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.
Highlights
Virus-induced diseases cause significant reductions in both crop quality and yield worldwide [1,2]
Following the programming with small RNAs, which may be either endogenously DCL-generated as described above or exogenously added as synthetic molecules, the functionality of in vitro-generated RNAinduced silencing complexes (RISC) can be tested in a ‘slicer assay’ with a chosen target RNA [23,28,37]
Considering its capability of acting as a silencing inducer and its homogeneous composition consisting of equal quantities of (+)- and (−)-strand RNA, we performed all of the experiments described below with Tomato bushy stunt virus (TBSV) dsRNA molecules
Summary
Virus-induced diseases cause significant reductions in both crop quality and yield worldwide [1,2]. A major component of the plant’s immune response against viral infections is the RNA silencing process [4,5]. Structured regions of viral mRNAs and genomes and/or double-stranded (ds) RNA molecules induce RNA silencing in plants [6,7,8,9]. DsRNAs are produced, for example, during infections with (+)-strand RNA viruses, which represent the vast majority of plant-infecting viruses [10]. Genome replication of these viruses occurs in the cell’s cytoplasm and involves a two-step process via (−)-strand RNA and dsRNA replication intermediates. Associated with the removal of the vsiRNA’s passenger strand, the remaining guide strand is incorporated into Argonaute (AGO) endonucleases [13], the core components of RNAinduced silencing complexes (RISC)
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