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

Authors: HILY, JEAN-MICHEL - Institut National De La Recherche Agronomique (INRA); CANDRESSE, THIERRY - Institut National De La Recherche Agronomique (INRA); GARCIA, SHAHINEZ - Institut National De La Recherche Agronomique (INRA); VIGNE, EMMANUELLE - Institut National De La Recherche Agronomique (INRA); TANNIERES, MELANIE - European Biological Control Laboratory (EBCL); KOMAR, VERONIQUE - Institut National De La Recherche Agronomique (INRA); BARNABE, GUILLAUME - Institut National De La Recherche Agronomique (INRA); ALLIAUME, ANTOINE - Institut National De La Recherche Agronomique (INRA); GILG, SOPHIE - Institut National De La Recherche Agronomique (INRA); HOMMAY, GERARD - Institut National De La Recherche Agronomique (INRA)

Submitted to: Frontiers in Microbiology
Publication Type: Peer Reviewed Journal
Publication Acceptance Date: 7/16/2018
Publication Date: 8/29/2018
Citation: Hily, J., Candresse, T., Garcia, S., Vigne, E., Tannieres, M., Komar, V., Barnabe, G., Alliaume, A., Gilg, S., Hommay, G. 2018. 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. Frontiers in Microbiology. 9:1782. https://doi.org/10.3389/fmicb.2018.01782.
DOI: https://doi.org/10.3389/fmicb.2018.01782

Interpretive Summary: Technology has enabled the discovery of a wide range of novel plant viruses, greatly enriching our vision of the the viral community associated with a particular host or ecosystem. The goal of this study was to better characterize the sanitary status of Gewurztraminer scions grafted onto Kober 5BB rootstocks mono-infected with specific grapevine fanleaf virus (GFLV) isolates using RNAseq methods. Surprisingly, analysis of datasets revealed the presence of a new virus. Phylogenetic analyses showed this new virus clusters within the Tymovirales order but the sequence of this virus present in the grapevine environment is divergent enough to consider it as a new species typifying a new genus. Viruses belonging to the Tymovirales order infect many different species covering different Kingdoms. They are mostly found in Plantae, but lately many have been described infecting Fungi as well as the class of Insecta in the Animalia Kingdom. The virus was detected only in samples collected at the end of the summer/early autumn season, which corresponds to a period when fungicide/pesticide treatments are generally discontinued. All the results suggest that GaTLV is likely a surface contaminant on grapevine leaves and might therefore instead be a virus infecting insects or fungi. Even though technologies produce an invaluable sum of information describing the sanitary status of a plant, a careful etiological and epidemiological study is necessary before assigning a new virus to a host. Nonetheless, in this work, even after a careful scientific investigation, it is still not possible to designate without any doubt the host of an infectious entity. Our study also confirm that grapevine phytobiome is probably richer than anticipated, with the use of technology allowing for the detection of not only grapevine pathogens but also grapevine associated-ecosystem. This work will be used by scientists or industry interested in the phytosanitary status of grapevine.

Technical 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 andmore 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.