title: Efficient and specific inhibition of plant microRNA function by anti-microRNA oligonucleotides (AMOs) in vitro and in vivo.
abstract: Anti-microRNA oligonucleotides (AMOs) are efficient and sequence-specific inhibitors of plant miRNA function both in vitro and in vivo. MicroRNAs (miRNAs) are small non-coding RNAs that play critical roles in developmental and physiological processes in plants and animals. Although miRNA knockdown by chemically modified antisense oligonucleotides prevails in animal and therapeutic studies, no such application has ever been reported in plants. Here, we show that sucrose-mediated delivery of 2'-O-methyl (2'-O-Me) anti-miRNA oligonucleotides (AMOs) is an efficient and sequence-specific way of inhibiting plant miRNA activity both in vitro and in vivo. Administration of AMOs to rice protoplasts and intact leaves resulted in efficient inhibition of miRNAs with concurrent de-repression of their target genes. AMOs caused simultaneous inhibition of miRNAs from the same family but exerted negligible effects on miRNAs from different families. In rice seedlings, a single-dose AMO treatment conferred long-lasting miRNA inhibition for at least 7 days. Although simultaneous dysregulation of multiple miRNAs by an AMO-and-miRNA-mimic mixture resulted in severe root defects, the phenotypic effects of individual AMOs and miRNA mimics were negligible, suggesting that those miRNAs function together in regulatory networks to ensure homeostasis. Our results validate the utility of AMOs as an efficient tool for plant miRNA loss-of-function studies in vivo, and this approach may prove to be a highly promising general method for unraveling miRNA-mediated gene-regulatory networks.
authors.affiliation: State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.|Institute of Biosciences and Technology, Texas Agriculture and Mechanics University, Houston, TX, 77030, USA.
authors.forename: Lian
authors.initials: L
authors.lastname: He
authors.affiliation: State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.
authors.forename: Munan
authors.initials: M
authors.lastname: Xie
authors.affiliation: State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.
authors.forename: Jianhua
authors.initials: J
authors.lastname: Huang
authors.affiliation: State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.|Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.
authors.forename: Tianyuan
authors.initials: T
authors.lastname: Zhang
authors.affiliation: State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.|Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China.
authors.forename: Suhua
authors.initials: S
authors.lastname: Shi
authors.affiliation: State Key Laboratory of Biocontrol and Key Laboratory of Gene Engineering of Ministry of Education, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China. lsstt@mail.sysu.edu.cn.|Guangdong Key Laboratory of Plant Resources, School of Life Sciences, Sun Yat-Sen University, Guangzhou, 510275, People's Republic of China. lsstt@mail.sysu.edu.cn.
authors.forename: Tian
authors.initials: T
authors.lastname: Tang
mesh_terms: D020868:Gene Silencing / Q000187:drug effects; D035683:MicroRNAs / Q000037:antagonists & inhibitors* / Q000235:genetics*; D009841:Oligonucleotides / Q000494:pharmacology*; D012275:Oryza / Q000187:drug effects / Q000235:genetics*; D010641:Phenotype; D059828:Plant Cells / Q000187:drug effects / Q000378:metabolism; D018515:Plant Leaves / Q000187:drug effects / Q000378:metabolism; D018517:Plant Roots / Q000187:drug effects / Q000254:growth & development / Q000378:metabolism; D011523:Protoplasts / Q000187:drug effects / Q000378:metabolism; D013997:Time Factors
chemical_list: D035683:MicroRNAs; D009841:Oligonucleotides
keywords: Anti-microRNA oligonucleotides (AMOs); Loss of function; Regulatory networks; microRNA