An improved cucumber mosaic virus-based vector for efficient decoying of plant microRNAs.
Carr, John P
Springer Science and Business Media LLC
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Liao, Q., Tu, Y., Carr, J. P., & Du, Z. (2015). An improved cucumber mosaic virus-based vector for efficient decoying of plant microRNAs.. Sci Rep, 5 (13178) https://doi.org/10.1038/srep13178
We previously devised a cucumber mosaic virus (CMV)-based vector system carrying microRNA target mimic sequences for analysis of microRNA function in Arabidopsis thaliana. We describe an improved version in which target mimic cloning is achieved by annealing two partly-overlapping complementary DNA oligonucleotides for insertion into an infectious clone of CMV RNA3 (LS strain) fused to the cauliflower mosaic virus-derived 35S promoter. LS-CMV variants carrying mimic sequences were generated by co-infiltrating plants with Agrobacterium tumefaciens cells harboring engineered RNA3 with cells carrying RNA1 and RNA2 infectious clones. The utility of using agroinfection to deliver LS-CMV-derived microRNA target mimic sequences was demonstrated using a miR165/166 target mimic and three solanaceous hosts: Nicotiana benthamiana, tobacco (N. tabacum), and tomato (Solanum lycopersicum). In all three hosts the miR165/166 target mimic induced marked changes in developmental phenotype. Inhibition of miRNA accumulation and increased target mRNA (HD-ZIP III) accumulation was demonstrated in tomato. Thus, a CMV-derived target mimic delivered via agroinfection is a simple, cheap and powerful means of launching virus-based miRNA mimics and is likely to be useful for high-throughput investigation of miRNA function in a wide range of plants.
microRNA sponge, target mimic, cucumber mosaic virus, RNA silencing, RNAi, miR159
This work was supported by the National Natural Science Foundation of China (grants 31170141 and 31470007), a Marie Curie International Incoming Fellowship (PIIF-GA-2009-236443), the 521 Talents Development Project (grant no.11610032521303) to ZD, the Leverhulme Trust (F/09741/F and RPG-2012-667) and the UK Biotechnology and Biological Sciences Research Council (BB/D014376/1 and BB/J011762/1) to JPC.
Isaac Newton Trust (1207(1))
Biotechnology and Biological Sciences Research Council (BB/J011762/1)
External DOI: https://doi.org/10.1038/srep13178
This record's URL: https://www.repository.cam.ac.uk/handle/1810/248947
Attribution 2.0 UK: England & Wales
Licence URL: http://creativecommons.org/licenses/by/2.0/uk/