An improved cucumber mosaic virus-based vector for efficient decoying of plant microRNAs

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.

Establishing RNA virus resistance in plants by harnessing CRISPR immune system

“…Arabidopsis thaliana ecotype Col‐0 and Nicotiana benthamiana plants used in this study were grown as previously described (Zhang et al ., , ). Agro‐infectious clones of TMV‐GFP (Shivprasad et al ., ) and CMV (Fny strain) (Liao et al ., ) and were kindly provided by Prof. Xueping Zhou (Zhejiang University) and Dr. Zhouhang Gu (Zhejiang Sci‐Tech University), respectively.…”

An efficient Potato virus X -based microRNA silencing in Nicotiana benthamiana

“…We previously developed a plant virus-based miRNA expression system, in which a Tobacco rattle virus (TRV)-based vector was used to express artificial TMs and cause virus-based miRNA silencing/suppression (VbMS) 31 32 . Several research groups have developed VbMS systems based on different plant viruses, including Cotton leaf crumple virus (CLCrV) 33 and Cucumber mosaic virus (CMV) 34 35 and have demonstrated good miRNA suppression. VbMS enables a prompt elucidation of miRNA function and has proved to be effective in Nicotiana benthamiana 31 34 , N. tabacum 34 , Solanum lycopersicum 31 34 , Arabiopsis 32 35 and Gossypium hirsutum 33 .…”

The interaction between endogenous 30S ribosomal subunit protein S11 and Cucumber mosaic virus LS2b protein affects viral replication, infection and gene silencing suppressor activity

“…The Arabidopsis cDNA library (BioTech, GER) was prepared in plasmid pGADT7-Rec and transformed into yeast strain Y187. The bait plasmid pGBKT7-LS2b [ 17 ] was transformed into yeast strain Y2H.Gold. To confirm that the bait protein LS2b did not autonomously activate the reporter genes without a prey protein, we transformed the pGBKT7-LS2b construct into previously prepared competent Y2HGold cells.…”

“…Activated agrobacteria containing plasmids pCB301-LS109, pCB301-LS209, and pCB301-LS309∆CP-GFP [ 17 ] were systemically infected leaves 7-day post pTRV-RNA2-NbRPS11inoculation. LS-CMV was transported from the infected N .…”