Abstract
Cucumber mosaic virus (CMV) is a destructive plant virus with worldwide distribution and the broadest host range of any known plant virus, as well as a model plant virus for understanding plant–virus interactions. Since the discovery of RNA interference (RNAi) as a major antiviral defense, RNAi-based technologies have been developed for plant protection against viral diseases. In plants and animals, a key trigger of RNAi is double-stranded RNA (dsRNA) processed by Dicer and Dicer-like (DCL) family proteins in small interfering RNAs (siRNAs). In the present study, dsRNAs for coat protein (CP) and 2b genes of CMV were produced in vitro and in vivo and applied onto tobacco plants representing a systemic solanaceous host as well as on a local host plant Chenopodium quinoa. Both dsRNA treatments protected plants from local and systemic infection with CMV, but not against infection with unrelated viruses, confirming sequence specificity of antiviral RNAi. Antiviral RNAi was effective when dsRNAs were applied simultaneously with or four days prior to CMV inoculation, but not four days post inoculation. In vivo-produced dsRNAs were more effective than the in vitro-produced; in treatments with in vivo dsRNAs, dsRNA-CP was more effective than dsRNA-2b, while the effects were opposite with in vitro dsRNAs. Illumina sequencing of small RNAs from in vivo dsRNA-CP treated and non-treated tobacco plants revealed that interference with CMV infection in systemic leaves coincides with strongly reduced accumulation of virus-derived 21- and 22-nucleotide (nt) siRNAs, likely generated by tobacco DCL4 and DCL2, respectively. While the 21-nt class of viral siRNAs was predominant in non-treated plants, 21-nt and 22-nt classes accumulated at almost equal (but low) levels in dsRNA treated plants, suggesting that dsRNA treatment may boost DCL2 activity. Taken together, our findings confirm the efficacy of topical application of dsRNA for plant protection against viruses and shed more light on the mechanism of antiviral RNAi.
Highlights
RNA interference (RNAi) is an evolutionarily conserved mechanism, present in eukaryotic organisms, that regulates gene expression via mRNA degradation, repression of translation, and chromatin remodeling [1]
2.1. double-stranded RNA (dsRNA) Treatments of Tobacco Plants Resulted in Variable Levels of Protection against Cucumber mosaic virus (CMV)
All N. tabacum plants treated with CMV inoculum alone (n = 157) or CMV inoculum in mixture with in vivo produced dsRNA-MalE (n = 19), used as a control for sequence specificity of dsRNA, became infected after 14 days post inoculation, showing typical CMV symptoms including leaf curl/bump-like symptoms, mosaic symptoms, dwarfing of plants and necrosis of leaves
Summary
RNA interference (RNAi) is an evolutionarily conserved mechanism, present in eukaryotic organisms, that regulates gene expression via mRNA degradation, repression of translation, and chromatin remodeling [1] It is involved in developmental regulation, stress response, or defense against invading nucleic acids like transposons or viruses [2]. The main research focus in this direction has been the development of transgenic plants aiming at induction of RNAi that led to attenuation or elimination of viral disease symptoms To this respect, diverse plant–virus pathosystems have been studied and crops engineered for virus resistance have been developed, using among others so-called RNAi transgenes designed to express dsRNA as a key trigger of RNAi [9,10]. Certain engineered crops such as squash resistant to Watermelon mosaic virus 2, Zucchini yellow mosaic virus (ZYMV) and Cucumber mosaic virus (CMV), papaya resistant to Papaya ringspot virus, potato resistant to Potato virus Y, bean resistant to
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