Grapevine fanleaf virus (GFLV) and Arabis mosaic virus (ArMV) from the genus Nepovirus, family Secoviridae, cause a severe degeneration of grapevines. GFLV and ArMV have a bipartite RNA genome and are transmitted specifically by the ectoparasitic nematodes Xiphinema index and Xiphinema diversicaudatum, respectively. The transmission specificity of both viruses maps to their respective RNA2-encoded coat protein (CP). To further delineate the GFLV CP determinants of transmission specificity, three-dimensional (3D) homology structure models of virions and CP subunits were constructed based on the crystal structure of Tobacco ringspot virus, the type member of the genus Nepovirus. The 3D models were examined to predict amino acids that are exposed at the external virion surface, highly conserved among GFLV isolates but divergent between GFLV and ArMV. Five short amino acid stretches that matched these topographical and sequence conservation criteria were selected and substituted in single and multiple combinations by their ArMV counterparts in a GFLV RNA2 cDNA clone. Among the 21 chimeric RNA2 molecules engineered, transcripts of only three of them induced systemic plant infection in the presence of GFLV RNA1. Nematode transmission assays of the three viable recombinant viruses showed that swapping a stretch of (i) 11 residues in the ␤B-␤C loop near the icosahedral 3-fold axis abolished transmission by X. index but was insufficient to restore transmission by X. diversicaudatum and (ii) 7 residues in the ␤E-␣B loop did not interfere with transmission by the two Xiphinema species. This study provides new insights into GFLV CP determinants of nematode 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.

Grapevine fanleaf virus (GFLV) is transmitted specifically from grapevine to grapevine by the ectoparasitic root-feeding nematode Xiphinema index. Limited information is available on the survival of X. index in vineyard soil and on the retention of GFLV by X. index over extended periods of time. We addressed these two issues by quantifying the numbers of living X. index recovered from soil samples that were collected in three naturally GFLV-infected vineyards in France and subsequently stored at 7 or 20 degrees C in the absence of host plants. Our data indicated a two- to eightfold decrease in X. index numbers but the recovery of 8 to 10 living fourth-stage juveniles (J4) and adults per kilogram of soil after 4 years of storage regardless of temperature. In addition, GFLV was detected readily in all groups of 20 isolated X. index adults and J4 (except for J4 that were kept 4 years at 20 degrees C) by reverse transcription-polymerase chain reaction using total nematode RNAs and a primer set located in conserved regions at the 3' end of viral genomic RNA 2. Our findings on the long-term survival of viruliferous X. index under adverse conditions emphasize the need for new control strategies against GFLV.

SummarySince 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.

The viral determinants involved in the specific transmission of Grapevine fanleaf virus (GFLV) by its nematode vector Xiphinema index are located within the 513 C-terminal residues of the RNA2-encoded polyprotein, that is, the 9 C-terminal amino acids of the movement protein (2BMP) and contiguous 504 amino acids of the coat protein (2CCP) [Virology 291 (2001) 161]. To further delineate the viral determinants responsible for the specific spread, the four amino acids that are different within the 9 C-terminal 2BMP residues between GFLV and Arabis mosaic virus (ArMV), another nepovirus which is transmitted by Xiphinema diversicaudatum but not by X. index, were subjected to mutational analysis. Of the recombinant viruses derived from transcripts of GFLV RNA1 and RNA2 mutants that systemically infected herbaceous host plants, all with the 2CCP of GFLV were transmitted by X. index unlike none with the 2CCP of ArMV, regardless of the mutations within the 2BMP C-terminus. These results demonstrate that the coat protein is the sole viral determinant for the specific spread of GFLV by X. index.

The 3a protein, encoded by RNA 3 of cucumber mosaic virus (CMV), is the putative movement protein of viral progeny in infected plants. An analysis of transgenic tobacco plants constitutively expressing the CMV 3a protein showed that the protein is accumulated in leaves at every stage of development. In fully expanded leaves the protein is immunodetectable mostly in a cell-wallenriched fraction. Dye-coupling experiments using fluorescent-dextran probes were performed on fully expanded leaves to study the modifying effect of CMV 3a protein on the gating capacity of plasmodesmata. Movement of fluorescein-isothiocyanate-labelled dextran with a mean molecular mass of 10000 Da, and an approximate Stokes' radius of 2-3 nm, was detected between cells of the 3a protein transgenic plants, but not in the control plants. These results are consistent with the idea that the CMV 3a protein is involved in the modification of plasmodesmata and, therefore, in the cell-to-cell spread of the virus.

SummaryFor some crops, the only possible approach to gain a specific trait requires genome modification. The development of virus‐resistant transgenic plants based on the pathogen‐derived resistance strategy has been a success story for over three decades. However, potential risks associated with the technology, such as horizontal gene transfer (HGT) of any part of the transgene to an existing gene pool, have been raised. Here, we report no evidence of any undesirable impacts of genetically modified (GM) grapevine rootstock on its biotic environment. Using state of the art metagenomics, we analysed two compartments in depth, the targeted Grapevine fanleaf virus (GFLV) populations and nontargeted root‐associated microbiota. Our results reveal no statistically significant differences in the genetic diversity of bacteria that can be linked to the GM trait. In addition, no novel virus or bacteria recombinants of biosafety concern can be associated with transgenic grapevine rootstocks cultivated in commercial vineyard soil under greenhouse conditions for over 6 years.

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.