A Transient RNA Interference Assay System UsingArabidopsisProtoplasts

Abstract

Double-stranded RNA (dsRNA) induces sequence-specific gene silencing in eukaryotes through a process known as RNA interference (RNAi). RNAi is now used as a powerful tool for functional genomics in many eukaryotes, including plants. We herein report a dsRNA-mediated transient RNAi assay system using protoplasts from Arabidopsis mesophyll cells and suspension-cultured cells (cell line T87). Introduction of dsRNA into protoplasts led to marked silencing of target transgenes. Our assay system would provide a convenient and efficient way to induce RNAi in protoplasts of the model plant Arabidopsis thaliana.

Transient RNAi Induction against Endogenous Genes in Arabidopsis Protoplasts Using in Vitro-Prepared Double-Stranded RNA

“…We have been attempting to develop a simple and effective system for inducing RNAi in plant cells, and recently reported that introduction of in vitro-prepared dsRNA into Arabidopsis protoplasts with polyethylene glycol (PEG) caused effective and specific interference with transgene activity. 4) Although this method has the potential to knock down endogenous genes, its applicability to endogenous genes has not been proved. Here we show that introduction of in vitro-prepared dsRNA into Arabidopsis T87 protoplasts also leads to marked silencing of endogenous genes.…”

“…Comparison of their C T values indicated that their relative expression levels to Actin2/8 (internal standard, see below) were 0.1-2.1 for PAT1, 0.3-4.0 for sdh2-1, and 0.1-2.1 for sdh2-2 (n ¼ 3; expression of sdh2-3 was not detected, see below), confirming that they are surely expressed in T87 protoplasts except for sdh2-3. dsRNAs were prepared as described previously, 4) except that the T7 RiboMAX Express RNAi System (Promega, Madison, U.S.A.) was used for in vitro transcription. The sequence of the sGFP(S65T) gene (GFP) was used as a negative control.…”

“…4) Protoplasts were isolated and transfected as previously described. 4) Typically, 4 Â 10 4 protoplasts were transfected with 10 mg of dsRNA encoding a target gene sequence, and incubated in the modified LS medium 4) supplemented with 0.4 M mannitol at 22…”

“…These results indicate that introduction of dsRNA into Arabidopsis protoplasts caused specific interference with PAT1 expression in a dose-dependent manner, as observed in transgene (luciferase)-targeted RNAi. 4) As shown in T87 protoplasts (4 Â 10 4 cells) were transfected with increasing amounts of dsRNA (GFP: 103 bp, PAT1: 101 bp), and incubated at 22 C for 24 h. PAT1 expressions were calculated by the relative standard curve method using ACTIN2/8 as an internal standard. Expression levels determined in the presence of dsRNA were normalized to those obtained in the absence of dsRNA (Mock).…”

“…The relatively short duration of RNAi was inconsistent with our previous report that transgenetargeted RNAi lasted two weeks in T87 protoplasts. 4) This disparity might be due to differences in RNAdirected DNA methylation 9) and/or dsRNA amplification by RNA-dependent RNA polymerase (RdRp) 10) between transgenes and endogenous genes.…”