High-Throughput Sequencing and the Viromic Study of Grapevine Leaves: From the Detection of Grapevine-Infecting Viruses to the Description of a New Environmental Tymovirales Member

Abstract

In the past decade, high-throughput sequencing (HTS) has had a major impact on virus diversity studies as well as on diagnosis, providing an unbiased and more comprehensive view of the virome of a wide range of organisms. Rather than the serological and molecular-based methods, with their more “reductionist” view focusing on one or a few known agents, HTS-based approaches are able to give a “holistic snapshot” of the complex phytobiome of a sample of interest. In grapevine for example, HTS is powerful enough to allow for the assembly of complete genomes of the various viral species or variants infecting a sample of known or novel virus species. In the present study, a total RNAseq-based approach was used to determine the full genome sequences of various grapevine fanleaf virus (GFLV) isolates and to analyze the eventual presence of other viral agents. From four RNAseq datasets, a few complete grapevine-infecting virus and viroid genomes were de-novo assembled: (a) three GFLV genomes, 11 grapevine rupestris stem-pitting associated virus (GRSPaV) and six viroids. In addition, a novel viral genome was detected in all four datasets, consisting of a single-stranded, positive-sense RNA molecule of 6033 nucleotides. This genome displays an organization similar to Tymoviridae family members in the Tymovirales order. Nonetheless, the new virus shows enough differences to be considered as a new species defining a new genus. Detection of this new agent in the original grapevines proved very erratic and was only consistent at the end of the growing season. This virus was never detected in the spring period, raising the possibility that it might not be a grapevine-infecting virus, but rather a virus infecting a grapevine-associated organism that may be transiently present on grapevine samples at some periods of the year. Indeed, the Tymoviridae family comprises isometric viruses infecting a wide range of hosts in different kingdoms (Plantae, Fungi, and Animalia). The present work highlights the fact that even though HTS technologies produce invaluable data for the description of the sanitary status of a plant, in-depth biological studies are necessary before assigning a new virus to a particular host in such metagenomic approaches.

Structural basis of nanobody-recognition of grapevine fanleaf virus and of virus resistance loss

“…To determine whether RB mutations preexisted within natural viral populations or were acquired in Nb23-expressing plants, we analyzed HTS datasets (2×150pb RNAseq performed on an Illumina Hiseq 3000, Illumina, San Diego, CA) from a previous study Hily, et al 35 . We specifically focused on 12 repetitions of grafted and non-grafted grapevines naturally infected by a vineyard population of GFLV.…”

“…We specifically focused on 12 repetitions of grafted and non-grafted grapevines naturally infected by a vineyard population of GFLV. In addition, total RNA was extracted from a single sample containing 300 viruliferous nematodes previously fed on GFLV-F13 infected plant according to Demangeat, et al 36 and cDNA library were made as described in Hily, et al 35 . Analyses of datasets were performed using CLC Genomics Workbench 8.5.1 software (Qiagen), with mapping parameters set at length fraction of 0.5 and similarity of 0.7.…”

Detection of Multiple Variants of Grapevine Fanleaf Virus in Single Xiphinema index Nematodes

“…The virome of each grapevine sample was obtained by (i) mapping reads onto a curated list of grapevine-infecting virus references, and (ii) performing de novo assembly followed by BLAST [36] analyses, as previously described [37]. Complete to near-complete consensus genomes were obtained after extension of contigs by successive rounds of residual reads mapping.…”

“…The presence of viruses other than GFLV was further assessed in the three grapevines by analyzing the same RNAseq dataset. This was done by directly mapping total cleaned reads onto a curated collection of grapevine-infecting viruses' reference sequences as previously described [37]. For the three grapevines, multiple variants of grapevine rupestris stem pitting-associated virus (GRSPaV), grapevine fleck virus (GFkV), and viroids (grapevine yellow speckle viroid-1, GYSVd-1 and hop stunt viroid, HSVd) were detected ( Table 1).…”

Unravelling the virome in birch: RNA-Seq reveals a complex of known and novel viruses

“…Diseased grapevine plants infected by Grapevine Pinot gris virus are found based on HTS data to acquire very complex viromes [15]. The total RNA-Seq approach concretely in grapevine resulted in complete virus and viroid genomes through de-novo assembly [16–17]. Building on this success, the movement to apply HTS for routine virus detection is gaining momentum.…”