Topically delivered 22 nt siRNAs enhance RNAi silencing of endogenous genes in two species

Abstract

Main conclusion 22 nt siRNAs applied to leaves induce production of transitive sRNAs for targeted genes and can enhance local silencing. Systemic silencing was only observed for a GFP transgene. Abstract RNA interference (RNAi) is a gene silencing mechanism important in regulating gene expression during plant development, response to the environment and defense. Better understanding of the molecular mechanisms of this pathway may lead to future strategies to improve crop traits of value. An abrasion method to deliver siRNAs into leaf cells of intact plants was used to investigate the activities of 21 and 22 nt siRNAs in silencing genes in Nicotiana benthamiana and Amaranthus cruentus. We confirmed that both 21 and 22 nt siRNAs were able to silence a green fluorescent protein (GFP) transgene in treated leaves of N. benthamiana, but systemic silencing of GFP occurred only when the guide strand contained 22 nt. Silencing in the treated leaves of N. benthamiana was demonstrated for three endogenous genes: magnesium cheletase subunit I (CHL-I), magnesium cheletase subunit H (CHL-H), and GENOMES UNCOUPLED4 (GUN4). However, systemic silencing of these endogenous genes was not observed. Very high levels of transitive siRNAs were produced for GFP in response to treatment with 22 nt siRNAs but only low levels were produced in response to a 21 nt siRNA. The endogenous genes tested also produced transitive siRNAs in response to 22 nt siRNAs. 22 nt siRNAs produced greater local silencing phenotypes than 21 nt siRNAs for three of the genes. These special properties of 22 nt siRNAs were also observed for the CHL-H gene in A. cruentus. These experiments suggest a functional role for transitive siRNAs in amplifying the RNAi response.

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

“…(1) All sprayed 22-nt siRNAs triggered transitivity towards both their 5ʹ and 3ʹ, demonstrating that siRNAmediated transitivity is bi-directional and not unidirectional in agreement with previous studies (Hendrix et al, 2021a;Vaistij et al, 2002). ( 2) Despite targeting different regions on the GFP transcript, the three applied 22-nt siR-NAs produced very similar transitivity patterns, suggesting that they were not used as primers for RDR6 (otherwise transitivity would have spread only towards the 5ʹ upstream region of each siRNA trigger), corroborating previous reports (Hendrix et al, 2021b;Moissiard et al, 2007).…”

Spray-Induced Silencing of Pathogenicity Gene MoDES1 via Exogenous Double-Stranded RNA Can Confer Partial Resistance Against Fungal Blast in Rice

“…Some reports have shown that topically applied dsRNA can move systemically across plant tissues to prevent pathogen colonization even in the unsprayed regions (Koch et al, 2016;Mitter et al, 2017;Song et al, 2018). Systemic silencing was also demonstrated (Hendrix et al, 2021), where it was hypothesized that 22-bp siRNAs could induce silencing in systemic regions of Nicotiana benthamiana by secondary siRNA generation. The key to a prolonged and robust silencing induced by sprayed dsRNA can be attributed to their systemic nature.…”

Nanovector-mediated exogenous delivery of dsRNA induces silencing of target genes in very young tomato flower buds

“…However, the examples documented in the literature are limited. What has emerged from these studies is that exogenous RNAs are more efficient at suppressing transgenes than endogenous ones 36,58,61,62,65 . It has been suggested that the high level of transgene transcription, which is quite often regulated by strong promoters, can cause aberrant transcripts that produce secondary dsRNAs 58 .…”