Designing Effective amiRNA and Multimeric amiRNA Against Plant Viruses

Abstract

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.

Recent Biotechnological Advances in the Improvement of Cassava

“…In this approach, the endogenous miRNA-miRNA duplex in a native miRNA precursor is replaced with a customized sequence designed from the target gene. Upon processing, the amiRNA redirects the miRNA-induced silencing complex to silence the targeted mRNA, thereby generating a loss-of-function phenotype for the gene of interest [62][63][64][65][66]. The amiRNA strategy has especially been used in targeting plant viruses (reviewed in Fondong et al [59].…”

Virus Diseases of Cereal and Oilseed Crops in Australia: Current Position and Future Challenges

“…WSMV research for Australian wheat breeding programs has focused on using both conventional and molecular genetic modification approaches to develop new wheat cultivars with resistance to this virus and to WCM [74,[155][156][157][158][159]. In glasshouse experiments, the resistances conferred by genes Wsm1 originally from Thinopyrum intermedium and Wsm2 from wheat breeding line C0960293-2, and the resistance present in wheat breeding line c2652 were all effective against WSMV [156].…”

“…When an RNA interference (RNAi) construct consisting of hairpin RNA derived from the nuclear inclusion protein 'a' (NIa) gene of WSMV was introduced, it conferred immunity to this virus in transgenic wheat plants [155]. Similarly, when an artificial miRNA strategy targeting conserved regions of WSMV was used, some transgenic wheat plants immune to WSMV were obtained [157,158].…”

Genetic recombination in plant-infecting messenger-sense RNA viruses: overview and research perspectives

“…For example, measures could be taken to reduce recombination while designing the antiviral resistance in transgenic plants with artificial micro RNAs (Fahim and Larkin, 2013) or with double stranded RNA-expressing transgenes (Zhang et al, 2011). Also, the potential instability and recovery of the wild-type virus via recombination can be reduced during construction of plant RNA viral vectors (Nagyová and Subr, 2007).…”