Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic. The specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index is solely determined by the viral coat protein. There are plenty of plant viruses that no one has heard of, but few are as widely known as grapevine fanleaf virus. Learn how to identify a sick.
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Due to the observed variation in protoplast viability, we chose to test the effect of the drugs on viral replication by calculating the percentage of infected cells as previously described elsewhere 11 rather than by Northern blot analysis.
Fuchs Colmar, FranceR.
Although some replication centers were found in the ER-rich perinuclear region, many of them remained peripheral. The perfect colocalization of dsRNA and fanlead RNA signals within ER aggregates, together with the exclusion from these aggregates of the Golgi which remained essentially unaffected, leads us to conclude that the ER is the major contributor to the morphogenesis of the viral compartment where virus replication occurs.
Whereas the center of the rosettes was rather amorphous and electron dense, their periphery was made of vesicles similar in size to the isolated vesicles seen in Fig. This page was last edited on 22 Marchat Cells were fixed with glutaraldehyde, permeabilized, quenched with NaBH 4and treated with primary antibodies, followed by treatment with secondary antibodies coupled to fluorochromes Ab1 and Ab2.
The membranes were then incubated with goat anti-rabbit IgG A or goat anti-mouse IgG B and C coupled virue horseradish peroxidase and revealed by chemiluminescence.
RNA1 encodes polyprotein P1 kDawhich is processed by an embedded proteinase activity into five proteins required for replication, namely, 1A of unknown function1B probably the helicase1C VPg1D proteinaseand 1E polymerase 38 Grapevlne Read Edit View history.
No signal was detected in total protein extracts from healthy protoplasts Fig. Cloning and in vitro characterization of the grapevine fanleaf virus proteinase cistron.
In addition, Golgi stacks were restricted to the periphery of these aggregates Fig. Support Center Support Center. Upon uptake of the virus, a fankeaf can remain viruliferous for nearly a year. Fanleaf and LeafrollWashington State University. For FDA, the parameters were as follows: The specific cytopathological effect of GFLV infection culminated in the nearly complete depletion of the cortical ER accompanied by a progressive build-up of perinuclear virus-induced ER aggregates.
Journal List J Virol v. Finally, the CP-containing spikes in Fig. In the present study, the structural preservation of cells and the intensity of immunolabelings were markedly enhanced, and image resolution was further improved by using high-resolution confocal microscopy.
Grapevine Fanleaf Virus Replication Occurs on Endoplasmic Reticulum-Derived Membranes
Photo courtesy of William M. The structure and origin of these aggregates could now be resolved in much better detail by CLSM.
Enzyme cytochemical identification of single-stranded and double-stranded RNAs in virus-infected plant and insect cells. In an attempt to visualize the membranes that cosedimented with the VPg precursors, ISEM was performed on sucrose gradient fractions with immunoaffinity-purified anti-VPg antibodies. Received Dec 27; Accepted Jun 3.
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To compare the distribution of the viral proteins involved in replication with that of proteins involved in movement and encapsidation, we analyzed the intracellular distribution of the movement protein 2B MP and the coat protein 2C CPboth of which are dispensable for replication Preparation and infection of protoplasts. In the first syndrome, infectious malformations, the vines may be stunted or show reduced vigor. Inhibition of grapevins RNA synthesis by brefeldin A. Using epifluorescence microscopy, we have previously described the formation of a perinuclear complex where viral RNA was synthesized and viral proteins accumulated 23but this could not be further analyzed due to technical limitations.
Evidence for the involvement of virus-induced vesicles in poliovirus replication was obtained already in the sixties 10and danleaf molecular mechanisms underlying the formation of virus-induced vesicles, their origin, and their role in viral replication were recently unraveled 1958 To demonstrate whether the ER-derived aggregates were involved in viral replication, double-labeling experiments were performed with anti-dsRNA and anti-VPg antibodies.
Grapevine fanleaf nepovirus P38 putative movement protein is located on tubules in vivo.
Grapevine fanleaf virus
Detection of viral proteins in cytopathic structures in cowpea protoplasts infected with cowpea mosaic virus. Arrowheads, Golgi; N, nucleus; P, plastids; M, mitochondria. Association of cowpea mosaic virus-induced double-stranded RNA in a cytopathological structure in infected cells. BFA and cerulenin treatment. The number of infected cells, as revealed by the anti-VPg or anti-CP signal, was counted versus the total number of protoplasts determined under UV illumination after staining the nucleus with Hoechst and corrected for cell viability.
Excitation and emission wavelengths were and to nm, respectively, for either GFP or A and and to nm, respectively, for A Infected berries are uneven in size with numerous small and seedless individuals, some of which may not mature.
Fanleaf Degeneration of Grape
Altogether, these results clearly confirm that GFLV replication occurs at numerous discrete sites associated with condensed ER but not GA -derived perinuclear aggregates. These typical foliar symptoms resemble a fan, hence, the name of the virus and the disease Figure 1.
In view of the close resemblance between both systems, we suggest that GFLV could use a similar mechanism to recruit membranes for replication purposes. Recently, it was demonstrated that poliovirus vesicles are formed at the ER by the cellular COPII budding mechanism and are thus homologous to vesicles of the anterograde transport pathway Brefeldin A inhibits cell-free.
A monoclonal antibody, JIM84, recognizes the Golgi apparatus and plasma membrane in plant cells. Such bundles of filamentous structures could not correspond to the previously described tubules made of the grapevind movement protein and containing virions 54 since they were occasionally observed in healthy protoplasts data not shown.
Formation of plant RNA virus replication complexes on membranes: In line with the proposed association of P2 famleaf RNA2 via the 2A region 23some label corresponding to the movement protein 2B presumably as the polyprotein or a proteolytic maturation precursor and the coat protein were occasionally detected in the vicinity of the replication compartment.