Targeted gene suppression through double-stranded RNA application using easy-to-use methods in Arabidopsis thaliana

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.

Suppression of Phytophthora capsici using double-stranded RNAs targeting NLP effector genes in Nicotiana benthamiana

“…In our previous study [12,13], treating plants with dsR-NAs targeting different regions in the target mRNA sequence yielded different effects on the target gene. Likewise, we first examined which region of the PcNLP2 and PcNLP6 mRNAs should be targeted to most effectively suppress P. capsici infection.…”

“…The guide strand of siRNAs is incorporated into the Argonaute protein to form RNAinduced silencing complex (RISC), which post-transcriptionally silences target genes through sequence-specific base pairing [6][7][8]. Target gene silencing via exogenous dsRNA is influenced by the length of dsRNA, regions in the target mRNA, and the method of application [9][10][11][12][13][14].…”

“…In previous studies, we suppressed green fluorescent protein and pepper mottle virus in plants using dsRNAs varying in their length and position of their targets [12][13][14].…”

“…The aim of this study was to suppress P. capsici via RNAi using dsRNAs targeting PcNLP2 and PcNLP6. For efficient application of dsRNAs, location on the target gene and time of application are crucial factors [12,13]. Therefore, we designed and synthesized two distinct dsRNAs targeting PcNLP2 and PcNLP6, respectively, and applied them at the optimum time for effective P. capsici suppression.…”

Sequence optimization and multiple gene-targeting improve the inhibitory efficacy of exogenous double-stranded RNA against pepper mottle virus in Nicotiana benthamiana

“…Exogenous treatment of dsRNA can also induce RNAi in plants [3][4][5], opening up new applications of dsRNAs in protecting crops from plant pathogens and pests [6][7][8][9][10]. Since dsRNA-based RNAi technology does not require the host genome to be modified, it is free from issues surrounding genetically modified organisms.…”