Designing effective amiRNA and multimeric amiRNA against plant viruses.

RNA-mediated virus resistance is increasingly becoming a method of choice for antiviral defense in plants when effective natural resistance is unavailable. In this chapter we discuss the design principles of artificial micro RNA (amiRNA), in which a natural miRNA precursor gene is modified to target a different species of RNA, in particular viral RNA. In addition, we explore the advantages and effectiveness of multiple amiRNAs within one polycistronic amiRNA precursor against a virus, as illustrated with Wheat streak mosaic virus, WSMV. The judicious selection of amiRNAs, which are sequences of short length as compared to other related methodologies of RNA interference, greatly assists in avoiding unintended off-targets in the host plant. The viral sequences targeted can be genomic or replicative and should be derived from conserved regions of the published WSMV genome. In short, using published folding and miRNA selection rules and algorithms, candidate miRNA sequences are selected from conserved regions between a number of WSMV genomes, and are BLASTed against wheat TIGR ESTs. Five miRNAs are selected that are least likely to interfere with the expression of transcripts from the wheat host. Then, the natural miRNA in each of the five arms of the polycistronic rice miR395 is replaced in silico with the chosen artificial miRNAs. This artificial precursor is transformed into wheat behind a ubiquitin promoter, and its integration into transformed wheat plants is confirmed by PCR and Southern blot analysis. We have demonstrated the effectiveness of this methodology using an amiRNA precursor that we have termed Fanguard. The processing of amiRNAs in transgenic leaves is verified through splinted ligation assay, and the functionality of the transgene in preventing viral replication is verified by virus bioassay. Resistance is confirmed using mechanical virus inoculation over two subsequent generations. This example demonstrates the potential of polycistronic amiRNA to achieve stable immunity to economically important viruses.