Improved insect‐proofing: expressing double‐stranded RNA in chloroplasts

Abstract

RNA interference (RNAi) was discovered almost 20 years ago and has been exploited worldwide to silence genes in plants and animals. A decade later, it was found that transforming plants with an RNAi construct targeting an insect gene could protect the plant against feeding by that insect. Production of double‐stranded RNA (dsRNA) in a plant to affect the viability of a herbivorous animal is termed trans‐kingdom RNAi (TK‐RNAi). Since this pioneering work, there have been many further examples of successful TK‐RNAi, but also reports of failed attempts and unrepeatable experiments. Recently, three laboratories have shown that producing dsRNA in a plant's chloroplast, rather than in its cellular cytoplasm, is a very effective way of delivering TK‐RNAi. Our review examines this potentially game‐changing approach and compares it with other transgenic insect‐proofing schemes. © 2018 The Authors. Pest Management Science published by John Wiley & Sons Ltd on behalf of Society of Chemical Industry.

Genetically Modified Organism-Free RNA Interference: Exogenous Application of RNA Molecules in Plants

“…Should the plant-applied dsRNAs avoid plant DCL processing, they will be taken up intact by the insects or fungi and be processed therein by the pathogen/pest DICER proteins; the resulting siR-NAs will conceivably exhibit greater biological activity against insect or fungal genes. To achieve this, recent approaches rely on the expression of dsRNA transgenes in chloroplasts (Bally et al, 2016(Bally et al, , 2018. Chloroplasts lack DCLs and thus the chloroplast-expressed dsRNAs remain intact for pathogen/pest uptake.…”

Bacterium-Mediated RNA Interference: Potential Application in Plant Protection

“…As said, endogenous RNAi requires the often lengthy and difficult process of transforming and screening plant lines that optimally and stably express active RNA [16], which is further compounded by RNA-induced self-silencing of dsRNA transgenes [17]. Furthermore, RNAi-induced insect pest resistance requires the accumulation of long dsRNA, which occurs at low levels in transgenic plants, due to processing by plant RNAi machineries [18]. These difficulties can be bypassed in Exo-RNAi by using either chemical [19], in-vitro [20], or biological syntheses of dsRNA with a desired length [21,22], which are simpler to customize and prepare.…”

Assessing Environmental RNAi in a Non-Model Organism

“…To explore the greater accumulation of Dicer products after dsRNA feeding, we focused specifically on alignments of mite derived libraries to plastids as these organelles do not have Dicer activity. Potential dsRNA/shRNA substrates are not processed prior to ingestion 23 . Plastids also lack well defined transcriptional units, leading to potential widespread formation of dsRNA 24 .…”

“…A python-based algorithm was used to find overlapping read pairs that represent Dicer cleavage produced small RNA duplexes 37 . Small RNAs of [15][16][17][18][19][20][21][22][23][24][25][26][27][28][29][30][31] nts were used to query target pairs, also 15-31 nts long, that exhibited an overlap of 2 nts less than the size of query RNA. The number of reads for each pair was plotted to show a heatmap matrix.…”