An improved cucumber mosaic virus-based vector for efficient decoying of plant microRNAs.
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.
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Qiansheng
Q
Liao
College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China.
Yifei
Y
Tu
Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom.
John P
JP
Carr
1] College of Life Sciences, Zhejiang Sci-Tech University, Hangzhou 310018, China [2] Department of Plant Sciences, University of Cambridge, Cambridge CB2 3EA, United Kingdom.
Zhiyou
Z
Du
D017360:Arabidopsis / Q000235:genetics* / Q000378:metabolism; D017796:Caulimovirus / Q000235:genetics; D017799:Cucumovirus / Q000235:genetics*; D005822:Genetic Vectors / Q000235:genetics / Q000378:metabolism*; D018551:Solanum lycopersicum / Q000235:genetics / Q000378:metabolism; D035683:MicroRNAs / Q000037:antagonists & inhibitors / Q000235:genetics / Q000378:metabolism*; D016376:Oligonucleotides, Antisense / Q000235:genetics / Q000378:metabolism; D010641:Phenotype; D018515:Plant Leaves / Q000235:genetics / Q000378:metabolism; D011401:Promoter Regions, Genetic; D018749:RNA, Plant / Q000032:analysis; D012335:RNA, Ribosomal / Q000235:genetics; D012367:RNA, Viral / Q000235:genetics / Q000378:metabolism; D060888:Real-Time Polymerase Chain Reaction; D014026:Nicotiana / Q000235:genetics / Q000378:metabolism
C583141:MIRN165 microRNA, Arabidopsis; C580969:MIRN166 microRNA, Arabidopsis; D035683:MicroRNAs; D016376:Oligonucleotides, Antisense; D018749:RNA, Plant; D012335:RNA, Ribosomal; D012367:RNA, Viral