High-pressure sprayed siRNAs influence the efficiency but not the profile of transitive silencing

In plants, small interfering RNAs (siRNAs) are a quintessential class of RNA interference (RNAi)-inducing molecules produced by the endonucleolytic cleavage of double-stranded RNAs (dsRNAs). In order to ensure robust RNAi, siRNAs are amplified through a positive feedback mechanism called transitivity. Transitivity relies on RNA-DIRECTED RNA POLYMERASE 6 (RDR6)-mediated dsRNA synthesis using siRNA-targeted RNA. The newly synthesized dsRNA is subsequently cleaved into secondary siRNAs by DICER-LIKE (DCL) endonucleases. Just like primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex reinforcing the cleavage of the target RNA. Although the molecular players underlying transitivity are well established, the mode of action of transitivity remains elusive. In this study, we investigated the influence of primary target sites on transgene silencing and transitivity using the green fluorescent protein (GFP)-expressing Nicotiana benthamiana 16C line, high-pressure spraying protocol, and synthetic 22-nucleotide (nt) long siRNAs. We found that the 22-nt siRNA targeting the 3' of the GFP transgene was less efficient in inducing silencing when compared with the siRNAs targeting the 5' and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs is shaped by the occupancy of the primary siRNA triggers on the target RNA. Our findings suggest that RDR6-mediated dsRNA synthesis is not primed by primary siRNAs and that dsRNA synthesis appears to be generally initiated at the 3'-end of the target RNA.