Potential functional pathways of plant RNA virus-derived small RNAs in a vector insect.

During infection, RNA viruses can produce two types of virus-derived small RNAs (vsRNAs), small interfering RNA (siRNA) and microRNA (miRNA), that play a key role in RNA silencing-mediated antiviral mechanisms in various hosts by associating with different Argonaute (Ago) proteins. Ago1 has been widely identified as an essential part of the miRNA pathway, while Ago2 is required for the siRNA pathway. Thus, analysis of the interaction between vsRNAs and Ago proteins can provide a clue about which pathway the vsRNA may be involved in. In this study, using rice stripe virus (RSV)-small brown planthoppers (Laodelphax striatellus, Fallen) as an infection model, the interactions of eight vsRNAs derived from four viral genomic RNA fragments and Ago1 or Ago2 were detected via the RNA immunoprecipitation (RIP) method. vsRNA4-1 and vsRNA4-2 derived from RSV RNA4 were significantly enriched in Ago1-immunoprecipitated complexes, whereas vsRNA2-1 and vsRNA3-2 seemed enriched in Ago2-immunoprecipitated complexes. vsRNA1-2 and vsRNA2-2 were detected in both of the two Ago-immunoprecipitated complexes. In contrast, vsRNA1-1 and vsRNA3-1 did not accumulate in either Ago1- or Ago2-immunoprecipitated complexes, indicating that regulatory pathways other than miRNA or siRNA pathways might be employed. In addition, two conserved L. striatellus miRNAs were analysed via the RIP method. Both miRNAs accumulated in Ago1-immunoprecipitated complexes, which was consistent with previous studies, suggesting that our experimental system can be widely used. In conclusion, our study provides an accurate and convenient detection system to determine the potential pathway of vsRNAs, and this method may also be suitable for studying other sRNAs.