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

Thien Ho, Denise Pallett, Rachel Rusholme, Tamas Dalmay, Hui Wang

Research output: Contribution to journalArticlepeer-review

52 Citations (Scopus)

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.
Original languageEnglish
Pages (from-to)217-223
Number of pages7
JournalJournal of Virological Methods
Volume136
Issue number1-2
DOIs
Publication statusPublished - Sep 2006

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