Plant Plasmodesmata Research Articles

Plant plasmodesmata bridges form through ER-dependent incomplete cytokinesis.

The ER is important for stabilizing nascent plasmodesmata, a process integral to incomplete cytokinesis in plants.

Multicellularity and the Need for Communication-A Systematic Overview on (Algal) Plasmodesmata and Other Types of Symplasmic Cell Connections.

In the evolution of eukaryotes, the transition from unicellular to simple multicellular organisms has happened multiple times. For the development of complex multicellularity, characterized by sophisticated body plans and division of labor between specialized cells, symplasmic intercellular communication is supposed to be indispensable. We review the diversity of symplasmic connectivity among the eukaryotes and distinguish between distinct types of non-plasmodesmatal connections, plasmodesmata-like structures, and 'canonical' plasmodesmata on the basis of developmental, structural, and functional criteria. Focusing on the occurrence of plasmodesmata (-like) structures in extant taxa of fungi, brown algae (Phaeophyceae), green algae (Chlorophyta), and streptophyte algae, we present a detailed critical update on the available literature which is adapted to the present classification of these taxa and may serve as a tool for future work. From the data, we conclude that, actually, development of complex multicellularity correlates with symplasmic connectivity in many algal taxa, but there might be alternative routes. Furthermore, we deduce a four-step process towards the evolution of canonical plasmodesmata and demonstrate similarity of plasmodesmata in streptophyte algae and land plants with respect to the occurrence of an ER component. Finally, we discuss the urgent need for functional investigations and molecular work on cell connections in algal organisms.

An RNA exosome subunit mediates cell-to-cell trafficking of a homeobox mRNA via plasmodesmata.

Messenger RNAs (mRNAs) function as mobile signals for cell-to-cell communication in multicellular organisms. The KNOTTED1 (KN1) homeodomain family transcription factors act non–cell autonomously to control stem cell maintenance in plants through cell-to-cell movement of their proteins and mRNAs through plasmodesmata; however, the mechanism of mRNA movement is largely unknown. We show that cell-to-cell movement of a KN1 mRNA requires ribosomal RNA–processing protein 44A (AtRRP44A), a subunit of the RNA exosome that processes or degrades diverse RNAs in eukaryotes. AtRRP44A can interact with plasmodesmata and mediates the cell-to-cell trafficking of KN1 mRNA, and genetic analysis indicates that AtRRP44A is required for the developmental functions of SHOOT MERISTEMLESS, an Arabidopsis KN1 homolog. Our findings suggest that AtRRP44A promotes mRNA trafficking through plasmodesmata to control stem cell–dependent processes in plants.

Plasmodesmata Ultrastructure Determination Using Electron Tomography.

Plant plasmodesmata (PD) are complex intercellular channels consisting of a thin endoplasmic reticulum (ER) tubule enveloped by the plasma membrane (PM). PD were first observed by electron microscopy about 50years ago and, since, numerous studies in transmission and scanning electron microscopy have provided important information regarding their overall organization, revealing at the same time their diversity in terms of structure and morphology. However, and despite the fact that PD cell-cell communication is of critical importance for plant growth, development, cellular patterning, and response to biotic and abiotic stresses, linking their structural organization to their functional state has been proven difficult. This is in part due to their small size (20-50nm in diameter) and the difficulty to resolve these structures in three dimensions at nanometer resolution to provide details of their internal organization.In this protocol, we provide in detail a complete process to produce high-resolution transmission electron tomograms of PD. We describe the preparation of the plant sample using high-pressure cryofixation and cryo-substitution. We also describe how to prepare filmed grids and how to cut and collect the sections using an ultramicrotome. We explain how to acquire a tilt series and how to reconstruct a tomogram from it using the IMOD software. We also give a few guidelines on segmentation of the reconstructed tomogram.

Penicillium chrysogenum polypeptide extract protects tobacco plants from tobacco mosaic virus infection through modulation of ABA biosynthesis and callose priming.

The polypeptide extract of the dry mycelium of Penicillium chrysogenum (PDMP) can protect tobacco plants from tobacco mosaic virus (TMV), although the mechanism underlying PDMP-mediated TMV resistance remains unknown. In our study, we analysed a potential mechanism via RNA sequencing (RNA-seq) and found that the abscisic acid (ABA) biosynthetic pathway and β-1,3-glucanase, a callose-degrading enzyme, might play an important role in PDMP-induced priming of resistance to TMV. To test our hypothesis, we successfully generated a Nicotiana benthamiana ABA biosynthesis mutant and evaluated the role of the ABA pathway in PDMP-induced callose deposition during resistance to TMV infection. Our results suggested that PDMP can induce callose priming to defend against TMV movement. PDMP inhibited TMV movement by increasing callose deposition around plasmodesmata, but this phenomenon did not occur in the ABA biosynthesis mutant; moreover, these effects of PDMP on callose deposition could be rescued by treatment with exogenous ABA. Our results suggested that callose deposition around plasmodesmata in wild-type plants is mainly responsible for the restriction of TMV movement during the PDMP-induced defensive response to TMV infection, and that ABA biosynthesis apparently plays a crucial role in PDMP-induced callose priming for enhancing defence against TMV.

Pathogenic Bacteria Target Plant Plasmodesmata to Colonize and Invade Surrounding Tissues.

A hallmark of multicellular organisms is their ability to maintain physiological homeostasis by communicating among cells, tissues, and organs. In plants, intercellular communication is largely dependent on plasmodesmata (PD), which are membrane-lined channels connecting adjacent plant cells. Upon immune stimulation, plants close PD as part of their immune responses. Here, we show that the bacterial pathogen Pseudomonas syringae deploys an effector protein, HopO1-1, that modulates PD function. HopO1-1 is required for P. syringae to spread locally to neighboring tissues during infection. Expression of HopO1-1 in Arabidopsis (Arabidopsis thaliana) increases the distance of PD-dependent molecular flux between neighboring plant cells. Being a putative ribosyltransferase, the catalytic activity of HopO1-1 is required for regulation of PD. HopO1-1 physically interacts with and destabilizes the plant PD-located protein PDLP7 and possibly PDLP5. Both PDLPs are involved in bacterial immunity. Our findings reveal that a pathogenic bacterium utilizes an effector to manipulate PD-mediated host intercellular communication for maximizing the spread of bacterial infection.

Phytosphinganine Affects Plasmodesmata Permeability via Facilitating PDLP5-Stimulated Callose Accumulation in Arabidopsis

Plant plasmodesmata (PDs) are specialized channels that enable communication between neighboring cells. The intercellular permeability of PDs, which affects plant development, defense, and responses to stimuli, must be tightly regulated. However, the lipid compositions of PD membrane and their impact on PD permeability remain elusive. Here, we report that the Arabidopsis sld1 sld2 double mutant, lacking sphingolipid long-chain base 8 desaturases 1 and 2, displayed decreased PD permeability due to a significant increase in callose accumulation. PD-located protein 5 (PDLP5) was significantly enriched in the leaf epidermal cells of sld1 sld2 and showed specific binding affinity to phytosphinganine (t18:0), suggesting that the enrichment of t18:0-based sphingolipids in sld1 sld2 PDs might facilitate the recruitment of PDLP5 proteins to PDs. The sld1 sld2 double mutant seedlings showed enhanced resistance to the fungal-wilt pathogen Verticillium dahlia and the bacterium Pseudomonas syringae pv. tomato DC3000, which could be fully rescued in sld1 sld2 pdlp5 triple mutant. Taken together, these results indicate that phytosphinganine might regulate PD functions and cell-to-cell communication by modifying the level of PDLP5 in PD membranes.

Metagenomics of plant and fungal viruses reveals an abundance of persistent lifestyles.

Most studies of plant viruses have focused on the acute viruses that cause disease in crop and ornamental plants. These viruses are transmitted horizontally, often by insect vectors, and are occasionally transmitted vertically. Although known for at least four decades, the persistent viruses of plants are very poorly studied. These viruses were previously called “cryptic” because they did not appear to illicit any symptoms in infected plants (Boccardo et al., 1987). Persistent plant viruses are not known to be transmitted horizontally, although phylogenetic evidence suggests some level of transmission (Roossinck, 2010). They are vertically transmitted at nearly 100% levels through both ova and pollen (Valverde and Gutierrez, 2007). They have been identified in metagenomic studies by their similarity to known persistent viruses, and because they lack any movement protein, a feature of all known acute viruses that must move through the plant plasmodesmata to establish a systemic infection. Persistent viruses do not move between plant cells, but rather infect every cell and move by cell division. Most plant persistent viruses have double-stranded (ds) RNA genomes, and encode only an RNA dependent RNA polymerase (RdRp) and a coat protein. Of the well-characterized persistent plant viruses, those in the Endornaviridae are the exception. These viruses have a single-stranded (ss) RNA genome, based on their RdRp, and encode a large polyprotein that does not have any apparent coat protein, but encodes a number of additional domains that appear to be derived from diverse sources (Roossinck et al., 2011). They are usually found as dsRNA replicative intermediates. Viruses of fungi have very similar lifestyles to plant persistent viruses, and several virus families are shared between plants and fungi. Phylogenetic evidence indicates that virus transmission has occurred within and between the two kingdoms (Roossinck, 2010; Roossinck et al., 2011). Fungal viruses are even less well-studied than plant viruses, and the diversity of these viruses remains mostly unknown. A majority of known fungal viruses have dsRNA genomes, some have ssRNA genomes, and a few examples of DNA viruses are known (Yu et al., 2010). Recently a negative sense ssRNA virus was characterized from a fungus (Liu et al., 2014). Similar to plant viruses, most fungal viruses have been studied in the context of pathogenic fungi. The discovery of the hypovirulence phenotype of Cryphonectria hypovirus 1 that suppresses the disease phenotype of the chestnut blight fungus led to a search for other examples that could be exploited to mitigate the effects of plant pathogenic fungi [reviewed in Dawe and Nuss (2013)].

Actin and Myosin Co-Localize in Plasmodesmata and Ectodesmata-Like Structure

Actin and myosin were found to be associated with the cytoplasmic sleeve of plasmodesmata. As cytoskeletal proteins, actin and myosin are believed to regulate the conductivity of plasmodesmata (PDs) in higher plants. Using immunocytochemical methods, we found the two proteins to be co-localized - and closely linked to each other - in plasmodesmata and ectodesmata-like structure in ageing parenchymatous cells of Allium sativum L. We suggest that intercellular communication is affected by the interaction between actin and myosin.

Identification of distinct steps during tubule formation by the movement protein of Cowpea mosaic virus.

The movement protein (MP) of Cowpea mosaic virus (CPMV) forms tubules through plasmodesmata in infected plants thus enabling virus particles to move from cell to cell. Localization studies of mutant MPs fused to GFP in protoplasts and plants identified several functional domains within the MP that are involved in distinct steps during tubule formation. Coinoculation experiments and the observation that one of the C-terminal deletion mutants accumulated uniformly in the plasma membrane suggest that dimeric or multimeric MP is first targeted to the plasma membrane. At the plasma membrane the MP quickly accumulates in peripheral punctuate spots, from which tubule formation is initiated. One of the mutant MPs formed tubules containing virus particles on protoplasts, but could not support cell-to-cell movement in plants. The observations that this mutant MP accumulated to a higher level in the cell than wt MP and did not accumulate in the cell wall opposite infected cells suggest that breakdown or disassembly of tubules in neighbouring, uninfected cells is required for cell-to-cell movement.

Cloning of a tobacco cDNA coding for a putative transcriptional coactivator MBF1 that interacts with the tomato mosaic virus movement protein

Viral movement through plasmodesmata in host plants depends on the interaction between virus-encoded movement protein (MP) and host proteins. To search for MP-interacting protein (MIP), far-western screening of a tobacco cDNA library was carried out using a recombinant MP of tomato mosaic virus (ToMV) as a probe. One of the positive cDNA clones, designated MIP204, was highly homologous to a class of transcriptional coactivators commonly referred to as multiprotein bridging factor 1 (MBF1). ToMV MP could also bind to the Arabidopsis homologues of MBF1. The recombinant MIP204 bound to MPs of ToMV and a crucifer tobamovirus CTMV-W, but not of cucumber mosaic virus. MPs of ToMV and the related virus may interact with MBF1-like proteins to modulate host gene expression.

The Cytoskeleton and the Secretory Pathway Are Not Involved in Targeting the Cowpea Mosaic Virus Movement Protein to the Cell Periphery

The movement protein (MP) of cowpea mosaic virus (CPMV) forms tubules on infected protoplasts and through plasmodesmata in infected plants. In protoplasts the MP fused to GFP (MP-GFP) was shown to localize in peripheral punctate structures and in long tubular structures extending from the protoplast surface. Using cytoskeletal assembly inhibitors (latrunculin B and oryzalin) and an inhibitor of the secretory pathway (brefeldin A), targeting of the MP to the peripheral punctate structures was demonstrated not to be dependent on an intact cytoskeleton or functional secretion pathway. Furthermore it was shown that a disrupted cytoskeleton had no effect on tubule formation but that the addition of brefeldin A severely inhibited tubule formation. The results presented in this paper suggest a role for a plasma membrane host factor in tubule formation of plant viral MPs.

Towards reconciliation of structure with function in plasmodesmata—who is the gatekeeper?

Whilst the structure of higher plant plasmodesmata was first described by Robards (1963. Desmotubule—a plasmodesmatal substructure. Nature 218, 784), and despite many subsequent intensive investigations, there is still much that remains unclear relating to their ultrastructure and functioning in higher plants. We have examined chemically fixed plant material, and suggest that the conformational changes seen in plasmodesmatal substructure, particularly the deposition of electron-dense extra-plasmodesmal material, is linked to either manipulation of the hormonal balance (as in Avocado fruit), or of osmotic potential in leaf blade material. These changes result in the deposition of β 1,3-glucan (callose) at the neck region of these plasmodesmata. This electron-dense material is deposited at the neck region of plasmodesmata, and forms a collar-like structure. The formation of a collar is shown to be coupled with loss of lucence within the cytoplasmic sleeve. The formation of a collar at the plasmodesmatal orifice thus results in encapsulation and closure of the plasmodesmatal orifice. Closure of the orifice coincides with a loss of electron-lucence and a lack of resolution of the desmotubule. These ultrastructural changes are potentially significant and could contribute to, result in, or assist in the down-regulation of cell to cell trafficking via plasmodesmata.

Physiological elevations in cytoplasmic free calcium by cold or ion injection result in transient closure of higher plant plasmodesmata.

The concentration of cytoplasmic free calcium ([Ca(2+)](cyt)) required to close higher plant plasmodesmata was investigated using corn (Zea mays L. cv. Black Mexican Sweet) suspension-culture cells. Physiological elevations of [Ca(2+)](cyt) were applied by cold treatment, and ion injection was also used to increase [Ca(2+)](cyt), by diffusion (for small increases) or by iontophoresis (for larger increases). The impact of such treatments on [Ca(2+)](cyt) was measured by ratiometric ion imaging. Intercellular communication during treatments was monitored using our recently developed electrophysiological technique that allows the electrical resistance of plasmodesmata and the plasma membranes of a sister-cell pair to be measured. A 4-fold increase in the calculated resistance of single plasmodesmata was observed in response to cold treatment that caused a 2-fold increase in average [Ca(2+)](cyt) (from 107 to 210 nM). In response to iontophoresis of Ca(2+), plasmodesmata were observed to go from "open" (low resistance) to "shut" (high resistance) and then back "open" within 10 s. Our results thus indicate that higher plant plasmodesmata respond quickly to physiological changes in [Ca(2+)](cyt).

Localization of a centrin-like protein to higher plant plasmodesmata

Antibodies against centrin, the ubiquitous calcium-binding contractile protein, recognized a 17 kDa protein in extracts of onion root tips and cauliflower florets. Using immunofluorescence microscopy, anti-centrin antibodies were localized to the developing cell plate of onion and cauliflower root tip cells. In cauliflower florets, these antibodies localized to the walls in a punctate manner, consistent with the distribution of plasmodesmata as shown by colocalization with callose. Anti-centrin antibodies were localized to plasmodesmata of onion root tips and cauliflower florets with immunogold electron microscopy. Furthermore, this label was concentrated around the necks of plasmodesmata. In contrast, an antibody against calmodulin, which is a closely related calcium-binding protein, did not label plasmodesmata. We propose that centrin is a component of calcium-sensitive contractile nanofilaments in the neck region of plasmodesmata and facilitates the calcium-induced regulation of intercellular transport.

A 45 kDa protein isolated from the nodal walls ofChara corallinais localised to plasmodesmata

Summary The cellular anatomy of the green alga, Chara corallina, was exploited to isolate putative plasmodesmataassociated proteins. In C. corallina , large internodal cells are symplastically connected via intervening nodal complexes of smaller cells which have plasmodesmata in their cell walls. Comparison of proteins extracted from walls with plasmodesmata (nodal complexes) with those from walls without plasmodesmata (external internodal walls) identified four putative plasmodesmata‐associated proteins. These putative plasmodesmata‐associated proteins were approximately 95, 45, 44 and 33 kDa. A monoclonal antibody (MAB45/22) was raised against the 45 kDa putative plasmodesmata‐associated protein (CPAP45). Using immunofluorescence, this antibody co‐localised with aniline blue induced fluorescence of callose in the source cell walls. MAB45/22 was localised to the plasmodesmata of C. corallina and, in particular, to the central cavity using immunogold cytochemistry. In contrast, a monoclonal antibody to callose specifically labelled the mouth of C. corallina plasmodesmata. MAB45/22 also labelled higher plant plasmodesmata.

Comparative ultrastructure of plasmodesmata of Chara and selected bryophytes: toward an elucidation of the evolutionary origin of plant plasmodesmata

We have used transmission electron microscopy to examine plasmodesmata of the charophycean green alga Chara zeylanica, and of the putatively early divergent bryophytes Monoclea gottschei (liverwort), Notothylas orbicularis (hornwort), and Sphagnum fimbriatum (moss), in an attempt to learn when seed plant plasmodesmata may have originated. The three bryophytes examined have desmotubules. In addition, Monoclea was found to have branched plasmodesmata, and plasmodesmata of Sphagnum displayed densely staining regions around the neck region, as well as ring-like wall specializations. In Chara, longitudinal sections revealed endoplasmic reticulum (ER) that sometimes appeared to be associated with plasmodesmata, but this was rare, despite abundant ER at the cell periphery. Across all three fixation methods, cross-sectional views showed an internal central structure, which in some cases appeared to be connected to the plasma membrane via spoke-like structures. Plasmodesmata were present even in the incompletely formed reticulum of forming cell plates, from which we conclude that primary plasmodesmata are formed at cytokinesis in Chara zeylanica. Based on these results it appears that plasmodesmata of Chara may be less specialized than those of seed plants, and that complex plasmodesmata probably evolved in the ancestor of land plants before extant lineages of bryophytes diverged.

Regulation of electrical coupling between Arabidopsis root hairs

Voltage clamp was used to measure the voltage dependence of cell-to-cell coupling via plasmodesmata between higher-plant cells (root hairs of Arabidopsis thaliana (L.) Heynh.). In addition, ionophoresis was used to introduce a variety of ions [Ca2+, inositol-trisphosphate, Li+, K+, Mg2+, ethylene glycol-bis(β-aminoethyl ether)-N,N,N′, N′-tetraacetic acid (EGTA), 1,2-bis(2-aminophenoxy)ethane-N,N,N′,N′-tetraacetic acid (BAPTA), H+, and OH−] to examine whether they regulate cell-to-cell coupling. Electrical coupling showed high variability in this single cell type at the same developmental stage; the coupling ratio ranged from near 0% to about 90% with a mean value of 32%. It was voltage independent for intracellular voltage gradients (transplasmodesmatal) of -163 to 212 mV. While Ca2+ closes the plasmodesmatal connections (at concentrations higher than those causing cessation of cytoplasmic streaming), inositol-trisphosphate and lithium are without effect. Apparently, inositol-trisphosphate may not cause increased cytosolic Ca2+ in root hairs. Alkalinization by OH ionophoresis caused a modest decline in cell-to-cell coupling, as did acidification by H+ ionophoresis (to an extent causing the cell to become flacid). Increases in cytosolic K+, Mg2+, and the calcium chelator BAPTA by ionophoresis had no effect on cell-to-cell coupling. The regulation (and lack thereof) reported here for plant plasmodesmata is quite similar to that of gap junctions.

Cell-to-Cell Trafficking of Macromolecules through Plasmodesmata Potentiated by the Red Clover Necrotic Mosaic Virus Movement Protein.

Direct evidence is presented for cell-to-cell trafficking of macromolecules via plasmodesmata in higher plants. The fluorescently labeled 35-kD movement protein of red clover necrotic mosaic virus (RCNMV) trafficked rapidly from cell to cell when microinjected into cowpea leaf mesophyll cells. Furthermore, this protein potentiated rapid cell-to-cell trafficking of RCNMV RNA, but not DNA. Electron microscopic studies demonstrated that the 35-kD movement protein does not unfold the RCNMV RNA molecules. Thus, if unfolding of RNA is necessary for cell-to-cell trafficking, it may well involve participation of endogenous cellular factors. These findings support the hypothesis that trafficking of macromolecules is a normal plasmodesmal function, which has been usurped by plant viruses for their cell-to-cell spread.

Reinvestigation of intracellular localization of the 30K protein in tobacco protoplasts infected with tobacco mosaic virus RNA

It has been shown that the 30K protein of tobacco mosaic virus (TMV) is responsible for the cell-to-cell movement function of the virus. It is still obscure how the protein is involved in this function at the molecular level. We formerly found that the 30K protein is localized to the plasnyiodesmata of TMV-infected plants. We also reported that the 30K protein was detected in a nuclei-rich fraction of TMV-infected protoplasts after biochemical fractionation. To clarify the inconsistency, the 30K protein was immunocytologically localized in TMV-infected protoplasts using a newly prepared antibody against the 30K protein. On some sections, the 30K protein was found near the nucleus but not in or on the nucleus. At later stages of infection a novel electron-transparent structure was detected in the cytoplasm where the 30K proteins were localized. This structure might reflect an intermediate form between its synthesis in the cytoplasm and its targeting to the plasmodesmata in whole plants.