Abstract
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.
Highlights
We previously devised a cucumber mosaic virus (CMV)-based vector system carrying microRNA target mimic sequences for analysis of microRNA function in Arabidopsis thaliana
In A. thaliana, inhibition of miR399 occurs by interaction with a non-cleavable target mimic sequence in transcripts of the gene Induced by Phosphate Starvation[1] (IPS1)[11]
We developed a Target mimic (TM) vector based on cucumber mosaic virus (CMV) strain LS, in which miRNA TM sequences were introduced at a point in the RNA3 sequence immediately downstream of the coat protein (CP) gene[19]
Summary
We previously devised a cucumber mosaic virus (CMV)-based vector system carrying microRNA target mimic sequences for analysis of microRNA function in Arabidopsis thaliana. The tobacco rattle virus (TRV)-based vector[17] to express TMs or STTMs to inhibit specific miRNAs in Nicotiana benthamiana and tomato (Solanum lycopersicum).
Sign-in/Register to view highlights & summary 
Published Version (
Free)
Talk to us
Join us for a 30 min session where you can share your feedback and ask us any queries you have
Schedule a callDisclaimer: All third-party content on this website/platform is and will remain the property of their respective owners and is provided on "as is" basis without any warranties, express or implied. Use of third-party content does not indicate any affiliation, sponsorship with or endorsement by them. Any references to third-party content is to identify the corresponding services and shall be considered fair use under The CopyrightLaw.
