Comparison of Serological and Molecular Methods With High-Throughput Sequencing for the Detection and Quantification of Grapevine Fanleaf Virus in Vineyard Samples

Grapevine fanleaf virus (GFLV) is the main causal agent of fanleaf degeneration, the most damaging viral disease of grapevine. GFLV is included in most grapevine certification programs that rely on robust diagnostic tools such as biological indexing, serological methods, and molecular techniques, for the identification of clean stocks. The emergence of high throughput sequencing (HTS) offers new opportunities for detecting GFLV and other viruses in grapevine accessions of interest. Here, two HTS-based methods, i.e., RNAseq and smallRNAseq (focusing on the 21 to 27 nt) were explored for their potential to characterize the virome of grapevine samples from two 30-year-old GFLV-infected vineyards in the Champagne region of France. smallrnaseq was optimal for the detection of a wide range of viral species within a sample and RNAseq was the method of choice for full-length viral genome assembly. The implementation of a protocol to discriminate between low GFLV titer and in silico contamination (intra-lane contamination due to index misassignment) during data processing was critical for data analyses. Furthermore, we compared the performance of semi-quantitative DAS-ELISA (double antibody enzyme-linked immunosorbent assay), RT-qPCR (Reverse transcription-quantitative polymerase chain reaction), Immuno capture (IC)-RT-PCR, northern blot for viral small interfering RNA (vsiRNA) detection and RNAseq for the detection and quantification of GFLV. While detection limits were variable among methods, as expected, GFLV diagnosis was consistently achieved with all of these diagnostic methods. Together, this work highlights the robustness of DAS-ELISA, the current method routinely used in the French grapevine certification program, for the detection of GFLV and offers perspectives on the potential of HTS as an approach of high interest for certification

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

The specific transmission of Grapevine fanleaf virus by its nematode vector Xiphinema index is solely determined by the viral coat protein

AbstractThe 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 50 distal amino acids of the 2A HP homing protein of Grapevine fanleaf virus elicit a hypersensitive reaction on Nicotiana occidentalis

International audienceAvirulence factors are critical for the arm's race between a virus and its host in determining incompatible reactions. The response of plants to viruses from the genus Nepovirus in the family Secoviridae, including Grapevine fanleaf virus (GFLV), is well characterized, although the nature and characteristics of the viral avirulence factor remain elusive. By using infectious clones of GFLV strains F13 and GHu in a reverse genetics approach with wild-type, assortant and chimeric viruses, the determinant of necrotic lesions caused by GFLV-F13 on inoculated leaves of Nicotiana occidentalis was mapped to the RNA2-encoded protein 2A(HP), particularly to its 50 C-terminal amino acids. The necrotic response showed hallmark characteristics of a genuine hypersensitive reaction, such as the accumulation of phytoalexins, reactive oxygen species, pathogenesis-related protein 1c and hypersensitivity-related (hsr) 203J transcripts. Transient expression of the GFLV-F13 protein 2A(HP) fused to an enhanced green fluorescent protein (EGFP) tag in N.occidentalis by agroinfiltration was sufficient to elicit a hypersensitive reaction. In addition, the GFLV-F13 avirulence factor, when introduced in GFLV-GHu, which causes a compatible reaction on N.occidentalis, elicited necrosis and partially restricted the virus. This is the first identification of a nepovirus avirulence factor that is responsible for a hypersensitive reaction in both the context of virus infection and transient expression

Characterization of Grapevine Fanleaf Virus Isolates in ‘Chardonnay’ Vines Exhibiting Severe and Mild Symptoms in Two Vineyards

International audienceFanleaf degeneration is a complex viral disease of Vitis spp. that detrimentally impacts fruit yield and reduces the productive lifespan of most vineyards worldwide. In France, its main causal agent is grapevine fanleaf virus (GFLV). In the past, field experiments were conducted to explore cross-protection as a management strategy of fanleaf degeneration, but results were unsatisfactory because the mild virus strain negatively impacted fruit yield. In order to select new mild GFLV isolates, we examined two old ‘Chardonnay’ parcels harbouring vines with distinct phenotypes. Symptoms and agronomic performances were monitored over the four-year study on 21 individual vines that were classified into three categories: asymptomatic GFLV-free vines, GFLV-infected vines severely diseased and GFLV-infected vines displaying mild symptoms. The complete coding genomic sequences of GFLV isolates in infected vines was determined by high-throughput sequencing. Most grapevines were infected with multiple genetically divergent variants. While no specific molecular features were apparent for GFLV isolates from vines displaying mild symptoms, a genetic differentiation of GFLV populations depending on the vineyard parcel was observed. The mild symptomatic grapevines identified during this study were established in a greenhouse to recover GFLV variants of potential interest for cross-protection studies

Biological Evidence and Molecular Modeling of a Grapevine Pinot gris Virus Outbreak in a Vineyard

International audienceSince its identification in 2003, little has been revealed about the spread of grapevine Pinot gris virus (GPGV), an emerging grapevine virus. According to studies from Italy, GPGV transmission in the vineyard can be fast but progressive over the years. To gain new insights into the spread of GPGV infections, we tested 67 grapevines in a single vineyard parcel in southern France. These vines were sampled over 8 years (2013 to 2020) and tested for GPGV by reverse-transcription PCR using a new primer pair designed from the recently described genetic diversity of GPGV worldwide. While focusing on a portion of the samples (n = 20), we observed a drastic increase in newly GPGV-infected vines from 2014 (5%, 1 of 20) to 2015 (80%, 16 of 20) and 2016 (90%, 18 of 20). Infected vines were scattered throughout the vineyard with no distinct pattern of distribution, and some rare vines remained negative through 2020. Using all available genomic information, we performed Bayesian-based phylogeographic analyses that identified a major intravineyard transmission in 2014 to 2015. To test our model, we analyzed 47 additional grapevines and confirmed the outbreak of GPGV in 2015, validating our in silico projection. Interestingly, some grapevines remained negative throughout the study, in spite of their close proximity to infected plants. These results raise questions about the dynamics of vector populations and environmental conditions that may be required for virus spread to occur in the vineyard

Evidence of differential spreading events of grapevine pinot Gris virus in Italy using datamining as a tool

International audienceSince its identification in 2003, grapevine Pinot gris virus (GPGV, Trichovirus) has now been detected in most grape-growing countries. So far, little is known about the epidemiology of this newly emerging virus. In this work, we used datamining as a tool to monitor in-silico the sanitary status of three vineyards in Italy. All data used in the study were recovered from a work that was already published and for which data were publicly available as SRA (Sequence Read Archive, NCBI) files. While incomplete, knowledge gathered from this work was still important, with evidence of differential accumulation of the virus in grapevine according to year, location, and variety-rootstock association. Additional data regarding GPGV genetic diversity were collected. Some advantages and pitfalls of datamining are discussed

Severe Stunting Symptoms upon Nepovirus Infection Are Reminiscent of a Chronic Hypersensitive-like Response in a Perennial Woody Fruit Crop

Virus infection of plants can result in various degrees of detrimental impacts and disparate symptom types and severities. Although great strides have been made in our understanding of the virus–host interactions in herbaceous model plants, the mechanisms underlying symptom development are poorly understood in perennial fruit crops. Grapevine fanleaf virus (GFLV) causes variable symptoms in most vineyards worldwide. To better understand GFLV-grapevine interactions in relation to symptom development, field and greenhouse trials were conducted with a grapevine genotype that exhibits distinct symptoms in response to a severe and a mild strain of GFLV. After validation of the infection status of the experimental vines by high-throughput sequencing, the transcriptomic and metabolomic profiles in plants infected with the two viral strains were tested and compared by RNA-Seq and LC-MS, respectively, in the differentiating grapevine genotype. In vines infected with the severe GFLV strain, 1023 genes, among which some are implicated in the regulation of the hypersensitive-type response, were specifically deregulated, and a higher accumulation of resveratrol and phytohormones was observed. Interestingly, some experimental vines restricted the virus to the rootstock and remained symptomless. Our results suggest that GFLV induces a strain- and cultivar-specific defense reaction similar to a hypersensitive reaction. This type of defense leads to a severe stunting phenotype in some grapevines, whereas others are resistant. This work is the first evidence of a hypersensitive-like reaction in grapevine during virus infection