Plasmodesma-mediated selective protein traffic between "symplasmically isolated" cells probed by a viral movement protein.
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Intercellular communication is essential for differentiation and development. In plants, plasmodesmata (PD) form cytoplasmic channels for direct communication. During plant development, programmed reduction in PD number and transport capacity creates the so-called symplasmic domains. Small fluorescent dyes and ions can diffuse among cells within a domain but not across domain boundaries. Such symplasmic isolation is thought to allow groups of cells to differentiate and develop into tissues with distinct structures and functions. Whether or how "symplasmically isolated" cells communicate with one another is poorly understood. One well-documented symplasmic domain is the sieve element-companion cell (SE-CC) complex in the phloem tissue. We report here that, when produced in the CC of transgenic tobacco, the 3a movement protein (3a MP) of Cucumber mosaic virus fused to green fluorescent protein (GFP) can traffic out of the SE-CC complex via PD. The extent of 3a MP:GFP traffic across the boundary between vascular and nonvascular tissues depends on organ type and developmental stage. Our findings provide experimental evidence that endogenous machinery exists for protein traffic between the symplasmically isolated SE-CC complex and neighboring cells. We suggest that PD-mediated traffic of selected macromolecules can be a mechanism for symplasmically isolated cells to communicate with one another. (+info)
A subclass of plant heat shock cognate 70 chaperones carries a motif that facilitates trafficking through plasmodesmata.
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Plasmodesmata establish a pathway for the trafficking of non-cell-autonomously acting proteins and ribonucleoprotein complexes. Plasmodesmal enriched cell fractions and the contents of enucleate sieve elements, in the form of phloem sap, were used to isolate and characterize heat shock cognate 70 (Hsc70) chaperones associated with this cell-to-cell transport pathway. Three Cucurbita maxima Hsc70 chaperones were cloned and functional and sequence analysis led to the identification of a previously uncharacterized subclass of non-cell-autonomous chaperones. The highly conserved nature of the heat shock protein 70 (Hsp70) family, in conjunction with mutant analysis, permitted the characterization of a motif that allows these Hsc70 chaperones to engage the plasmodesmal non-cell-autonomous translocation machinery. Proof of concept that this motif is necessary for Hsp70 gain-of-movement function was obtained through the engineering of a human Hsp70 that acquired the capacity to traffic through plasmodesmata. These results are discussed in terms of the roles likely played by this subclass of Hsc70 chaperones in the trafficking of non-cell-autonomous proteins. (+info)
Selective trafficking of non-cell-autonomous proteins mediated by NtNCAPP1.
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In plants, cell-to-cell communication is mediated by plasmodesmata and involves the trafficking of non-cell-autonomous proteins (NCAPs). A component in this pathway, Nicotiana tabacum NON-CELL-AUTONOMOUS PATHWAY PROTEIN1 (NtNCAPP1), was affinity purified and cloned. Protein overlay assays and in vivo studies showed that NtNCAPP1 is located on the endoplasmic reticulum at the cell periphery and displays specificity in its interaction with NCAPs. Deletion of the NtNCAPP1 amino-terminal transmembrane domain produced a dominant-negative mutant that blocked the trafficking of specific NCAPs. Transgenic tobacco plants expressing this mutant form of NtNCAPP1 and plants in which the NtNCAPP1 gene was silenced were compromised in their ability to regulate leaf and floral development. These results support a model in which NCAP delivery to plasmodesmata is both selective and regulated. (+info)
Immunodetection and fluorescent microscopy of transgenically expressed hordeivirus TGBp3 movement protein reveals its association with endoplasmic reticulum elements in close proximity to plasmodesmata.
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The subcellular localization of the hydrophobic TGBp3 protein of Poa semilatent virus (PSLV, genus Hordeivirus) was studied in transgenic plants using fluorescent microscopy to detect green fluorescent protein (GFP)-tagged protein and immunodetection with monoclonal antibodies (mAbs) raised against the GFP-based fusion expressed in E. coli. In Western blot analysis, mAbs efficiently recognized the wild-type and GFP-fused PSLV TGBp3 proteins expressed in transgenic Nicotiana benthamiana, but failed to detect TGBp3 in hordeivirus-infected plants. It was found that PSLV TGBp3 and GFP-TGBp3 had a tendency to form large protein complexes of an unknown nature. Fractionation studies revealed that TGBp3 represented an integral membrane protein and probably co-localized with an endoplasmic reticulum-derived domain. Microscopy of epidermal cells in transgenic plants demonstrated that GFP-TGBp3 localized to cell wall-associated punctate bodies, which often formed pairs of opposing discrete structures that co-localized with callose, indicating their association with the plasmodesmata-enriched cell wall fields. After mannitol-induced plasmolysis of the leaf epidermal cells in the transgenic plants, TGBp3 appeared within the cytoplasm and not at cell walls. Although TGBp3-induced bodies were normally static, most of them became motile after plasmolysis and displayed stochastic motion in the cytoplasm. (+info)
Dispatch. Intercellular signaling: an elusive player steps forth.
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Plasmodesmata play a central role in cell-to-cell communication in plants, allowing transport of regulatory proteins and mRNAs. A recent study has identified a specific protein that regulates the intercellular transport of macromolecules in plants, known as non-cell autonomous pathway protein 1. (+info)
High-throughput viral expression of cDNA-green fluorescent protein fusions reveals novel subcellular addresses and identifies unique proteins that interact with plasmodesmata.
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A strategy was developed for the high-throughput localization of unknown expressed proteins in Nicotiana benthamiana. Libraries of random, partial cDNAs fused to the 5' or 3' end of the gene for green fluorescent protein (GFP) were expressed in planta using a vector based on Tobacco mosaic virus. Viral populations were screened en masse on inoculated leaves using a confocal microscope fitted with water-dipping lenses. Each viral infection site expressed a unique cDNA-GFP fusion, allowing several hundred cDNA-GFP fusions to be screened in a single day. More than half of the members of the library carrying cDNA fusions to the 5' end of gfp that expressed fluorescent fusion proteins displayed discrete, noncytosolic, subcellular localizations. Nucleotide sequence determination of recovered cDNA sequences and subsequent sequence searches showed that fusions of GFP to proteins that had a predicted subcellular "address" became localized with high fidelity. In a subsequent screen of >20,000 infection foci, 12 fusion proteins were identified that localized to plasmodesmata, a subcellular structure for which very few protein components have been identified. This virus-based system represents a method for high-throughput functional genomic study of plant cell organelles and allows the identification of unique proteins that associate with specific subcompartments within organelles. (+info)
Limitations on geminivirus genome size imposed by plasmodesmata and virus-encoded movement protein: insights into DNA trafficking.
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Animals and plants evolved systems to permit non-cell-autonomous trafficking of RNA, whereas DNA plays a cell-autonomous role. In plants, plasmodesmata serve as the conduit for this phenomenon, and viruses have evolved to use this pathway for the spread of infectious nucleic acids. In this study, a plant DNA virus was used to explore the constraints imposed on the movement of DNA through this endogenous RNA trafficking pathway. The combined properties of the geminivirus-encoded movement protein and plasmodesmata were shown to impose a strict limitation on the size of the viral genome at the level of cell-to-cell movement. Size-increased viral genome components underwent homologous and nonhomologous recombination to overcome this strict limitation. Our results provide insights into the genetic mechanisms that underlie viral evolution and provide a likely explanation for why relatively few types of plant DNA viruses have evolved: they would have had to overcome the constraints imposed by an endogenous system operating to ensure that DNA acts in a cell-autonomous manner. (+info)
Nodule initiation involves the creation of a new symplasmic field in specific root cells of medicago species.
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The organogenesis of nitrogen-fixing nodules in legume plants is initiated in specific root cortical cells and regulated by long-distance signaling and carbon allocation. Here, we explore cell-to-cell communication processes that occur during nodule initiation in Medicago species and their functional relevance using a combination of fluorescent tracers, electron microscopy, and transgenic plants. Nodule initiation induced symplasmic continuity between the phloem and nodule initials. Macromolecules such as green fluorescent protein could traffic across short or long distances from the phloem into these primordial cells. The created symplasmic field was regulated throughout nodule development. Furthermore, Medicago truncatula transgenic plants expressing a viral movement protein showed increased nodulation. Hence, the establishment of this symplasmic field may be a critical element for the control of nodule organogenesis. (+info)