and GVA MP-RFP in protoplasts resulted in co-localization of these proteins into the same punctate structures, indicating that the MP is not accumulated randomly onto the cell surface, but targeted to particular sites at the cell periphery, where punctate and tubule-like structures are likely formed. With the use of cytoskeleton and secretory pathway inhibitors, we showed that the cytoskeletal elements are not likely to be involved in targeting of the MP-GFP to the punctate cellular structures. In addition to MP, a functional coat protein was found to be essential for virus spread within inoculated leaves.

Histone H3 interacts and colocalizes with the nuclear shuttle protein and the movement protein of a geminivirus.

Geminiviruses are plant-infecting viruses with small circular single-stranded DNA genomes. These viruses utilize nuclear shuttle proteins (NSPs) and movement proteins (MPs) for trafficking of infectious DNA through the nuclear pore complex and plasmodesmata, respectively. Here, a biochemical approach was used to identify host factors interacting with the NSP and MP of the geminivirus Bean dwarf mosaic virus (BDMV). Based on these studies, we identified and characterized a host nucleoprotein, histone H3, which interacts with both the NSP and MP. The specific nature of the interaction of histone H3 with these viral proteins was established by gel overlay and in vitro and in vivo coimmunoprecipitation (co-IP) assays. The NSP and MP interaction domains were mapped to the N-terminal region of histone H3. These experiments also revealed a direct interaction between the BDMV NSP and MP, as well as interactions between histone H3 and the capsid proteins of various geminiviruses. Transient-expression assays revealed the colocalization of histone H3 and NSP in the nucleus and nucleolus and of histone H3 and MP in the cell periphery and plasmodesmata. Finally, using in vivo co-IP assays with a Myc-tagged histone H3, a complex composed of histone H3, NSP, MP, and viral DNA was recovered. Taken together, these findings implicate the host factor histone H3 in the process by which an infectious geminiviral DNA complex forms within the nucleus for export to the cell periphery and cell-to-cell movement through plasmodesmata.

Plasmodesmata viewed as specialised membrane adhesion sites.

A significant amount of work has been expended to identify the elusive components of plasmodesmata (PD) to help understand their structure, as well as how proteins are targeted to them. This review focuses on the role that lipid membranes may play in defining PD both structurally and as subcellular targeting addresses. Parallels are drawn to findings in other areas of research which focus on the lateral segregation of membrane domains and the generation of three-dimensional organellar shapes from flat lipid bilayers. We conclude that consideration of the protein-lipid interactions in cell biological studies of PD components and PD-targeted proteins may yield new insights into some of the many open questions regarding these unique structures.

Plasmodesmal targeting and intercellular movement of potato mop-top pomovirus is mediated by a membrane anchored tyrosine-based motif on the lumenal side of the endoplasmic reticulum and the C-terminal transmembrane domain in the TGB3 movement protein.

Live-cell fluorescence microscopy was used to investigate the third triple gene block protein (TGB3) of potato mop-top pomovirus and its role in assisted targeting of TGB2 to plasmodesmata (PD). Wild-type and mutant TGB3 proteins were expressed under the control of the 35S promoter or from a virus reporter clone. Assisted targeting of TGB2 to PD was optimal when the proteins were expressed from a bicistronic plasmid in the relative ratios expected in a virus infection, suggesting that excess TGB3 inhibited PD localisation. Contrary to the generally accepted view, bimolecular fluorescence complementation showed that the TGB3 N terminus is located in the cytosol. Mutational analysis to dissect TGB3 sub domain functions showed that PD targeting was mediated by a composite signal comprising an ER-lumenal tyrosine-based motif and the C-terminal transmembrane domain. Mutation of either of these domains also abolished cell-to-cell movement of the virus. The results are discussed in the context of TGB3 membrane topology.

Reflection across plant cell boundaries in confocal laser scanning microscopy.

The fluorescence patterns of proteins tagged with the green fluorescent protein (GFP) and its derivatives are routinely used in conjunction with confocal laser scanning microscopy to identify their sub-cellular localization in plant cells. GFP-tagged proteins localized to plasmodesmata, the intercellular junctions of plants, are often identified by single or paired punctate labelling across the cell wall. The observation of paired puncta, or 'doublets', across cell boundaries in tissues that have been transformed through biolistic bombardment is unexpected if there is no intercellular movement of the GFP-tagged protein, since bombardment usually leads to the transformation of single, isolated cells. We expressed a putative plasmodesmal protein tagged with GFP by bombarding Allium porrum epidermal cells and assessed the nature of the doublets observed at the cell boundaries. Doublets were formed when fluorescent spots were abutting a cell boundary and were only observable at certain focal planes. Fluorescence emitted from the half of a doublet lying outside the transformed cells was polarized. Optical simulations performed using finite-difference time-domain computations showed a dramatic distortion of the confocal microscope's point spread function when imaging voxels close to the plant cell wall due to refractive index differences between the wall and the cytosol. Consequently, axially and radially out-of-focus light could be detected. A model of this phenomenon suggests how a doublet may form when imaging only a single real fluorescent body in the vicinity of a plant cell wall using confocal microscopy. We suggest, therefore, that the appearance of doublets across cell boundaries is insufficient evidence for plasmodesmal localization due to the effects of the cell wall on the reflection and scattering of light.

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

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

Plasmodemos: estructura y funcion / Plasmodesmata: Structure and Function

Los plasmodesmos son canales que atraviesan la membrana y la pared celular. Estos canales especializados y no pasivos, actúan como compuertas que facilitan y regulan la comunicación y el transporte de sustancias como agua, nutrientes, metabolitos y macromoléculas entre las células vegetales. En los últimos años, una nueva visión sobre estos canales ha surgido y, estudios han demostrado que los plasmodesmos son más complejos de lo que anteriormente se pensaba. En esta nota, se pretende exponer el conocimiento actual sobre dichas estructuras, enfocándonos en su estructura y función.

The cysteine-histidine-rich region of the movement protein of Cucumber mosaic virus contributes to plasmodesmal targeting, zinc binding and pathogenesis.

Viral movement proteins (MPs) are central to the establishment of viral pathogenesis, and yet relatively little is understood about the structural and functional aspects of MPs or about the host factors on which they depend. Through chemical mutagenesis of transgenic Arabidopsis expressing Cucumber mosaic virus (CMV) MP fused with the green fluorescent protein, we have studied the function of a central region of the MP, defined by a number of conserved cysteine and histidine residues (Cys-His-rich region), which potentially functions as a zinc-binding domain. Transient expression of mutant MPs identified through an in planta screen for altered MP function or constructed with altered putative zinc ligands through site-directed mutagenesis showed that mutations in the Cys-His-rich region affected localization to and trafficking through plasmodesmata. In vitro zinc-binding analysis revealed that wild type (wt) CMV MP had the ability to bind zinc and that movement-defective mutants bound zinc with less affinity than wt MP. Furthermore, a correlation between the association of the MP with plasmodesmata and virus pathogenesis was shown. We discuss roles of the Cys-His region in biochemical and biological functions of the MP during virus movement.

Proteomic identification of putative plasmodesmatal proteins from Chara corallina.

Plasmodesmata are channels that bridge the cell walls of plant cells, allowing regulated transport of molecules between neighbouring cells. We have used a proteomic strategy to identify putative plasmodesmata-associated proteins in the giant-celled green alga Chara corallina. Proteins were extracted from the plasmodesmata-rich nodal complexes and the middle of the long internodal cells, which do not contain plasmodesmata. Comparison of protein spot patterns generated by two-dimensional gel electrophoresis of both the soluble and cell wall fractions from the two cell types was done. Fifty-eight spots that were common to the nodal and internodal soluble fractions were analysed by matrix assisted laser desorption/ionisation-time of flight mass spectrometry, and peptide mass fingerprint data were used to search the database. Matches were made to four of these spots, in each case to housekeeping proteins. Further, a number of nodal specific spots were identified, 11 from the soluble fraction and nine from the wall fraction. These spots were excised from the gels and analysed by liquid chromatography tandem mass spectrometry to obtain peptide sequence. Database searches suggest that these spots include homologues to previously identified plasmodesmata-associated proteins cp-wap13 and heat shock cognate 70, as well as RNA-binding proteins, eukaryotic initiation factor 4A and a beta-1,3-glucanase. Several spots remained unidentified providing exciting new candidate plasmodesmata-associated proteins.