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

Bill Hendrix,Michael J Bennett,Brian D Eads,Paul H Hoffer,Matthew J Bauer,Rick A Sanders,Janette Oakes,Jill Deikman,Wei Zheng,Jennifer T Mai,Alberto B Iandolino,Amy Caruano-Yzermans,Ericka R Havecker,Chelly Hresko,Danielle N Taylor

doi:10.1007/s00425-021-03708-y

Bill Hendrix, Michael J Bennett + Show 13 more

Open Access

https://doi.org/10.1007/s00425-021-03708-y

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Abstract

Main conclusion22 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.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.

Highlights

RNA interference (RNAi) is a gene silencing mechanism used by plants and other eukaryotes to regulate the expression of endogenous genes, to silence transposons, and defend against virus infection (Baulcombe 2004)
An efficacious dsRNA to silence green fluorescent protein (GFP) was selected by calculating a Reynolds score (Reynolds et al 2004) for all possible 19 mers, and testing high scoring sequences as 22 nt siRNAs with 2 nt 3′ overhangs in protoplasts prepared from tobacco BY-2 cells expressing a GFP transgene (Yang et al 2021)
GFP fluoresces green under UV light, and GFP gene suppression can be detected by the unmasking of red fluorescence emitted from chlorophyll

Summary

Introduction

RNA interference (RNAi) is a gene silencing mechanism used by plants and other eukaryotes to regulate the expression of endogenous genes, to silence transposons (and other heterochromatic regions), and defend against virus infection (Baulcombe 2004). It is achieved by the action of a small RNA (sRNA) with complementarity to the target gene sequence bound to an argonaute (AGO) protein to form an RNA-induced silencing complex (RISC). Topical delivery of sRNAs to plant cells provides a tool that can be used to investigate details of the RNAi mechanisms in plants

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