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1.
Grapevine fanleaf virus replication occurs on endoplasmic reticulum-derived membranes 总被引:2,自引:0,他引:2 下载免费PDF全文
Ritzenthaler C Laporte C Gaire F Dunoyer P Schmitt C Duval S Piéquet A Loudes AM Rohfritsch O Stussi-Garaud C Pfeiffer P 《Journal of virology》2002,76(17):8808-8819
Infection by Grapevine fanleaf nepovirus (GFLV), a bipartite RNA virus of positive polarity belonging to the Comoviridae family, causes extensive cytopathic modifications of the host endomembrane system that eventually culminate in the formation of a perinuclear "viral compartment." We identified by immunoconfocal microscopy this compartment as the site of virus replication since it contained the RNA1-encoded proteins necessary for replication, newly synthesized viral RNA, and double-stranded replicative forms. In addition, by using transgenic T-BY2 protoplasts expressing green fluorescent protein in the endoplasmic reticulum (ER) or in the Golgi apparatus (GA), we could directly show that GFLV replication induced a depletion of the cortical ER, together with a condensation and redistribution of ER-derived membranes, to generate the viral compartment. Brefeldin A, a drug known to inhibit vesicle trafficking between the GA and the ER, was found to inhibit GFLV replication. Cerulenin, a drug inhibiting de novo synthesis of phospholipids, also inhibited GFLV replication. These observations imply that GFLV replication depends both on ER-derived membrane recruitment and on de novo lipid synthesis. In contrast to proteins involved in viral replication, the 2B movement protein and, to a lesser extent, the 2C coat protein were not confined to the viral compartment but were transported toward the cell periphery, a finding consistent with their role in cell-to-cell movement of virus particles. 相似文献
2.
Schellenberger P Sauter C Lorber B Bron P Trapani S Bergdoll M Marmonier A Schmitt-Keichinger C Lemaire O Demangeat G Ritzenthaler C 《PLoS pathogens》2011,7(5):e1002034
Many animal and plant viruses rely on vectors for their transmission from host to
host. Grapevine fanleaf virus (GFLV), a picorna-like virus from
plants, is transmitted specifically by the ectoparasitic nematode
Xiphinema index. The icosahedral capsid of GFLV, which
consists of 60 identical coat protein subunits (CP), carries the determinants of
this specificity. Here, we provide novel insight into GFLV transmission by
nematodes through a comparative structural and functional analysis of two GFLV
variants. We isolated a mutant GFLV strain (GFLV-TD) poorly transmissible by
nematodes, and showed that the transmission defect is due to a glycine to
aspartate mutation at position 297 (Gly297Asp) in the CP. We next determined the
crystal structures of the wild-type GFLV strain F13 at 3.0 Å and of
GFLV-TD at 2.7 Å resolution. The Gly297Asp mutation mapped to an exposed
loop at the outer surface of the capsid and did not affect the conformation of
the assembled capsid, nor of individual CP molecules. The loop is part of a
positively charged pocket that includes a previously identified determinant of
transmission. We propose that this pocket is a ligand-binding site with
essential function in GFLV transmission by X. index. Our data
suggest that perturbation of the electrostatic landscape of this pocket affects
the interaction of the virion with specific receptors of the nematode''s
feeding apparatus, and thereby severely diminishes its transmission efficiency.
These data provide a first structural insight into the interactions between a
plant virus and a nematode vector. 相似文献
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Caroline Hemmer Samia Djennane Léa Ackerer Kamal Hleibieh Aurélie Marmonier Sophie Gersch Shahinez Garcia Emmanuelle Vigne Véronique Komar Mireille Perrin Claude Gertz Lorène Belval François Berthold Baptiste Monsion Corinne Schmitt‐Keichinger Olivier Lemaire Bernard Lorber Carlos Gutiérrez Serge Muyldermans Gérard Demangeat Christophe Ritzenthaler 《Plant biotechnology journal》2018,16(2):660-671
Since their discovery, single‐domain antigen‐binding fragments of camelid‐derived heavy‐chain‐only antibodies, also known as nanobodies (Nbs), have proven to be of outstanding interest as therapeutics against human diseases and pathogens including viruses, but their use against phytopathogens remains limited. Many plant viruses including Grapevine fanleaf virus (GFLV), a nematode‐transmitted icosahedral virus and causal agent of fanleaf degenerative disease, have worldwide distribution and huge burden on crop yields representing billions of US dollars of losses annually, yet solutions to combat these viruses are often limited or inefficient. Here, we identified a Nb specific to GFLV that confers strong resistance to GFLV upon stable expression in the model plant Nicotiana benthamiana and also in grapevine rootstock, the natural host of the virus. We showed that resistance was effective against a broad range of GFLV isolates independently of the inoculation method including upon nematode transmission but not against its close relative, Arabis mosaic virus. We also demonstrated that virus neutralization occurs at an early step of the virus life cycle, prior to cell‐to‐cell movement. Our findings will not only be instrumental to confer resistance to GFLV in grapevine, but more generally they pave the way for the generation of novel antiviral strategies in plants based on Nbs. 相似文献
5.
Transformation of grapevine rootstocks with the coat protein gene of grapevine fanleaf nepovirus 总被引:9,自引:0,他引:9
S. Krastanova M. Perrin P. Barbier G. Demangeat P. Cornuet N. Bardonnet L. Otten L. Pinck B. Walter 《Plant cell reports》1995,14(9):550-554
Summary Control of fanleaf disease induced by the Grapevine Fanleaf Nepovirus (GFLV) today is based on sanitary selection and soil disinfection with nematicides. This way of control is not always efficient and nematicides can be dangerous pollutants. Coat protein (CP) mediated protection could be an attractive alternative. We have transferred a chimeric CP gene of GFLV-F13 via Agrobacterium tumefaciens LBA4404 into two rootstock varieties: Vitis rupestris and 110 Richter (V. rupestris X V. Berlandieri). Transformation was performed on embryogenic callus obtained from anthers and on hypocotyl fragments from mature embryos. Success of the transformation was assessed by polymerase chain reaction and Southern analyses. Transformants with a number of copies of the CP gene varying from one to five were obtained. Enzyme-linked immunosorbent assay with virus-specific antibodies revealed various levels of expression of the coat protein in the different transformants.Abbreviations 2,4D
2,4 dichlorophenoxyacetic acid
- BAP
6-benzylaminopurine
- CP
coat protein
- EDTA
ethylene diamine tetraacetic acid
- ELISA
enzyme-linked immunosorbent assay
- GFLV
grapevine fanleaf virus
- GUS
glucuronidase
- IBA
indole-3-butyric acid
- NAA
1-naphthaleneacetic acid
- NOA
-naphthoxyacetic acid
- NOS
nopaline synthase
- NPTII
neomycin phosphotransferase II
- PCR
polymerase chain reaction 相似文献
6.
Primary structure and location of the genome-linked protein (VPg) of grapevine fanleaf nepovirus 总被引:1,自引:0,他引:1
The genome linked protein VPg covalently linked to the RNAs of grapevine fanleaf nepovirus has been sequenced. The VPg (Mr = 2931) composed of 24 residues is linked by its N-terminal Ser beta-OH group to the viral RNAs. The VPg mapped from residues 1218 to 1241 of the 253K polyprotein encoded by GFLV RNA1. 相似文献
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The antioxidative response of grapevine leaves (Vitis vinifera cv. Trebbiano) affected by the presence of grapevine fanleaf virus was studied during the summer of 2010 at three different harvest times (July 1st and 26th, and August 30th). At the first and second harvest, infected leaves showed increases in the concentration of superoxide radical and hydrogen peroxide, the latter increasing for enhanced activity of superoxide dismutase. In contrast, at the last harvest time, increases in the ascorbate pool and ascorbate peroxidase activity maintained hydrogen peroxide to control levels. The glutathione pool was negatively affected as summer progressed, showing a decrease in its total and reduced form amounts. At the same time, increases in the ascorbate pool were observed, making antioxidant defenses of grapevine effective also at the last harvest time. Increases in phenolic acids, and in particular in p-hydroxybenzoic acid, at the first and second harvest might have enhanced the efficiency of the antioxidant system through an interrelation between a peroxidase/phenol/ascorbate system and the NADPH/glutathione/ascorbate cycle. The lack of increase in p-hydroxybenzoic acid at the third harvest could be due instead to the enhanced utilization of this acid for hydrogen peroxide detoxification. With time, grapevine plants lost their capacity to contrast the spread of grapevine fanleaf virus, but acquired a greater ability to counteract pathogen-induced oxidative stress, being endowed with more reduced antioxidant pools. 相似文献
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Summary Grapevine fanleaf nepovirus (GFLV) is responsible for the economically significant court-noué disease in vineyards. Its genome is made up of two single-stranded RNA molecules (RNA1 and RNA2) which direct the synthesis of polyproteins P1 and P2 respectively. A chimeric coat protein gene derived from the C-terminal part of P2 was constructed and subsequently introduced into a binary transformation vector. Transgenic Nicotiana benthamiana plants expressing the coat protein under the control of the CaMV 35S promoter were engineered by Agrobacterium tumefaciens-mediated transformation. Protection against infection with virions or viral RNA was tested in coat protein-expressing plants. A significant delay of systemic invasion was observed in transgenic plants inoculated with virus compared to control plants. This effect was also observed when plants were inoculated with viral RNA. No coat protein-mediated cross-protection was observed when transgenic plants were infected with arabis mosaic virus (ArMV), a closely related nepovirus also responsible for a court-noué disease.Abbreviations GFLV-F13
grapevine fanleaf virus F13 isolate
- ArMV
arabis mosaic virus
- CP
coat protein
- MS
Murashige and Skoog
- NPTII
neomycin phosphotransferase II
- CaMV
cauliflower mosaic virus
- ELISA
enzyme linked immunosorbent assay
- VPg
genome linked viral protein
- TMV
tobacco mosaic virus
- PVX
potato virus X
- PVY
potato virus Y
- TRV
tobacco rattle virus
- +CP
CP expressing
- -CP
control plant, not expressing CP
- CPMP
coat protein-mediated protection
- CPMCP
coat crotein-mediated cross protection 相似文献
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Display of whole proteins on inner and outer surfaces of grapevine fanleaf virus‐like particles 下载免费PDF全文
Lorène Belval Caroline Hemmer Claude Sauter Catherine Reinbold Jean‐Daniel Fauny François Berthold Léa Ackerer Corinne Schmitt‐Keichinger Olivier Lemaire Gérard Demangeat Christophe Ritzenthaler 《Plant biotechnology journal》2016,14(12):2288-2299
Virus‐like particles (VLPs) derived from nonenveloped viruses result from the self‐assembly of capsid proteins (CPs). They generally show similar structural features to viral particles but are noninfectious and their inner cavity and outer surface can potentially be adapted to serve as nanocarriers of great biotechnological interest. While a VLP outer surface is generally amenable to chemical or genetic modifications, encaging a cargo within particles can be more complex and is often limited to small molecules or peptides. Examples where both inner cavity and outer surface have been used to simultaneously encapsulate and expose entire proteins remain scarce. Here, we describe the production of spherical VLPs exposing fluorescent proteins at either their outer surface or inner cavity as a result of the self‐assembly of a single genetically modified viral structural protein, the CP of grapevine fanleaf virus (GFLV). We found that the N‐ and C‐terminal ends of the GFLV CP allow the genetic fusion of proteins as large as 27 kDa and the plant‐based production of nucleic acid‐free VLPs. Remarkably, expression of N‐ or C‐terminal CP fusions resulted in the production of VLPs with recombinant proteins exposed to either the inner cavity or the outer surface, respectively, while coexpression of both fusion proteins led to the formation hybrid VLP, although rather inefficiently. Such properties are rather unique for a single viral structural protein and open new potential avenues for the design of safe and versatile nanocarriers, particularly for the targeted delivery of bioactive molecules. 相似文献
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Noémie S. Jelly Paul Schellenbaum Bernard Walter Pascale Maillot 《Transgenic research》2012,21(6):1319-1327
Grapevines are affected worldwide by viruses that compromise fruit yield and quality. Grapevine fanleaf virus (GFLV) causes fanleaf degeneration disease, a major threat to grapevine production. Transgenic approaches exploiting the RNA silencing machinery have proven suitable for engineering viral resistance in several crop species. However, the artificial microRNA (amiRNA)-based strategy has not yet been reported in grapevine. We developed two amiRNA precursors (pre-amiRNAs) targeting the coat protein (CP) gene of GFLV and characterised their functionality in grapevine somatic embryos. To create these pre-amiRNAs, natural pre-miR319a of Arabidopsis thaliana was modified by overlapping PCR in order to replace miR319a with two amiRNAs targeting different regions of the CP gene: amiRCP-1 or amiRCP-2. Transient expression of these two pre-amiRNA constructs was tested in grapevine somatic embryos after co-cultivation with Agrobacterium tumefaciens. Expression of amiRCP-1 and amiRCP-2 was detected in plant tissues by an endpoint stem-loop RT-PCR as early as 1?day after a 48-h co-cultivation, indicating active processing of pre-amiRNAs by the plant machinery. In parallel, GUS-sensor constructs (GCP-1 and GCP-2) were obtained by fusing the target sequence of amiRCP-1 or amiRCP-2 to the 3?? terminus of the GUS gene. Co-transformation assays with GUS-sensors and the pre-amiRNA constructs provided evidence for in vivo recognition and cleavage of the 21-nt target sequence of GUS-sensors by the corresponding amiRNA. This is the first report of amiRNA ectopic expression in grapevine. The constructs we developed could be useful for engineering GFLV-resistant grapes in the future. 相似文献
13.
Sami Fattouch Sonia M'hirsi Hajer Acheche Mohamed marrakchi Nejib Marzouki 《Plant Molecular Biology Reporter》2001,19(3):235-244
A simple, sensitive and specific RNA capture method is described for the detection of Grapevine fanleaf virus (GFLV) in infected
grapevines. This method consists of hybridizing GFLV-RNAs to oligoprobes immobilized on nylon membranes, followed by RT-PCR
amplification of targeted viral sequences. The RNA oligoprobe capture RT-PCR method is 10-fold more sensitive than IC-RT-PCR.
The efficiency of the RNA oligoprobe capture RT-PCR and the reuse of immobilized oligoprobe membranes without loss of efficiency
could make this procedure suitable for the routine diagnosis of GFLV in grapevines. 相似文献
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We report isolation, characterization and cross-species amplification of nine microsatellite loci from the phytoparasitic nematode Xiphinema index, the vector of grapevine fanleaf virus. Levels of polymorphism were evaluated in 62 individuals from two X. index populations. The number of alleles varies between two and 10 depending on locus and population. Observed heterozygosity on loci across both populations varied from 0.32 to 0.857 (mean 0.545). The primers were tested for cross-species amplification in three other species of phytoparasitic nematodes of the Xiphinema genus. These nine microsatellite loci constitute valuable markers for population genetics and phylogeographical studies of X. index. 相似文献
15.
Schellenberger P Demangeat G Lemaire O Ritzenthaler C Bergdoll M Oliéric V Sauter C Lorber B 《Journal of structural biology》2011,174(2):344-351
The small icosahedral plant RNA nepovirus Grapevine fanleaf virus (GFLV) is specifically transmitted by a nematode and causes major damage to vineyards worldwide. To elucidate the molecular mechanisms underlying the recognition between the surface of its protein capsid and cellular components of its vector, host and viral proteins synthesized upon infection, the wild type GFLV strain F13 and a natural mutant (GFLV-TD) carrying a Gly???Asp mutation were purified, characterized and crystallized. Subsequently, the geometry and volume of their crystals was optimized by establishing phase diagrams. GFLV-TD was twice as soluble as the parent virus in the crystallization solution and its crystals diffracted X-rays to a resolution of 2.7 ?. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3 ? by growth in agarose gel. Preliminary crystallographic analyses indicate that both types of crystals are suitable for structure determination. Keys for the successful production of GFLV crystals include the rigorous quality control of virus preparations, crystal quality improvement using phase diagrams, and crystal lattice reinforcement by growth in agarose gel. These strategies are applicable to the production of well-diffracting crystals of other viruses and macromolecular assemblies. 相似文献
16.
GRETA NÖLKE PASCAL COBANOV KERSTIN UHDE-HOLZEM GÖTZ REUSTLE RAINER FISCHER STEFAN SCHILLBERG 《Molecular Plant Pathology》2009,10(1):41-49
Grapevine fanleaf virus (GFLV) is one of the most destructive pathogens of grapevine. In this study, we generated monoclonal antibodies binding specifically to the coat protein of GFLV. Antibody FL3 , which bound most strongly to GFLV and showed cross-reactivity to Arabis mosaic virus (ArMV), was used to construct the single-chain antibody fragment scFvGFLVcp-55. To evaluate the potential of this single-chain variable fragment (scFv) to confer antibody-mediated virus resistance, transgenic Nicotiana benthamiana plants were generated in which the scFv accumulated in the cytosol. Recombinant protein levels of up to 0.1% total soluble protein were achieved. The T1 and T2 progenies conferred partial or complete protection against GFLV on challenge with the viral pathogen. The resistance to GFLV in transgenic plants was strictly related to scFvGFLVcp-55 accumulation levels, confirming that the antibody fragment was functional in planta and responsible for the GFLV resistance. In addition, transgenic plants conferring complete protection to GFLV showed substantially enhanced tolerance to ArMV. We demonstrate the first step towards the control of grapevine fanleaf degeneration, as scFvGFLVcp-55 could be an ideal candidate for mediating nepovirus resistance. 相似文献
17.
The 50 distal amino acids of the 2AHP homing protein of Grapevine fanleaf virus elicit a hypersensitive reaction on Nicotiana occidentalis 下载免费PDF全文
Isabelle R. Martin Emmanuelle Vigne François Berthold Véronique Komar Olivier Lemaire Marc Fuchs Corinne Schmitt‐Keichinger 《Molecular Plant Pathology》2018,19(3):731-743
18.
Involvement of the secretory pathway and the cytoskeleton in intracellular targeting and tubule assembly of Grapevine fanleaf virus movement protein in tobacco BY-2 cells 下载免费PDF全文
Laporte C Vetter G Loudes AM Robinson DG Hillmer S Stussi-Garaud C Ritzenthaler C 《The Plant cell》2003,15(9):2058-2075
Grapevine fanleaf virus (GFLV) is one of a large class of plant viruses whose cell-to-cell transport involves the passage of virions through tubules composed of virus-encoded movement protein (MP). The tubules are embedded within modified plasmodesmata, but the mechanism of targeting of MP to these sites is unknown. To study intracellular GFLV MP trafficking, a green fluorescent protein-MP fusion (GFP:MP) was expressed in transgenic tobacco BY-2 suspension cells under the control of an inducible promoter. We show that GFP:MP is targeted preferentially to calreticulin-labeled foci within the youngest cross walls, where it assembles into tubules. During cell division, GFP:MP colocalizes in the cell plate with KNOLLE, a cytokinesis-specific syntaxin, and both proteins are linked physically, as shown by coimmunoprecipitation of the two proteins from the same microsomal fraction. In addition, treatment with various drugs has revealed that a functional secretory pathway, but not the cytoskeleton, is required for tubule formation. However, correct GFP:MP targeting to calreticulin-labeled foci seems to be cytoskeleton dependent. Finally, biochemical analyses have revealed that at least a fraction of the MP behaves as an intrinsic membrane protein. These findings support a model in which GFP:MP would be transported to specific sites via Golgi-derived vesicles along two different pathways: a microtubule-dependent pathway in normal cells and a microfilament-dependent default pathway when microtubules are depolymerized. 相似文献
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