A simplified method for cloning of short interfering RNAs from Brassica juncea infected with Turnip mosaic potyvirus and Turnip crinkle carmovirus.

Ho, T, Pallett, D, Rusholme, R, Dalmay, T and Wang, H (2006) A simplified method for cloning of short interfering RNAs from Brassica juncea infected with Turnip mosaic potyvirus and Turnip crinkle carmovirus. Journal of Virological Methods, 136 (1-2). pp. 217-223. ISSN 1879-0984

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Abstract

RNA silencing is a plant defense mechanism in which virus infected plants produce short interfering RNAs (siRNAs) derived from viral RNA, that attack the virus at the post-transcriptional level. In a previous study on Cymbidium ringspot tombusvirus (CymRSV) infection in Nicotiana benthamiana, siRNAs (determined by cloning and sequencing) predominantly originated from the sense (+) strand of the viral RNA, suggesting that the majority of siRNAs are produced through the direct cleavage of the virus single strand (ss) RNA by the plant Dicer-like enzyme. To test whether this asymmetry in strand polarity is a generic rule for all plant viruses, siRNAs from Brassica juncea, either singly infected by Turnip mosaic potyvirus (TuMV, the family Potyviridae), or doubly infected with TuMV and Turnip crinkle carmovirus (TCV, the family Tombusviridae) were investigated. A simplified siRNA cloning method was developed, using a single ligation reaction to attach both 5' and 3' adapters to the target short RNAs followed by one-step RT-PCR amplification. In the TCV infection, as for the CymRSV infection, siRNAs were produced predominantly (97.6%) from the +ss RNA. However, for TuMV infections, siRNAs were derived from both strands (+/-, 58.1–41.9%), indicating the presence of alternative siRNA production mechanisms.

Item Type: Article
Faculty \ School: Faculty of Science > School of Biological Sciences
Related URLs:
Depositing User: EPrints Services
Date Deposited: 01 Oct 2010 13:38
Last Modified: 14 Oct 2019 15:30
URI: https://ueaeprints.uea.ac.uk/id/eprint/1241
DOI: 10.1016/j.jviromet.2006.05.016

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