Topical Application of Double-Stranded RNA Targeting 2b and CP Genes of Cucumber mosaic virus Protects Plants against Local and Systemic Viral Infection

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.

The Specificity of Transgene Suppression in Plants by Exogenous dsRNA

“…The exogenously applied dsRNAs entered not only fungal cells but also plant cells and plant vascular system. They were processed to siRNAs and initiated RNA silencing, supporting the conclusion that observed resistance is an RNAi-mediated process [ 11 , 16 ]. The exogenous dsRNAs and siRNAs have been shown to spread systemically from the treated leaves to non-treated ones [ 14 , 15 ].…”

“…Recent studies increasingly demonstrate the ability of plants to uptake and process exogenous RNAs and the effectiveness of exogenous RNAs against plant pathogens and pests [ 9 , 44 , 46 ]. Recent studies reported on the substantial induction of plant viral [ 14 , 15 , 16 , 17 ], fungal [ 11 , 12 , 13 ], and insect [ 47 , 48 ] resistance after external application of dsRNAs, hpRNAs, and siRNAs targeting essential genes of the pathogens. The exogenously applied dsRNAs entered not only fungal cells but also plant cells and plant vascular system.…”

“…In recent years, there has been an increasing number of studies showing that plants can uptake and process exogenous dsRNAs, hpRNAs, and siRNAs that were designed to complement important genes of plant pathogenic fungi or viruses [ 9 , 10 ]. These RNAs can be transferred to the fungi and induce RNAi processes, negatively affecting the fungal viability [ 11 , 12 , 13 ] or inducing plant virus resistance [ 14 , 15 , 16 , 17 ]. As a result, a new GM-free and eco-friendly strategy has emerged to protect plants from microbial infections, viral and insect attacks, and was termed as spray-induced gene silencing (SIGS) or ‘RNA vaccination’ [ 4 , 18 ].…”

External dsRNA Downregulates Anthocyanin Biosynthesis-Related Genes and Affects Anthocyanin Accumulation in Arabidopsis thaliana

“…It is well established that spraying plants with dsRNAs and siRNAs encoding key genes of plant pathogenic fungi [16][17][18][19][20][21] and viruses [22][23][24][25][26][27] effectively reduces development of the pathogens and suppresses the infection process. These externally applied dsR-NAs and siRNAs have been shown to spread systemically into plant tissues and were uptaken by the fungal cells inducing a RNAi-mediated silencing of the targeted genes of the pathogens [16,17,21,27].…”

“…There is an increasing number of studies that show induction of plant fungal [16][17][18][19][20][21] and viral [22][23][24][25][26][27] resistance after external application (spraying or mechanical inoculation) of dsRNAs, siRNAs, or hpRNAs designed to target virulence-related genes of the pathogens. Recent studies have also provided evidence that both plants and infecting pathogens were capable of the RNA uptake, and this eventually triggered RNAi-mediated silencing of the pathogen virulence-related genes [16,17,21,22,27].…”