Subcelullar localization of proteins associated with Prune dwarf virus replication

Abstract

Prune dwarf virus (PDV) is one of the most dangerous pathogens of fruit trees worldwide. One of the most important proteins required for PDV infection is replicase. (P1 protein) which anchored viral RNA and builds replication complex along with RNA depended polymerase. Despite the importance of PDV as a pathogen, our knowledge regarding tissue/cellular localization and structure of PDV P1 protein is still incomplete. The aim of this work was to localize replicase distribution in leaf tissues and cells by immunofluorescent and immunogold labeling of Nicotiana tobaccum cv Samsun and development of a 3D model of PDV replicase. In this paper we demonstrate that PDV replication, is similar to that of Alfalfa mosaic virus and is strongly connected with tonoplasts. In addition, PDV replicase and coat protein (CP) were also found to be strongly associated with membranes of endoplasmic reticulum and, indicating the potential involvement of these membrane structures in the processes related to viral infection. Bioinformatic analyzes based on 3D modeling and structure prediction revealed that P1 protein has a potential transmembrane domain which enables protein anchoring to tonoplast during replication complex assembly.

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

“…We demonstrate that PDV generates chlorotic lesions on inoculated leaves at 7 dpi, also previously reported by Fulton [11] and Kozieł et al [8] for cucumber cotyledons or inoculated leaves of tobacco [10]. However, for the latter two hosts, infection symptoms were also observed on upper leaves at 14 dpi by Fulton [11] and Kozieł et al [9]. Moreover, Kozieł et al [9] also observed deformation of leaf blades resulting from PDV infection.…”

“…According to the Plant Virus Online database [13], this species is not susceptible to a broad spectrum of different isolates/strains of PDV. Fifty quinoa plants were mechanically inoculated as was presented in [9]. Mock-and PDV-inoculated leaves along with leaves and stems above were checked for the presence of PDV by using DAS-ELISA in 3 repeats for each time point, with primary antibodies against the PDV CP (Bioreba, Reinach, Switzerland), followed by purified anti-rabbit antibodies conjugated with alkaline phosphatase (Bioreba, Reinach, Switzerland) [14].…”

“…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. Purified rabbit polyclonal antibody anti-P1-PDV (GeneCust, Boynes, Luxemburg) served as primary antibody [9], while the secondary one was anti-rabbit antibody IgG with the attached AlexaFluor ® 488 (Abcam, Cambridge, UK).…”

“…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. Purified rabbit polyclonal antibody anti-P1-PDV (GeneCust, Boynes, Luxemburg) served as primary antibody [9], while the secondary one was anti-rabbit antibody IgG with the attached AlexaFluor ® 488 (Abcam, Cambridge, UK). For better contrast of plant tissues, we added 4 ,6-diamidine-2 -phenylindole dihydrochloride (DAPI).…”

“…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].…”