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Marc Fuchs - One of the best experts on this subject based on the ideXlab platform.
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identification of protein interactions of Grapevine Fanleaf Virus rna dependent rna polymerase during infection of by affinity purification and tandem mass spectrometry
2021Co-Authors: Larissa J. Osterbaan, Victoria Hoyle, Michelle Curtis, Stacy L Deblasio, Keith D Rivera, Michelle Heck, Marc FuchsAbstract:The RNA-dependent RNA polymerase (1E) is involved in replication of Grapevine Fanleaf Virus (GFLV, , ) and causes vein clearing symptoms in . Information on protein 1E interaction with other viral and host proteins is scarce. To study protein 1E biology, three GFLV infectious clones, i.e. GHu (a symptomatic wild-type strain), GHu-1E (an asymptomatic GHu mutant) and F13 (an asymptomatic wild-type strain), were engineered with protein 1E fused to a V5 epitope tag at the C-terminus. Following -mediated delivery of GFLV clones in and protein extraction at seven dpi, when optimal 1E:V5 accumulation was detected, two viral and six plant putative interaction partners of V5-tagged protein 1E were identified for the three GFLV clones by affinity purification and tandem mass spectrometry. This study provides insights into the protein interactome of 1E during GFLV systemic infection in and lays the foundation for validation work.
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identification of protein interactions of Grapevine Fanleaf Virus rna dependent rna polymerase during infection of nicotiana benthamiana by affinity purification and tandem mass spectrometry
Journal of General Virology, 2021Co-Authors: Larissa J. Osterbaan, Victoria Hoyle, Michelle Curtis, Stacy L Deblasio, Keith D Rivera, Michelle Heck, Marc FuchsAbstract:The RNA-dependent RNA polymerase (1EPol) is involved in replication of Grapevine Fanleaf Virus (GFLV, NepoVirus, Secoviridae) and causes vein clearing symptoms in Nicotiana benthamiana. Information on protein 1EPol interaction with other viral and host proteins is scarce. To study protein 1EPol biology, three GFLV infectious clones, i.e. GHu (a symptomatic wild-type strain), GHu-1EK802G (an asymptomatic GHu mutant) and F13 (an asymptomatic wild-type strain), were engineered with protein 1EPol fused to a V5 epitope tag at the C-terminus. Following Agrobacterium tumefaciens-mediated delivery of GFLV clones in N. benthamiana and protein extraction at seven dpi, when optimal 1EPol:V5 accumulation was detected, two viral and six plant putative interaction partners of V5-tagged protein 1EPol were identified for the three GFLV clones by affinity purification and tandem mass spectrometry. This study provides insights into the protein interactome of 1EPol during GFLV systemic infection in N. benthamiana and lays the foundation for validation work.
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Optimal systemic Grapevine Fanleaf Virus infection in Nicotiana benthamiana following agroinoculation
Journal of Virological Methods, 2018Co-Authors: Larissa J. Osterbaan, Emmanuelle Vigne, Corinne Schmitt-keichinger, Marc FuchsAbstract:One of the greatest hindrances to the study of Grapevine Fanleaf Virus (GFLV) is the dearth of robust protocols for reliable, scalable, and cost-effective inoculation of host plants, especially methods which allow for rapid and targeted manipulation of the Virus genome. Agroinoculation fulfills these requirements: it is a relatively rapid, inexpensive, and reliable method for establishing infections, and enables genetic manipulation of viral sequences by modifying plasmids. We designed a system of binary plasmids based on the two genomic RNAs [RNA1 (1) and RNA2 (2)] of GFLV strains F13 (F) and GHu (G) and optimized parameters to maximize systemic infection frequency in Nicotiana benthamiana via agroinoculation. The genomic make-up of the inoculum (G1–G2 and reassortant F1–G2), the identity of the co-infiltrated silencing suppressor (Grapevine leafroll associated Virus 2 p24), and temperature at which plants were maintained (25 °C) significantly increased systemic infection, while high optical densities of infiltration cultures (OD600nm of 1.0 or 2.0) increased the consistency of systemic infection frequency in N. benthamiana. In contrast, acetosyringone in the bacterial culture media, regardless of concentration, had no effect. Plasmids in this system are amenable to rapid and reliable manipulation by one-step site-directed mutagenesis, as shown by the creation of infectious RNA1 chimeras of the GFLV-F13 and GHu strains. The GFLV agroinoculation plasmids described here, together with the optimized protocol for bacterial culturing and plant maintenance, provide a robust system for the establishment of systemic GFLV infection in N. benthamiana and the rapid generation of GFLV mutants, granting a much-needed tool for investigations into GFLV-host interactions.
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genetic variability evolution and biological effects of Grapevine Fanleaf Virus satellite rnas
Phytopathology, 2013Co-Authors: J Gottula, D Lapato, Keiran K Cantilina, S Saito, B Bartlett, Marc FuchsAbstract:Large satellite RNAs (type B satRNAs) of Grapevine Fanleaf Virus (GFLV) from the genus NepoVirus, family Secoviridae were identified in a naturally infected vineyard and a Grapevine germplasm collection. These GFLV satRNA variants had a higher nucleotide sequence identity with satRNAs of Arabis mosaic Virus (ArMV) strains NW and J86 (93.8 to 94.6%) than with the satRNA of GFLV strain F13 and those of other ArMV strains (68.3 to 75.0%). Phylogenetic analyses showed no distinction of GFLV and ArMV satRNAs with respect to the identity of the helper Virus. Seven stretches of 8 to 15 conserved nucleotides (I-VII) were identified in the 5' region of subgroup A nepoVirus genomic RNAs GFLV, ArMV, and Grapevine deformation Virus) and nepoVirus type B satRNAs, including previously reported motif I, suggesting that large satRNAs might have originated from recombination between an ancestral subgroup A nepoVirus RNA and an unknown RNA sequence with the 5' region acting as a putative cis-replication element. A comparative analysis of two GFLV strains carrying or absent of satRNAs showed no discernable effect on Virus accumulation and symptom expression in Chenopodium quinoa, a systemic herbaceous host. This work sheds light on the origin and biological effects of large satRNAs associated with subgroup A nepoViruses.
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transmission competency of single female xiphinema index lines for Grapevine Fanleaf Virus
Phytopathology, 2010Co-Authors: Gerard Demangeat, Veronique Komar, Cyril Vanghelder, R Voisin, Olivier Lemaire, Daniel Esmenjaud, Marc FuchsAbstract:Grapevine Fanleaf Virus (GFLV) is vectored specifically from Grapevine to Grapevine by the ectoparasitic nematode Xiphinema index. Limited information is available on the vector competency of X. index populations from diverse geographical origins. We determined the transmissibility of two GFLV strains showing 4.6% amino acid divergence within their coat protein (e.g., strains F13 and GHu) by seven clonal lines of X. index developed from seven distinct populations from the Mediterranean basin (Cyprus, southern France, Israel, Italy, and Spain), northern France, and California. X. index lines derived from single adult females were produced on fig (Ficus carica) plants to obtain genetically homogenous aviruliferous clones. A comparative reproductive rate analysis on Vitis rupestris du Lot and V. vinifera cv. Cabernet Sauvignon showed significant differences among clones, with the single-female Cyprus line showing the highest rate (30-fold the initial population) and the Spain and California lines showing the lowest rate (10-fold increase), regardless of the Grapevine genotype. However, there was no differential vector competency among the seven X. index lines for GFLV strains F13 and GHu. The implications of our findings for the dynamic of GFLV transmission in vineyards and screening of Vitis spp. for resistance to GFLV are discussed.
Gerard Demangeat - One of the best experts on this subject based on the ideXlab platform.
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a single resistance factor to solve vineyard degeneration due to Grapevine Fanleaf Virus
Communications Biology, 2021Co-Authors: Samia Djennane, Gerard Demangeat, Olivier Lemaire, Claude Gertz, Sophie Gersch, Emilce Prado, Vincent Dumas, Anne Alais, Monique Beuve, Didier MerdinogluAbstract:Grapevine Fanleaf disease, caused by Grapevine Fanleaf Virus (GFLV), transmitted by the soil-borne nematode Xiphinema index, provokes severe symptoms and economic losses, threatening vineyards worldwide. As no effective solution exists so far to control Grapevine Fanleaf disease in an environmentally friendly way, we investigated the presence of resistance to GFLV in Grapevine genetic resources. We discovered that the Riesling variety displays resistance to GFLV, although it is susceptible to X. index. This resistance is determined by a single recessive factor located on Grapevine chromosome 1, which we have named rgflv1. The discovery of rgflv1 paves the way for the first effective and environmentally friendly solution to control Grapevine Fanleaf disease through the development of new GFLV-resistant Grapevine rootstocks, which was hitherto an unthinkable prospect. Moreover, rgflv1 is putatively distinct from the Virus susceptibility factors already described in plants.
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From a Movement-Deficient Grapevine Fanleaf Virus to the Identification of a New Viral Determinant of Nematode Transmission
Viruses, 2019Co-Authors: Lorène Belval, Veronique Komar, Olivier Lemaire, Emmanuelle Vigne, Aurelie Marmonier, Sophie Gersch, Christophe Ritzenthaler, Corinne Schmitt-keichinger, Peggy Andret-link, Gerard DemangeatAbstract:Grapevine Fanleaf Virus (GFLV) and arabis mosaic Virus (ArMV) are nepoViruses responsible for Grapevine degeneration. They are specifically transmitted from Grapevine to Grapevine by two distinct ectoparasitic dagger nematodes of the genus Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two Viruses, were previously defined and exchanged to test their involvement in Virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the Virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in Virus transmission, thus representing a new viral determinant of nematode transmission.
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detection of multiple variants of Grapevine Fanleaf Virus in single xiphinema index nematodes
Viruses, 2019Co-Authors: Shahinez Garcia, Gerard Demangeat, Veronique Komar, Olivier Lemaire, Jeanmichel Hily, Claude Gertz, Emmanuelle VigneAbstract:Grapevine Fanleaf Virus (GFLV) is responsible for a widespread disease in vineyards worldwide. Its genome is composed of two single-stranded positive-sense RNAs, which both show a high genetic diversity. The Virus is transmitted from Grapevine to Grapevine by the ectoparasitic nematode Xiphinema index. Grapevines in diseased vineyards are often infected by multiple genetic variants of GFLV but no information is available on the molecular composition of Virus variants retained in X. index following nematodes feeding on roots. In this work, aviruliferous X. index were fed on three naturally GFLV-infected Grapevines for which the virome was characterized by RNAseq. Six RNA-1 and four RNA-2 molecules were assembled segregating into four and three distinct phylogenetic clades of RNA-1 and RNA-2, respectively. After 19 months of rearing, single and pools of 30 X. index tested positive for GFLV. Additionally, either pooled or single X. index carried multiple variants of the two GFLV genomic RNAs. However, the full viral genetic diversity found in the leaves of infected Grapevines was not detected in viruliferous nematodes, indicating a genetic bottleneck. Our results provide new insights into the complexity of GFLV populations and the putative role of X. index as reservoirs of Virus diversity.
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strategies for the crystallization of Viruses using phase diagrams and gels to produce 3d crystals of Grapevine Fanleaf Virus
Journal of Structural Biology, 2011Co-Authors: Pascale Schellenberger, Gerard Demangeat, Olivier Lemaire, Claude Sauter, Christophe Ritzenthaler, Marc Bergdoll, Vincent Olieric, Bernard LorberAbstract:Abstract 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 Gly297Asp 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 A. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3 A 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.
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Structural insights into viral determinants of nematode mediated Grapevine Fanleaf Virus transmission
PLoS Pathogens, 2011Co-Authors: P. Schellenberger, C. Sauter, B. Lorber, P. Bron, S. Trapani, M. Bergdoll, A. Marmonier, C. Schmitt-keichinger, O. Lemaire, Gerard DemangeatAbstract: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.
Daniel Esmenjaud - One of the best experts on this subject based on the ideXlab platform.
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transmission competency of single female xiphinema index lines for Grapevine Fanleaf Virus
Phytopathology, 2010Co-Authors: Gerard Demangeat, Veronique Komar, Cyril Vanghelder, R Voisin, Olivier Lemaire, Daniel Esmenjaud, Marc FuchsAbstract:ABSTRACT Grapevine Fanleaf Virus (GFLV) is vectored specifically from Grapevine to Grapevine by the ectoparasitic nematode Xiphinema index. Limited information is available on the vector competency of X. index populations from diverse geographical origins. We determined the transmissibility of two GFLV strains showing 4.6% amino acid divergence within their coat protein (e.g., strains F13 and GHu) by seven clonal lines of X. index developed from seven distinct populations from the Mediterranean basin (Cyprus, southern France, Israel, Italy, and Spain), northern France, and California. X. index lines derived from single adult females were produced on fig (Ficus carica) plants to obtain genetically homogenous aviruliferous clones. A comparative reproductive rate analysis on Vitis rupestris du Lot and V. vinifera cv. Cabernet Sauvignon showed significant differences among clones, with the single-female Cyprus line showing the highest rate (30-fold the initial population) and the Spain and California lines sh...
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transmission competency of single female xiphinema index lines for Grapevine Fanleaf Virus
Phytopathology, 2010Co-Authors: Gerard Demangeat, Veronique Komar, Cyril Vanghelder, R Voisin, Olivier Lemaire, Daniel Esmenjaud, Marc FuchsAbstract:Grapevine Fanleaf Virus (GFLV) is vectored specifically from Grapevine to Grapevine by the ectoparasitic nematode Xiphinema index. Limited information is available on the vector competency of X. index populations from diverse geographical origins. We determined the transmissibility of two GFLV strains showing 4.6% amino acid divergence within their coat protein (e.g., strains F13 and GHu) by seven clonal lines of X. index developed from seven distinct populations from the Mediterranean basin (Cyprus, southern France, Israel, Italy, and Spain), northern France, and California. X. index lines derived from single adult females were produced on fig (Ficus carica) plants to obtain genetically homogenous aviruliferous clones. A comparative reproductive rate analysis on Vitis rupestris du Lot and V. vinifera cv. Cabernet Sauvignon showed significant differences among clones, with the single-female Cyprus line showing the highest rate (30-fold the initial population) and the Spain and California lines showing the lowest rate (10-fold increase), regardless of the Grapevine genotype. However, there was no differential vector competency among the seven X. index lines for GFLV strains F13 and GHu. The implications of our findings for the dynamic of GFLV transmission in vineyards and screening of Vitis spp. for resistance to GFLV are discussed.
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development of nine polymorphic microsatellite markers for the phytoparasitic nematode xiphinema index the vector of the Grapevine Fanleaf Virus
Molecular Ecology Resources, 2009Co-Authors: L Villate, S Coedel, Daniel Esmenjaud, Olivier PlantardAbstract:: 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.
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permanent genetic resources note development of nine polymorphic microsatellite markers for the phytoparasitic nematode xiphinema index the vector of the Grapevine Fanleaf Virus
2009Co-Authors: L Villate, S Coedel, Daniel Esmenjaud, Olivier PlantardAbstract: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.
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spatial distribution of the dagger nematode xiphinema index and its associated Grapevine Fanleaf Virus in french vineyard
Phytopathology, 2008Co-Authors: L Villate, Virgil Fievet, B Hanse, F Delemarre, Daniel Esmenjaud, Olivier Plantard, M Van HeldenAbstract:The nematode Xiphinema index is, economically, the major Virus vector in viticulture, transmitting specifically the Grapevine Fanleaf Virus (GFLV), the most severe Grapevine Virus disease worldwide. Increased knowledge of the spatial distribution of this nematode, both horizontally and vertically, and of correlative GFLV plant infections, is essential to efficiently control the disease. In two infested blocks of the Bordeaux vineyard, vertical distribution data showed that the highest numbers of individuals occurred at 40 to 110 cm depth, corresponding to the two layers where the highest densities of fine roots were observed. Horizontal distribution based on a 10 x 15 m grid sampling procedure revealed a significant aggregative pattern but no significant neighborhood structure of nematode densities. At a finer scale ( approximately 2 x 2 m), nematode sampling performed in a third block confirmed a significant aggregative pattern, with patches of 6 to 8 m diameter, together with a significant neighborhood structure of nematode densities, thus identifying the relevant sampling scale to describe the nematode distribution. Nematode patches correlate significantly with those of GFLV-infected Grapevine plants. Finally, nematode and Virus spread were shown to extend preferentially parallel to vine rows, probably due to tillage during mechanical weeding.
Veronique Komar - One of the best experts on this subject based on the ideXlab platform.
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From a Movement-Deficient Grapevine Fanleaf Virus to the Identification of a New Viral Determinant of Nematode Transmission
Viruses, 2019Co-Authors: Lorène Belval, Veronique Komar, Olivier Lemaire, Emmanuelle Vigne, Aurelie Marmonier, Sophie Gersch, Christophe Ritzenthaler, Corinne Schmitt-keichinger, Peggy Andret-link, Gerard DemangeatAbstract:Grapevine Fanleaf Virus (GFLV) and arabis mosaic Virus (ArMV) are nepoViruses responsible for Grapevine degeneration. They are specifically transmitted from Grapevine to Grapevine by two distinct ectoparasitic dagger nematodes of the genus Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two Viruses, were previously defined and exchanged to test their involvement in Virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the Virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in Virus transmission, thus representing a new viral determinant of nematode transmission.
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detection of multiple variants of Grapevine Fanleaf Virus in single xiphinema index nematodes
Viruses, 2019Co-Authors: Shahinez Garcia, Gerard Demangeat, Veronique Komar, Olivier Lemaire, Jeanmichel Hily, Claude Gertz, Emmanuelle VigneAbstract:Grapevine Fanleaf Virus (GFLV) is responsible for a widespread disease in vineyards worldwide. Its genome is composed of two single-stranded positive-sense RNAs, which both show a high genetic diversity. The Virus is transmitted from Grapevine to Grapevine by the ectoparasitic nematode Xiphinema index. Grapevines in diseased vineyards are often infected by multiple genetic variants of GFLV but no information is available on the molecular composition of Virus variants retained in X. index following nematodes feeding on roots. In this work, aviruliferous X. index were fed on three naturally GFLV-infected Grapevines for which the virome was characterized by RNAseq. Six RNA-1 and four RNA-2 molecules were assembled segregating into four and three distinct phylogenetic clades of RNA-1 and RNA-2, respectively. After 19 months of rearing, single and pools of 30 X. index tested positive for GFLV. Additionally, either pooled or single X. index carried multiple variants of the two GFLV genomic RNAs. However, the full viral genetic diversity found in the leaves of infected Grapevines was not detected in viruliferous nematodes, indicating a genetic bottleneck. Our results provide new insights into the complexity of GFLV populations and the putative role of X. index as reservoirs of Virus diversity.
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Nanobody-mediated resistance to Grapevine Fanleaf Virus in plants
Plant Biotechnology Journal, 2018Co-Authors: Caroline Hemmer, Veronique Komar, Shahinez Garcia, Emmanuelle Vigne, Aurelie Marmonier, Lea Ackerer, Kamal Hleibieh, Samia Djennane, Sophie Gersch, Mireille PerrinAbstract:Summary 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.
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transmission competency of single female xiphinema index lines for Grapevine Fanleaf Virus
Phytopathology, 2010Co-Authors: Gerard Demangeat, Veronique Komar, Cyril Vanghelder, R Voisin, Olivier Lemaire, Daniel Esmenjaud, Marc FuchsAbstract:ABSTRACT Grapevine Fanleaf Virus (GFLV) is vectored specifically from Grapevine to Grapevine by the ectoparasitic nematode Xiphinema index. Limited information is available on the vector competency of X. index populations from diverse geographical origins. We determined the transmissibility of two GFLV strains showing 4.6% amino acid divergence within their coat protein (e.g., strains F13 and GHu) by seven clonal lines of X. index developed from seven distinct populations from the Mediterranean basin (Cyprus, southern France, Israel, Italy, and Spain), northern France, and California. X. index lines derived from single adult females were produced on fig (Ficus carica) plants to obtain genetically homogenous aviruliferous clones. A comparative reproductive rate analysis on Vitis rupestris du Lot and V. vinifera cv. Cabernet Sauvignon showed significant differences among clones, with the single-female Cyprus line showing the highest rate (30-fold the initial population) and the Spain and California lines sh...
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transmission competency of single female xiphinema index lines for Grapevine Fanleaf Virus
Phytopathology, 2010Co-Authors: Gerard Demangeat, Veronique Komar, Cyril Vanghelder, R Voisin, Olivier Lemaire, Daniel Esmenjaud, Marc FuchsAbstract:Grapevine Fanleaf Virus (GFLV) is vectored specifically from Grapevine to Grapevine by the ectoparasitic nematode Xiphinema index. Limited information is available on the vector competency of X. index populations from diverse geographical origins. We determined the transmissibility of two GFLV strains showing 4.6% amino acid divergence within their coat protein (e.g., strains F13 and GHu) by seven clonal lines of X. index developed from seven distinct populations from the Mediterranean basin (Cyprus, southern France, Israel, Italy, and Spain), northern France, and California. X. index lines derived from single adult females were produced on fig (Ficus carica) plants to obtain genetically homogenous aviruliferous clones. A comparative reproductive rate analysis on Vitis rupestris du Lot and V. vinifera cv. Cabernet Sauvignon showed significant differences among clones, with the single-female Cyprus line showing the highest rate (30-fold the initial population) and the Spain and California lines showing the lowest rate (10-fold increase), regardless of the Grapevine genotype. However, there was no differential vector competency among the seven X. index lines for GFLV strains F13 and GHu. The implications of our findings for the dynamic of GFLV transmission in vineyards and screening of Vitis spp. for resistance to GFLV are discussed.
Olivier Lemaire - One of the best experts on this subject based on the ideXlab platform.
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a single resistance factor to solve vineyard degeneration due to Grapevine Fanleaf Virus
Communications Biology, 2021Co-Authors: Samia Djennane, Gerard Demangeat, Olivier Lemaire, Claude Gertz, Sophie Gersch, Emilce Prado, Vincent Dumas, Anne Alais, Monique Beuve, Didier MerdinogluAbstract:Grapevine Fanleaf disease, caused by Grapevine Fanleaf Virus (GFLV), transmitted by the soil-borne nematode Xiphinema index, provokes severe symptoms and economic losses, threatening vineyards worldwide. As no effective solution exists so far to control Grapevine Fanleaf disease in an environmentally friendly way, we investigated the presence of resistance to GFLV in Grapevine genetic resources. We discovered that the Riesling variety displays resistance to GFLV, although it is susceptible to X. index. This resistance is determined by a single recessive factor located on Grapevine chromosome 1, which we have named rgflv1. The discovery of rgflv1 paves the way for the first effective and environmentally friendly solution to control Grapevine Fanleaf disease through the development of new GFLV-resistant Grapevine rootstocks, which was hitherto an unthinkable prospect. Moreover, rgflv1 is putatively distinct from the Virus susceptibility factors already described in plants.
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From a Movement-Deficient Grapevine Fanleaf Virus to the Identification of a New Viral Determinant of Nematode Transmission
Viruses, 2019Co-Authors: Lorène Belval, Veronique Komar, Olivier Lemaire, Emmanuelle Vigne, Aurelie Marmonier, Sophie Gersch, Christophe Ritzenthaler, Corinne Schmitt-keichinger, Peggy Andret-link, Gerard DemangeatAbstract:Grapevine Fanleaf Virus (GFLV) and arabis mosaic Virus (ArMV) are nepoViruses responsible for Grapevine degeneration. They are specifically transmitted from Grapevine to Grapevine by two distinct ectoparasitic dagger nematodes of the genus Xiphinema. GFLV and ArMV move from cell to cell as virions through tubules formed into plasmodesmata by the self-assembly of the viral movement protein. Five surface-exposed regions in the coat protein called R1 to R5, which differ between the two Viruses, were previously defined and exchanged to test their involvement in Virus transmission, leading to the identification of region R2 as a transmission determinant. Region R4 (amino acids 258 to 264) could not be tested in transmission due to its requirement for plant systemic infection. Here, we present a fine-tuning mutagenesis of the GFLV coat protein in and around region R4 that restored the Virus movement and allowed its evaluation in transmission. We show that residues T258, M260, D261, and R301 play a crucial role in Virus transmission, thus representing a new viral determinant of nematode transmission.
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detection of multiple variants of Grapevine Fanleaf Virus in single xiphinema index nematodes
Viruses, 2019Co-Authors: Shahinez Garcia, Gerard Demangeat, Veronique Komar, Olivier Lemaire, Jeanmichel Hily, Claude Gertz, Emmanuelle VigneAbstract:Grapevine Fanleaf Virus (GFLV) is responsible for a widespread disease in vineyards worldwide. Its genome is composed of two single-stranded positive-sense RNAs, which both show a high genetic diversity. The Virus is transmitted from Grapevine to Grapevine by the ectoparasitic nematode Xiphinema index. Grapevines in diseased vineyards are often infected by multiple genetic variants of GFLV but no information is available on the molecular composition of Virus variants retained in X. index following nematodes feeding on roots. In this work, aviruliferous X. index were fed on three naturally GFLV-infected Grapevines for which the virome was characterized by RNAseq. Six RNA-1 and four RNA-2 molecules were assembled segregating into four and three distinct phylogenetic clades of RNA-1 and RNA-2, respectively. After 19 months of rearing, single and pools of 30 X. index tested positive for GFLV. Additionally, either pooled or single X. index carried multiple variants of the two GFLV genomic RNAs. However, the full viral genetic diversity found in the leaves of infected Grapevines was not detected in viruliferous nematodes, indicating a genetic bottleneck. Our results provide new insights into the complexity of GFLV populations and the putative role of X. index as reservoirs of Virus diversity.
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Display of whole proteins on inner and outer surfaces of Grapevine Fanleaf Virus-like particles.
Plant Biotechnology Journal, 2016Co-Authors: Lorène Belval, Olivier Lemaire, Caroline Hemmer, Claude Sauter, Francois Berthold, Corinne Schmitt-keichinger, C Reinbold, Jean-daniel Fauny, L Ackerer, G DemangeatAbstract:Virus-like particles (VLPs) derived from non-enveloped Viruses result from the self-assembly of capsid proteins (CPs). They generally display similar structural features to viral particles but are non-infectious 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 either to 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.
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strategies for the crystallization of Viruses using phase diagrams and gels to produce 3d crystals of Grapevine Fanleaf Virus
Journal of Structural Biology, 2011Co-Authors: Pascale Schellenberger, Gerard Demangeat, Olivier Lemaire, Claude Sauter, Christophe Ritzenthaler, Marc Bergdoll, Vincent Olieric, Bernard LorberAbstract:Abstract 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 Gly297Asp 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 A. The diffraction limit of GFLV-F13 crystals was extended from 5.5 to 3 A 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.
