Nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants

Daai Zhang, Chengcheng Zhong, Neil A. Smith, Robert de Feyter, Ian K. Greaves, Steve M. Swain, Ren Zhang and Ming-Bo Wang ()
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Daai Zhang: CSIRO Agriculture and Food
Chengcheng Zhong: CSIRO Agriculture and Food
Neil A. Smith: CSIRO Agriculture and Food
Robert de Feyter: CSIRO Agriculture and Food
Ian K. Greaves: CSIRO Agriculture and Food
Steve M. Swain: CSIRO Agriculture and Food
Ren Zhang: University of Wollongong
Ming-Bo Wang: CSIRO Agriculture and Food

Nature Communications, 2022, vol. 13, issue 1, 1-16

Abstract: Abstract Hairpin RNA (hpRNA) transgenes are the most successful RNA interference (RNAi) method in plants. Here, we show that hpRNA transgenes are invariably methylated in the inverted-repeat (IR) DNA and the adjacent promoter, causing transcriptional self-silencing. Nucleotide substitutions in the sense sequence, disrupting the IR structure, prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine with thymine nucleotides, in a G:U hpRNA design, prevents self-silencing but still allows for the formation of hpRNA due to G:U wobble base-pairing. The G:U design induces effective RNAi in 90–96% of transgenic lines, compared to 57–65% for the traditional hpRNA design. While a traditional hpRNA transgene shows increasing self-silencing from cotyledons to true leaves, its G:U counterpart avoids this and induce RNAi throughout plant growth. Furthermore, siRNAs from G:U and traditional hpRNA show different characteristics and appear to function via different pathways to induce target DNA methylation.

Date: 2022
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DOI: 10.1038/s41467-022-31641-5

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