Abstract
RNA interference (RNAi) is an RNA-dependent gene silencing process that is regulated by the interaction between the RNA-induced silencing complex (RISC) and double-stranded RNA (dsRNA). Exogenous dsRNAs are imported directly into the cytoplasm, where they are cleaved by Dicer into short dsRNA fragments of 20–25 base pairs. These short dsRNA fragments, called small interfering RNAs (siRNAs) have sequence-specific interaction with target genes. The guide strand, onto which siRNAs are incorporated in the RISC interacts with the target mRNA sequence, thereby inducing cleavage and degradation of target messenger RNAs (mRNAs) by ribonucleases. Recent studies have shown that plant dsRNA treatments can induce RNAi. However, the dsRNA application methods and delivery systems involved have not been well examined. In this study, dsRNA was introduced to Arabidopsis thaliana by two methods: dipping and spray. We synthesized two dsRNAs designed to target mRNAs encoding enhanced green fluorescent protein (EGFP). After applying dsRNAs that target EGFP, we found an obvious reduction in GFP expression. This was determined using fluorescence microscopy and quantitative reverse transcription PCR to assess the mRNA levels of the auxin-sensitive reporter DR5-EGFP Arabidopsis thaliana. Our data revealed that applying target gene-specific exogenous dsRNAs can induce suppression of target genes of interest whether the dipping or spray method is used. This study therefore provides a foundation for understanding how to apply and deliver dsRNAs in plants.
Highlights
RNA interference (RNAi), a process that based on small interfering RNA has been employed to regulate plant growth, plant stress tolerance and other plant processes by inhibiting the expression of particular genes [1]
We designed two double-stranded RNA (dsRNA) to determine which were most effective in suppressing the enhanced green fluorescent protein (EGFP) coding sequence (CDS)
Our results showed that the application of exogenous dsRNAs targeting EGFP could suppress fluorescence and EGFP transcript levels after 1dpt (Figs. 3 and 4)
Summary
RNA interference (RNAi), a process that based on small interfering RNA (siRNA) has been employed to regulate plant growth, plant stress tolerance and other plant processes by inhibiting the expression of particular genes [1]. Double-stranded RNA (dsRNA) can induce RNAi; this is a biological gene silencing process capable of using sequence-specific gene targeting to inhibit gene translation or transcription [1]. The dsRNAs are cleaved by a ribonuclease called Dicer-like enzyme (DCL) [2] to produce small non-coding RNAs called siRNAs. Each siRNA is unwound into two single-stranded RNAs (ssRNAs), known as the passenger strand and the guide strand. The passenger strand is degraded, while the guide strand associates with Argonaute proteins (AGOs) [3] to form the RNA-induced silencing complex (RISC) [3]. The RISC drives the silencing of target mRNAs via sequencespecific base pairing, resulting in mRNA degradation and translation repression [4, 5]
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