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

Abstract

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, the 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. This secondary dsRNA is subsequently cleaved into secondary, mainly phased, siRNAs (phasiRNAs) by DICER-LIKE (DCL) endonucleases. As primary siRNAs, secondary siRNAs are also loaded into ARGONAUTE proteins (AGOs) to form an RNA-induced silencing complex (RISC) reinforcing 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 GFP-expressing Nicotiana benthamiana 16C line, high pressure spraying protocol (HPSP), and synthetic 22-nucleotide (nt) long siRNAs. We found that the siRNA targeting the 3 prime end of the GFP transgene was less efficient in inducing silencing when compared to the siRNAs targeting the 5 prime end and middle region of the GFP. Moreover, sRNA sequencing of locally silenced leaves showed that the amount but not the profile of secondary RNAs are 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 prime end of the target RNA.

Induction of promoter methylation and transcriptional gene silencing upon high pressure spraying of 24-nt small RNAs in Nicotiana benthamiana

“…Only 8 plants with at least one silenced leaf and 14 leaves with at least two silenced spots were detected (Fig 1b). The silenced spots were mostly very tiny, scattered, and had orange color, which was remarkably different from the 22nt-RNA induced PTGS (Fig 1c) (Uslu et al 2021). In order to find out whether PTGS underlies the mild silencing, sRNA sequencing was performed to detect gene body transitivity.…”

“…(a) The silencing phenotypes of 16C plants are shown. 22nt-siRNA164 by HPST (Uslu VV et al 2021) leads to consistent local silencing covering approximately 20% to 80% of the leaf surface. 22nt-siRNA164 by Carbon Dot and Low-Pressure Spraying (CD-LP) can lead to local silencing of the whole leaf area.…”

“…Airbrush pistol (CONRAD AFC-250A and CONRAD HP-200) is used to deliver the siRNAs from a distance of than 1cm. 24-nt siRNA was provided by Metabion, 22nt-siRNA is the siRNA164 in Uslu VV et al 2021. The RNA was dissolved in water for naked RNA spraying at high pressure and Silwett L77 + CD Spray buffer (10mM MES, 20mM glycerol, pH:5.7) is used for CD spray.…”

“…Three biological replicates of approximately 50mg leaf material were crashed using metal beads and small RNAs were extracted using mirVana miRNA extraction kit according to manufacturer’s instructions (www.thermofisher.com). sRNA-seq and bioinformatic analysis were performed by GenXPro, Frankfurt (http://genexpro.net) as indicated in the literature (Uslu VV et al 2021).…”

“…We have previously shown that high pressure spraying technology (HPST) is a very powerful tool to modify gene expression in plants by leading to PTGS of selected targets (Dalakouras et al ., 2016; Uslu et al ., 2021; Uslu and Wassenegger, 2020). Recently, the first example of spray-induced epigenetic modifications (SPIEM) has been reported, in which high pressure spraying of a 333 bp dsRNA triggered promoter methylation in N. benthamiana (Dalakouras and Ganopoulos, 2021).…”