Specific targeting of a plasmodesmal protein affecting cell-to-cell communication

Plasmodesmata provide the cytoplasmic conduits for cell-to-cell communication throughout plant tissues and participate in a diverse set of non–cell-autonomous functions. Despite their central role in growth and development and defence, resolving their modus operandi remains a major challenge in plant biology. Features of protein sequences and/or structure that determine protein targeting to plasmodesmata were previously unknown. We identify here a novel family of plasmodesmata-located proteins (called PDLP1) whose members have the features of type I membrane receptor-like proteins. We focus our studies on the first identified type member (namely At5g43980, or PDLP1a) and show that, following its altered expression, it is effective in modulating cell-to-cell trafficking. PDLP1a is targeted to plasmodesmata via the secretory pathway in a Brefeldin A–sensitive and COPII-dependent manner, and resides at plasmodesmata with its C-terminus in the cytoplasmic domain and its N-terminus in the apoplast. Using a deletion analysis, we show that the single transmembrane domain (TMD) of PDLP1a contains all the information necessary for intracellular targeting of this type I membrane protein to plasmodesmata, such that the TMD can be used to target heterologous proteins to this location. These studies identify a new family of plasmodesmal proteins that affect cell-to-cell communication. They exhibit a mode of intracellular trafficking and targeting novel for plant biology and provide technological opportunities for targeting different proteins to plasmodesmata to aid in plasmodesmal characterisation.     

Peptide antagonists of the plasmodesmal macromolecular trafficking pathway

In plants, cell-to-cell transport of endogenous and viral proteins and ribonucleoprotein complexes (RNPCs) occurs via plasmodesmata. Specificity of this transport pathway appears to involve interaction between such proteins/RNPCs and plasmodesmal chaperones/receptors. Here, KN1 and the cucumber mosaic virus movement protein (CMV-MP) were used, in a modified phage-display screening system, to identify peptides capable of interacting with proteins present in a plasmodesmal-enriched cell wall fraction. Binding/competition assays and microinjection experiments revealed that these phage-displayed peptides and homologous synthetic oligopeptides function as ligand-specific antagonists of macromolecular trafficking through plasmodesmata. A KN1 peptide antagonist had the capacity to interact with a motif involved in the dilation of plasmodesmal microchannels. Although KN1 could still achieve limited movement through plasmodesmata when this SEL motif was blocked, KN1-mediated transport of KN1-sense RNA was fully inhibited. These findings provide direct support for the hypothesis that KN1 requires, minimally, two physically separated signal motifs involved in the dilation of, and protein translocation through, plasmodesmal microchannels, and provide direct proof that plasmodesmal dilation is a prerequisite for the cell-to-cell transport of an RNPC.     

Dynamics of plasmodesmal connectivity in successive interfaces of the cambial zone

Frequency, density and branching of plasmodesmata were counted in successive tangential and transverse walls in the cambial zone of tomato stems in order to examine development of the plasmodesmal network in a chronological order. Coincident with progress of cell development, plasmodesmal connectivity increased, both at the xylem- and phloem-side. In transverse walls, the number of secondary plasmodesmata enhanced considerably. The same held for tangential walls, with a superimposed plasmodesmal doubling during the first phase of phloem development. This plasmodesmal doubling was interpreted to result from the deposition of wall material between branched plasmodesmal strands. Structural plasmodesmal development was correlated with production of hydroxyl radicals which control local cell wall alterations. Successive phases of plasmodesmal deployment and modification were distinguished which may coincide with differential functional capacities as documented by intracellular injection of fluorochromes. Diffusion-driven symplasmic transport appeared to be transiently interrupted during cell maturation.     

Developmental changes in plasmodesmata in transgenic tobacco expressing the movement protein of tobacco mosaic virus

Summary Cell-to-cell communication in plants occurs through plasmodesmata, cytoplasmic channels that traverse the cell wall between neighboring cells. Plasmodesmata are also exploited by many viruses as an avenue for spread of viral progeny. In the case of tobacco mosaic virus (TMV), a virally-encoded movement protein (MP) enables the virus to move through plasmodesmata during infection. We have used thin section electron microscopy and immunocytochemistry to examine the structure of plasmodesmata in transgenic tobacco plants expressing the TMV MP. We observed a change in structure of the plasmodesmata as the leaves age, both in control and MP expressing [MP(+)] plants. In addition, the plasmodesmata of older cells of MP(+) plants accumulate a fibrous material in the central cavity. The presence of the fibers is correlated with the ability to label plasmodesmata with anti-MP antibodies. The developmental stage of leaf tissue at which this material is observed is the stage at which an increase in the size exclusion limit of the plasmodesmata can be measured in MP(+) plants. Using cell fractionation and aqueous phase partitioning studies, we identified the plasma membrane and cell wall as the compartments with which the MP stably associates. The nature of the interaction between the MP and the plasma membrane was studied using sodium carbonate and Triton X-100 washes. The MP behaves as an integral membrane protein. Identifying the mechanism by which the MP associates with plasma membrane and plasmodesmata will lead to a better understanding of how the MP alters the function of the plasmodesmata.Abbreviations MP movement protein - TMV tobacco mosaic virus     

Plasmodesmata form and function

Intercellular transport via plasmodesmata controls cell fate decisions in plants, and is of fundamental importance in viral movement, disease resistance, and the spread of RNAi signals. Although plasmodesmata appear to be unique to plant cells, they may have structural and functional similarities to the newly discovered tunneling nanotubes that connect animal cells. Recently, proteins that localize to plasmodesmata have been identified, and a microtubule-associated protein was found to negatively regulate the trafficking of viral movement proteins. Other advances have delivered new insights into the function and molecular nature of plasmodesmata and have shown that protein trafficking through plasmodesmata is developmentally regulated, opening up the possibility that the genetic control of plasmodesmal function will soon be understood.     

Proteomic analysis of changes in the extracellular matrix of Arabidopsis cell suspension cultures induced by fungal elicitors

A proteomic approach has been applied to investigate changes in the extracellular matrix of Arabidopsis thaliana cell suspension cultures following treatments with two fungal pathogen elicitors, chitosan and extracts of Fusarium moniliforme. The oxidative burst and induction of glutathione S-transferase were used as markers for induction of the pathogen defence response. Changes in the cell wall and culture filtrate proteome were profiled. Proteins whose abundance changed reproducibly were analysed via matrix assisted laser desorption ionisation-time of flight (MALDI-TOF) mass spectrometry (MS). An increase in the level of two classical cell wall proteins (a putative endochitinase and a polygalacturonase inhibiting protein) and two novel proteins (a putative receptor-like protein kinase and a probable apospory-associated protein) were seen at 24 hours following elicitation. The level of an unknown protein and a hypothetical protein, which has some homology to serine carboxypeptidases, were decreased at 24 hours post-elicitation. In the culture filtrate extracts, we identified two pathogen elicitor responsive proteins, a xyloglucan endo-1,4-beta-D glucanases (XEG) and a peroxidase. Using a combination of two-dimensional polyacrylamide gel electrophoresis, immunoblotting with a phosphotyrosine-specific antibody, and MALDI-TOF MS we discovered that spots that represent putative lectin receptor-like kinase, a putative endochitinase and a XEG possess phosphorylated tyrosine residues. The identification of phosphorylated bona fide cell wall proteins and a putative extracellular receptor-like kinase with no transmembrane domain implicate the existence of an extracellular phosphorylation network which could be involved in intercellular communication.     

Cell-to-cell transport of proteins: requirement for unfolding and characterization of binding to a putative plasmodesmal receptor

Plasmodesmata and the nuclear pore complex (NPC) mediate the selective trafficking of proteins and protein-nucleic acid complexes. The events underlying the translocation of endogenous and viral proteins through plasmodesmata were investigated to further explore the parallels between these cell-to-cell and intracellular communication systems. Studies performed with crosslinked KNOTTED1 (KN1) revealed that a conformational change is required for the cell-to-cell movement of this protein. Microinjection of gold-conjugated KN1 established that, as with the NPC, a combination of protein unfolding and microchannel dilation appears to be involved in protein translocation. However, during this process the extent of microchannel dilation is much less than observed for the NPC, which may reflect a physical limitation imposed by the cell wall. Co-injection of KN1-gold with unbound KN1 or cucumber mosaic virus movement protein (CMV-MP) established that the KN1-gold probe is highly effective at blocking plasmodesmal transport of KN1 and CMV-MP. This result provided the foundation for competition experiments which demonstrated that KN1 and the viral movement proteins of CMV and tobacco mosaic virus likely utilize a common receptor in the pathway for cell-to-cell transport of proteins. A combination of biochemical fractionation methods, an in vitro binding assay founded on the high affinity between KN1-gold and the putative common plasmodesmal receptor, and microinjection techniques were used to isolate plasmodesmal constituents involved in cell-to-cell transport. A model describing the steps involved in protein transport through plasmodesmata is presented.     

Identification of glycosylphosphatidylinositol-anchored proteins in Arabidopsis. A proteomic and genomic analysis

In a recent bioinformatic analysis, we predicted the presence of multiple families of cell surface glycosylphosphatidylinositol (GPI)-anchored proteins (GAPs) in Arabidopsis (G.H.H. Borner, D.J. Sherrier, T.J. Stevens, I.T. Arkin, P. Dupree [2002] Plant Physiol 129: 486-499). A number of publications have since demonstrated the importance of predicted GAPs in diverse physiological processes including root development, cell wall integrity, and adhesion. However, direct experimental evidence for their GPI anchoring is mostly lacking. Here, we present the first, to our knowledge, large-scale proteomic identification of plant GAPs. Triton X-114 phase partitioning and sensitivity to phosphatidylinositol-specific phospholipase C were used to prepare GAP-rich fractions from Arabidopsis callus cells. Two-dimensional fluorescence difference gel electrophoresis and one-dimensional sodium dodecyl sulfate-polyacrylamide gel electrophoresis demonstrated the existence of a large number of phospholipase C-sensitive Arabidopsis proteins. Using liquid chromatography-tandem mass spectrometry, 30 GAPs were identified, including six beta-1,3 glucanases, five phytocyanins, four fasciclin-like arabinogalactan proteins, four receptor-like proteins, two Hedgehog-interacting-like proteins, two putative glycerophosphodiesterases, a lipid transfer-like protein, a COBRA-like protein, SKU5, and SKS1. These results validate our previous bioinformatic analysis of the Arabidopsis protein database. Using the confirmed GAPs from the proteomic analysis to train the search algorithm, as well as improved genomic annotation, an updated in silico screen yielded 64 new candidates, raising the total to 248 predicted GAPs in Arabidopsis.     

A plasmodesmata-associated beta-1,3-glucanase in Arabidopsis

Plasmodesmal conductivity is regulated in part by callose turnover, which is hypothesized to be determined by beta-1,3-glucan synthase versus glucanase activities. A proteomic analysis of an Arabidopsis thaliana plasmodesmata (Pd)-rich fraction identified a beta-1,3-glucanase as present in this fraction. The protein encoded by the putative plasmodesmal associated protein (ppap) gene, termed AtBG_ppap, had previously been found to be a post-translationally modified glycosylphosphatidylinositol (GPI) lipid-anchored protein. When fused to green fluorescent protein (GFP) and expressed in tobacco (Nicotiana tabacum) or Nicotiana benthamiana epidermal cells, this protein displays fluorescence patterns in the endoplasmic reticulum (ER) membrane system, along the cell periphery and in a punctate pattern that co-localizes with aniline blue-stained callose present around the Pd. Plasma membrane localization was verified by co-localization of AtBG_ppap:GFP together with a plasma membrane marker N-[3-triethylammoniumpropyl]-4-[p-diethylaminophenylhexatrienyl] pyridinium dibromide (FM4-64) in plasmolysed cells. In Arabidopsis T-DNA insertion mutants that do not transcribe AtBG_ppap, functional studies showed that GFP cell-to-cell movement between epidermal cells is reduced, and the conductivity coefficient of Pd is lower. Measurements of callose levels around Pd after wounding revealed that callose accumulation in the mutant plants was higher. Taken together, we suggest that AtBG_ppap is a Pd-associated membrane protein involved in plasmodesmal callose degradation, and functions in the gating of Pd.     

Identification of phytochrome-interacting protein candidates in Arabidopsis thaliana by co-immunoprecipitation coupled with MALDI-TOF MS

Phytochrome-interacting proteins have been extensively studied to elucidate light-signaling pathway in plants. However, most of these proteins have been identified by yeast two-hybrid screening using the C-terminal domain of phytochromes. We used co-immunoprecipitation followed by proteomic analysis in plant cell extracts in an attempt to screen for proteins interacting either directly or indirectly with native holophytochromes including the N-terminal domain as well as C-terminal domain. A total of 16 protein candidates were identified, and were selected from 2-DE experiments. Using MALDI-TOF MS analysis, 7 of these candidates were predicted to be putative phytochrome A-interacting proteins and the remaining ones to be phytochrome B-interacting proteins. Among these putative interacting proteins, protein phosphatase type 2C (PP2C) and a 66-kDa protein were strong candidates as novel phytochrome-interacting proteins, as knockout mutants for the genes encoding these two proteins had impaired light-signaling functions. A transgenic knockout Arabidopsis study showed that a 66-kDa protein candidate regulates hypocotyl elongation in a light-specific manner, and altered cotyledon development under white light during early developmental stages. The PP2C knockout plants also displayed light-specific changes in hypocotyl elongation. These results suggest that co-immunoprecipitation, followed by proteomic analysis, is a useful method for identifying novel interacting proteins and determining real protein-protein interactions in the cell.     

Rapid detection of plasmodesmata in purified cell walls

Summary Plasmodesmata are complex channels within the plant cell wall, which create plasma membrane and symplastic continuity between neighbouring cells. To detect plasmodesmata in cell wall preparations fromNicotiana cle elandii, we have used 3,3-dihexyl-oxacarbocyanine iodide (DiOC₆), a cationic amphiphilic fluorescent probe, widely employed for general studies of membrane structure and dynamics. Punctate fluorescent staining was readily seen in pit fields, small depressions within the cell wall known to be rich in plasmodesmata. Scanning electron microscopy was used to demonstrate that the punctate staining corresponded to plasmodesmata. Treatment of cell wall fragments with chloroform-methanol to remove lipids did not alter the staining of plasmodesmata. In contrast, pronase E-sodium dodecyl sulfate treatment completely abolished staining, indicating that the DiOC₆ labelling of plasmodesmata may be protein rather than lipid specific. Although not membrane mediated, DiOC₆ staining of plasmodesmata is a simple, rapid, and specific tool for the detection of plasmodesmata in isolated cell walls and will prove useful for studies of plasmodesmal location, structure, and composition.     

Comparative proteomic profiling of the choline transporter‐like1 (CHER1) mutant provides insights into plasmodesmata composition of fully developed Arabidopsis thaliana leaves

In plants, intercellular communication and exchange are highly dependent on cell wall bridging structures between adhering cells, so‐called plasmodesmata (PD). In our previous genetic screen for PD‐deficient Arabidopsis mutants, we described choline transporter‐like 1 (CHER1) being important for PD genesis and maturation. Leaves of cher1 mutant plants have up to 10 times less PD, which do not develop to complex structures. Here we utilize the T‐DNA insertion mutant cher1–4 and report a deep comparative proteomic workflow for the identification of cell‐wall‐embedded PD‐associated proteins. Analyzing triplicates of cell‐wall‐enriched fractions in depth by fractionation and quantitative high‐resolution mass spectrometry, we compared > 5000 proteins obtained from fully developed leaves. Comparative data analysis and subsequent filtering generated a list of 61 proteins being significantly more abundant in Col‐0. This list was enriched for previously described PD‐associated proteins. To validate PD association of so far uncharacterized proteins, subcellular localization analyses were carried out by confocal laser‐scanning microscopy. This study confirmed the association of PD for three out of four selected candidates, indicating that the comparative approach indeed allowed identification of so far undescribed PD‐associated proteins. Performing comparative cell wall proteomics of Nicotiana benthamiana tissue, we observed an increase in abundance of these three selected candidates during sink to source transition. Taken together, our comparative proteomic approach revealed a valuable data set of potential PD‐associated proteins, which can be used as a resource to unravel the molecular composition of complex PD and to investigate their function in cell‐to‐cell communication.     

Analysis of cytoplasmic membrane proteome of Streptococcus pneumoniae by shotgun proteomic approach

In this study, cytoplasmic membrane proteins of S. pneumoniae strain R6 (ATCC BBA-255) were effectively separated from cell wall or extracellular proteins by sodium carbonate precipitation (SCP) and ultracentrifugation. Forty seven proteins were analyzed as cytoplasmic membrane proteins from the 260 proteins identified by the shotgun proteomic method using SDS-PAGE/LC/MS-MS. ABC transporters for metabolites such as metals, oligopeptides, phosphate, sugar, and amino acids, and membrane proteins involved in phosphotransferse systems, were identified as the predominant and abundant, cytoplasmic membrane proteins that would be essential for nutrient uptake, antibiotic resistance and virulence mechanisms. Our result supports that gel-based shotgun proteomics combined with sodium carbonate precipitation and ultracentrifugation is an effective method for analysis of cytoplasmic membrane proteins of S. pneumoniae.     

胞间连丝研究的进展

胞间连丝为多细胞植物有机体提供了一个直接的细胞间物质运输和信息传递的细胞质通道,把一个个独立的“细胞王国”转变成相互连接的共质体,它是当今细胞生物学中十分活跃的研究领域。日益增多的研究结果揭示,胞间连丝协调基因表达和许多的细胞生理生化过程,对细胞的分裂与分化、形态发生、植物体的生长与发育,以及植物对环境的反应与适应等诸方面都起着十分重要的作用。本文仅就胞间连丝结构的多样性;胞间通道的调节因子;大分子蛋白质和核酸的胞间运输;胞间连丝阻断和共质体分区的形成及其与形态发生、休眠和抗逆性的关系等几个方面的新进展做一个简要的综述,借此例证胞间连丝在植物生命活动中的重要意义。     

胞间连丝研究的进展

胞间连丝为多细胞植物有机体提供了一个直接的细胞间物质运输和信息传递的细胞质通道,把一个个独立的“细胞王国”转变成相互连接的共质体,它是当今细胞生物学中十分活跃的研究领域。日益增多的研究结果揭示,胞间连丝协调基因表达和许多的细胞生理生化过程,对细胞的分裂与分化、形态发生、植物体的生长与发育,以及植物对环境的反应与适应等诸方面都起着十分重要的作用。本文仅就胞间连丝结构的多样性;胞间通道的调节因子;大分子蛋白质和核酸的胞间运输;胞间连丝阻断和共质体分区的形成及其与形态发生、休眠和抗逆性的关系等几个方面的新进展做一个简要的综述,借此例证胞间连丝在植物生命活动中的重要意义。     

Structure and development of cell connections in the phloem ofMetasequoia glyptostroboides needles I. Ultrastructural aspects of modified primary plasmodesmata in Strasburger cells

Summary Symplasmic contacts of Strasburger cells in the mature needle ofMetasequoia glyptostroboides were analysed with special regard to changes of plasmodesmata in fine structure and distribution. In meristematic cells simple primary plasmodesmata are evenly distributed throughout the entire wall, whereas in mature Strasburger cells plasmodesmata are aggregated in defined, dome-shaped wall thickenings. The elongated, often multiple-branched cytoplasmic strands show a distinct neck region besides a considerably dilated sleeve region confluent with cavities, which have formed at branching sites of plasmodesmata in various planes of the wall thickening. Most branches radiating from these cavities connect the protoplasts of the adjacent cells; occasionally some strands are discontinuous. The desmotubules of both, continuous and discontinuous plasmodesmal branches exhibit great variability in structure and number: they may be partially dilated, multiple-stranded and branched within single plasmodesmal branches. Fine structurally, plasmodesmata of Strasburger cells show great resemblance with developing sieve pores of conifers. This characteristic fine structure implicates a special role of the endomembrane system for phloem loading in theMetasequoia leaf.Dedicated to Prof. Dr. Dr. h.c. Eberhard Schnepf on the occasion of his retirement     

Modified plasmodesmata inSorghum (Sorghum bicolor L Moench) leaf tissues infected by maize dwarf mosaic virus

The early acute response (EAR), a type of hypersensitive response, is defined by small chlorotic spots at the base of the youngest leaf of sorghum (Sorghum bicolor L. Moench) cultivar HOK, and usually appears within five days after inoculation with maize dwarf mosaic virus strain A (MDMV-A). These chlorotic spots become necrotic one to two days later and the leaf tissues are rapidly killed. In leaf tissues showing EAR, plasmodesmal fields contained many modified plasmodesmata of various sizes and structures within thickened cell walls. The membranous vesicles and tubules, derived from the extended terminal structures of modified plasmodesmata, were blocked by callose-like deposits in the area between the cell wall and plasmalemma. Also observed were two opposite-directed channels united via a central cavity at the middle lamella of the cell wall, one end of which was connected to the plasmalemma, but the other end sealed off to form a bulbous extension. The localized structure, an extraprotoplasmic sac containing aggregates of elongated virus-like particles associated with the modified plasmodesmata, was located between the plasmalemma and the cell wall. The sac was bound by membranes, and appeared to be sealed and completely excluded from the protoplasm. Extraprotoplasmic sacs appeared to be derived from the terminal extension of modified plasmodesmata, and these modification seem to be related to restriction of the viral spread.     

Plasmodesmal widening accompanies the short-term increase in symplasmic phloem unloading in pea root tips under osmotic stress

Summary Root tips ofPisum sativum seedlings were exposed to 350 mM mannitol, which was shown to effect a transient but dramatic increase in phloem unloading, and investigated by electron microscopy. After chemical fixation and embedding, extremely thin sections of the root extension zone were examined. Outer, inner, and desmotubule diameters of 830 primary plasmodesmata in transverse walls of cortical cells were measured. Statistical analysis indicated that the majority of plasmodesmata had no neck constriction during osmoregulation. Compared to controls, a highly significant increase in mean plasmodesmata diameter was found, but the desmotubule diameter remained unchanged. Both loss of neck constriction and widening of the cytoplasmic sleeve indicate an increase in effective passage area of plasmodesmata. Spokes between plasma membrane and desmotubule were preserved. Continued exposure of the root tips to mannitol led to a return to control values for plasmodesmal diameters. In contrast to these responses, plasmolysis of cortical cells by 1,000 mM sucrose, diminishing phloem unloading, was accompanied by a reduction in those plasmodesmata classified as open. This is the first report showing a correlation between the ultrastructure of plasmodesmata and the rate of symplasmic transport. The role of the different plasmodesmal components in controlling the passage area of symplasmic transport is discussed.