Highly efficacious antiviral protection of plants by small interfering RNAs identified in vitro

Abstract

In response to a viral infection, the plant’s RNA silencing machinery processes viral RNAs into a huge number of small interfering RNAs (siRNAs). However, a very few of these siRNAs actually interfere with viral replication. A reliable approach to identify these immunologically effective siRNAs (esiRNAs) and to define the characteristics underlying their activity has not been available so far. Here, we develop a novel screening approach that enables a rapid functional identification of antiviral esiRNAs. Tests on the efficacy of such identified esiRNAs of a model virus achieved a virtual full protection of plants against a massive subsequent infection in transient applications. We find that the functionality of esiRNAs depends crucially on two properties: the binding affinity to Argonaute proteins and the ability to access the target RNA. The ability to rapidly identify functional esiRNAs could be of great benefit for all RNA silencing-based plant protection measures against viruses and other pathogens.

Turnip mosaic virus in oilseed rape activates networks of sRNA-mediated interactions between viral and host genomes

“…In our study, only relatively few vsiRNAs were linked to vRNA cleavage events, albeit vsiRNAs were abundantly produced from the entire vRNA. This observation has already been noted in previous studies 88 . We speculate that vsiRNAs may have little, if any, influence on vRNA cleavage in early colonized cells at the infection front.…”

Barriers to Efficient Foliar Uptake of dsRNA and Molecular Barriers to dsRNA Activity in Plant Cells

“…However, only approximately half of siRNAs that were selected based on such guidelines were efficacious in reducing mRNA concentration for a targeted gene that could be silenced (unpublished Bayer Crop Science research). More recently, an in vitro assay was developed to identify efficacious siRNAs (esiRNAs) targeting a plant virus gene (Gago-Zachert et al, 2019). The two critical features for the esiRNA were ability to bind Argonaute proteins and the ability to access the target RNA.…”

Artificial Small RNA-Based Silencing Tools for Antiviral Resistance in Plants

“…Another possibility is to express as art-sRNAs those immunologically effective small interfering RNAs (esiRNAs) that are generated by DCLs from viral dsRNAs produced during viral replication [39]. Because the majority of virus-derived sRNAs are ineffective against the producing virus [52][53][54][55], it has been difficult to distinguish esiRNAs reliably and efficiently until very recently.…”

“…Because the majority of virus-derived sRNAs are ineffective against the producing virus [52][53][54][55], it has been difficult to distinguish esiRNAs reliably and efficiently until very recently. Gago-Zachert and colleagues recently reported the identification of TBSV-derived esiRNAs using an in vitro system of cytoplasmic extracts from N. tabacum BY-2 protoplasts expressing TBSV dsRNAs and selected AGO members [39]. First, 21-nucleotide sRNAs derived from TBSV dsRNAs and loaded by AGO1 or AGO2 were identified by immunoprecipitation followed by sRNA sequencing.…”

“…Second, the cleavage efficiency of each of these sRNAs was analyzed in in vitro cleavage assays, and third, the protective effects of several of these siRNAs were analyzed in N. benthamiana plants by agroinfiltrating each sRNA as an amiRNA, and subsequently inoculating TBSV in the same leaf-sites. Results showed that the functionality of esiRNAs mainly depended on the binding affinity to AGO proteins and the ability to target RNA [39]. Thus, this methodology could be attractive to identify naturally occurring virus-derived sRNAs that are efficient in silencing viral RNAs.…”