From a movement-deficient grapevine fanleaf virus to the identification of a new viral determinant of nematode transmission

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

Structural Insights into Viral Determinants of Nematode Mediated Grapevine fanleaf virus Transmission

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

Cloning and characterization of XiR1, a locus responsible for dagger nematode resistance in grape

The dagger nematode, Xiphinemaindex, feeds aggressively on grape roots and in the process, vectors grapevine fanleaf virus (GFLV) leading to the severe viral disease known as fanleaf degeneration. Resistance to X. index and GFLV has been the key objective of grape rootstock breeding programs. A previous study found that resistance to X. index derived from Vitis arizonica was largely controlled by a major quantitative trait locus, XiR1 (X. index Resistance 1), located on chromosome 19. The study presented here develops high-resolution genetic and physical maps in an effort to identify the XiR1 gene(s). The mapping was carried out with 1,375 genotypes in three populations derived from D8909-15, a resistant selection from a cross of V. rupestris A. de Serres (susceptible) × V. arizonica b42-26 (resistant). Resistance to X. index was evaluated on 99 informative recombinants that were identified by screening the three populations with two markers flanking the XiR1 locus. The high-resolution genetic map of XiR1 was primarily constructed with seven DNA markers developed in this study. Physical mapping of XiR1 was accomplished by screening three bacterial artificial chromosome (BAC) libraries constructed from D8909-15, V. vinifera Cabernet Sauvignon and V. arizonica b42-26. A total of 32 BAC clones were identified and the XiR1 locus was delineated within a 115 kb region. Sequence analysis of three BAC clones identified putative nucleotide binding/leucine-rich repeat (NB-LRR) genes. This is the first report of a closely linked major gene locus responsible for ectoparasitic nematode resistance. The markers developed from this study are being used to expedite the breeding of resistant grape rootstocks

Partial characterization of two divergent variants of grapevine leafroll-associated virus 4

International audienceTwo non mechanically transmissible viruses with filamentous closterovirus-like particles were extracted and partially characterized from leafroll-affected grapevine accessions of cv. Koussan from Turkey (Y253) and cv. Koudsi from Israel (Y252), both from the reference collection of grapevine viruses and virus-like diseases at the Institut National de la Recherche Agronomique (INRA) in Colmar, France. These viruses, denoted Y253-TK and Y252-IL, were independently investigated in France (INRA-Colmar) and Italy (DPPMA). Y253-TK did not react in DAS-ELISA or RT-PCR assays that would have detected any of the Grapevine leafroll-associated viruses (GLRaVs) described so far; Y252-IL did give weak and inconsistent positive reactions in IEM only with an antiserum to strain LR106 of Grapevine leafroll-associated virus 4 (GLRaV-4). A polyclonal antiserum to isolate Y252-IL decorated the homologous particles and, those of Y253-TK and, at lower dilution, particles of isolate GLRaV-4 LR106. A segment of the heat shock protein p70 homologue (HSP70h) gene, was amplified by RT-PCR of RNA from denatured purified particles of Y253-TK by using a set of degenerate primers. The 183 amino acid polypeptide deduced from the 549 bp HSP70h gene fragment showed a high degree of identity with the cognate genes of GLRaV-4 (95 %), GLRaV-5 (91 %), GLRaV-6 (84 %), and GLRaV-9 (90 %), but low identity with those of other GLRaVs (33-35 %). The HSP70h of isolates Y252-IL and LR106 showed 95 % amino acid identity. The coat protein sequence of isolates Y253-TK showed 99 and 94 % amino acid identity with those of isolates Y252-IL and LR106, respectively, with changes concentrated in the N-terminus, a feature that might have a bearing an the reaction with their heterologous antisera. Based an serological and molecular data, it was concluded that Y253-TK and Y252-IL are distinct, though similar, isolates of GLRaV-4. In two independent surveys in which 110 and 320 grapevine accessions from germplasm collections at Colmar and Bari, respectively, were examined by DAS-ELISA, the antisera As-Y253-TK and As-Y252-IL cross-reacted with the homologous and heterologous viruses. Whereas As-Y253-TK did not react with any of the other accessions from the French collection tested, As-Y252-IL recognized a virus present in a total of eight grapevine accessions from the Mediterranean area, grown in the Italian germplasm collection