Ultrastructural Analysis of Prune Dwarf Virus Intercellular Transport and Pathogenesis

Abstract

Prune dwarf virus (PDV) is an important viral pathogen of plum, sweet cherry, peach, and many herbaceous test plants. Although PDV has been intensively investigated, mainly in the context of phylogenetic relationship of its genes and proteins, many gaps exist in our knowledge about the mechanism of intercellular transport of this virus. The aim of this work was to investigate alterations in cellular organelles and the cell-to-cell transport of PDV in Cucumis sativus cv. Polan at ultrastructural level. To analyze the role of viral proteins in local transport, double-immunogold assays were applied to localize PDV coat protein (CP) and movement protein (MP). We observe structural changes in chloroplasts, mitochondria, and cellular membranes. We prove that PDV is transported as viral particles via MP-generated tubular structures through plasmodesmata. Moreover, the computer-run 3D modeling reveals structural resemblances between MPs of PDV and of Alfalfa mosaic virus (AMV), implying similarities of transport mechanisms for both viruses.

Modifications in Tissue and Cell Ultrastructure as Elements of Immunity-Like Reaction in Chenopodium quinoa against Prune Dwarf Virus (PDV)

“…Two weeks after PDV inoculation, fragments of quinoa leaf blades that were analyzed with DAS-ELISA were treated, as previously described by Kozieł et al [8]. To check the distribution of PDV, P1 protein was localized by immunofluorescence according to a procedure described by Kozieł et al [9] with modification regarding secondary antibody.…”

“…Fragments of quinoa leaf tissue (at 7, 14, and 20 dpi) were cut out, fixed, and embedded with EPOXY resin exactly as described by Kozieł et al [8], followed by slicing into thin sections for light microscope or ultrathin sections (100 nm) for TEM, and mounted on slides or copper grids, respectively [19]. Slides were stained with crystal violet, while ultrathin sections were contrasted with uranyl acetate/lead citrate (Sigma-Aldrich, St. Louis, MO, USA) for TEM.…”

“…Fragments of quinoa leaf tissue was prepared according to Section 2.4. Immunogold localization was performed exactly according to the procedure presented by [8] with use of primary antibodies targeted PDV MP used previously in [8] and secondary anti mouse antibodies associated with 18 nm gold particles (JaksonImmunoResearch Europe, Cambridgeshire, UK).…”

“…CP supports the "genome activation" process, which is crucial for replication of the viral RNA genome and also creates capsid for viral particles [7]. MP enables generation of tubular structures during cell-to-cell transport and supports translocation of viral particles via plasmodesmata [8].…”

“…In an infected host plant, PDV is transported both cell-to-cell and systemically throughout the whole plant. During local spreading (cell-to-cell movement), the virus is transported as viral particles via MP-generated tubular structures through plasmodesmata, which modify their size exclusion limit (SEL) [8]. In the case of systemic movement, our new data from infected tobacco and cucumber reveal that the transport is mainly associated with phloem and xylem cells [8][9][10].…”