Abstract
Woody perennial plants like grapevine and fruit trees can be infected by several viruses even as multiple infections. Since they are propagated vegetatively, the phytosanitary status of the propagation material (both the rootstock and the variety) can have a profound effect on the lifetime and health of the new plantations. The fast evolution of sequencing techniques provides a new opportunity for metagenomics-based viral diagnostics. Viral derived small RNAs produced by the host immune system during viral infection can be sequenced by next-generation techniques and analyzed for the presence of viruses, revealing the presence of all known viral pathogens in the sample. This method is based on Illumina sequencing of short RNAs and bioinformatics analysis of virus-derived small RNAs in the host. Here we describe a protocol for this challenging technique step by step with notes, in order to ensure success for every user.
This is a preview of subscription content, log in via an institution to check access.
Access this chapter
Tax calculation will be finalised at checkout
Purchases are for personal use only
References
Navarro B, Pantaleo V, Gisel A, Moxon S, Dalmay T, Bisztray G, Di Serio F, Burgyan J (2009) Deep sequencing of viroid-derived small RNAs from grapevine provides new insights on the role of RNA silencing in plant-viroid interaction. PLoS One 4(11):e7686. https://doi.org/10.1371/journal.pone.0007686
Giampetruzzi A, Roumi V, Roberto R, Malossini U, Yoshikawa N, La Notte P, Terlizzi F, Credi R, Saldarelli P (2012) A new grapevine virus discovered by deep sequencing of virus- and viroid-derived small RNAs in Cv Pinot gris. Virus Res 163(1):262–268. https://doi.org/10.1016/j.virusres.2011.10.010
Pantaleo V, Saldarelli P, Miozzi L, Giampetruzzi A, Gisel A, Moxon S, Dalmay T, Bisztray G, Burgyan J (2010) Deep sequencing analysis of viral short RNAs from an infected Pinot Noir grapevine. Virology 408(1):49–56. https://doi.org/10.1016/j.virol.2010.09.001
Baulcombe D (2004) RNA silencing in plants. Nature 431(7006):356–363. https://doi.org/10.1038/nature02874
Coetzee B, Freeborough MJ, Maree HJ, Celton JM, Rees DJ, Burger JT (2010) Deep sequencing analysis of viruses infecting grapevines: virome of a vineyard. Virology 400(2):157–163. https://doi.org/10.1016/j.virol.2010.01.023
Kreuze JF, Perez A, Untiveros M, Quispe D, Fuentes S, Barker I, Simon R (2009) Complete viral genome sequence and discovery of novel viruses by deep sequencing of small RNAs: a generic method for diagnosis, discovery and sequencing of viruses. Virology 388(1):1–7. https://doi.org/10.1016/j.virol.2009.03.024
Wu Q, Wang Y, Cao M, Pantaleo V, Burgyan J, Li WX, Ding SW (2012) Homology-independent discovery of replicating pathogenic circular RNAs by deep sequencing and a new computational algorithm. Proc Natl Acad Sci U S A 109(10):3938–3943. https://doi.org/10.1073/pnas.1117815109
Massart S, Candresse T, Gil J, Lacomme C, Predajna L, Ravnikar M, Reynard JS, Rumbou A, Saldarelli P, Skoric D, Vainio EJ, Valkonen JP, Vanderschuren H, Varveri C, Wetzel T (2017) A framework for the evaluation of biosecurity, commercial, regulatory, and scientific impacts of plant viruses and viroids identified by NGS technologies. Front Microbiol 8:45. https://doi.org/10.3389/fmicb.2017.00045
Bolger AM, Lohse M, Usadel B (2014) Trimmomatic: a flexible trimmer for Illumina sequence data. Bioinformatics 30(15):2114–2120. https://doi.org/10.1093/bioinformatics/btu170
Li H, Durbin R (2009) Fast and accurate short read alignment with burrows-wheeler transform. Bioinformatics 25(14):1754–1760. https://doi.org/10.1093/bioinformatics/btp324
Li H, Handsaker B, Wysoker A, Fennell T, Ruan J, Homer N, Marth G, Abecasis G, Durbin R, Genome Project Data Processing S (2009) The sequence alignment/map format and SAMtools. Bioinformatics 25(16):2078–2079. https://doi.org/10.1093/bioinformatics/btp352
Robinson JT, Thorvaldsdottir H, Winckler W, Guttman M, Lander ES, Getz G, Mesirov JP (2011) Integrative genomics viewer. Nat Biotechnol 29(1):24–26. https://doi.org/10.1038/nbt.1754
Zerbino DR, Birney E (2008) Velvet: algorithms for de novo short read assembly using de Bruijn graphs. Genome Res 18(5):821–829. https://doi.org/10.1101/gr.074492.107
Gambino G, Perrone I, Gribaudo I (2008) A rapid and effective method for RNA extraction from different tissues of grapevine and other woody plants. Phytochem Anal 19(6):520–525. https://doi.org/10.1002/pca.1078
Nagy T, Kis A, Poliska S, Barta E, Havelda Z, Marincs F (2016) [Letter to the Editor] Comparison of small RNA next-generation sequencing with and without isolation of small RNA fraction. BioTechniques 60(6):273–278. https://doi.org/10.2144/000114423
Acknowledgments
Our work was supported by the Hungarian Ministry of Agriculture and the KTIA_AIK_12 -1-2013-0001 project and by a Hungarian Scientific Research Fund (OTKA; K119783) grant from the National Research, Development and Innovation Office. N.C. and D.B. participate in the Program for Reinforcement of Scientists of the Hungarian Ministry of Agriculture. N.C. is a Ph.D. student of the Festetics Doctoral School at the University of Pannonia. R.P., E.D. and D.B are Ph.D. students of the Doctoral School of Biological Sciences at Szent István University.
The authors acknowledge the contribution of the COST Action FA 1407–DIVAS (Deep Investigation of Virus Associated Sequences).
Author information
Authors and Affiliations
Corresponding author
Editor information
Editors and Affiliations
Rights and permissions
Copyright information
© 2018 Springer Science+Business Media, LLC
About this protocol
Cite this protocol
Czotter, N. et al. (2018). Use of siRNAs for Diagnosis of Viruses Associated to Woody Plants in Nurseries and Stock Collections. In: Pantaleo, V., Chiumenti, M. (eds) Viral Metagenomics. Methods in Molecular Biology, vol 1746. Humana Press, New York, NY. https://doi.org/10.1007/978-1-4939-7683-6_9
Download citation
DOI: https://doi.org/10.1007/978-1-4939-7683-6_9
Published:
Publisher Name: Humana Press, New York, NY
Print ISBN: 978-1-4939-7682-9
Online ISBN: 978-1-4939-7683-6
eBook Packages: Springer Protocols