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

Author

Listed:
  • Daai Zhang

    (CSIRO Agriculture and Food
    University of Wollongong)

  • 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)

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.

Suggested Citation

  • Daai Zhang & Chengcheng Zhong & Neil A. Smith & Robert de Feyter & Ian K. Greaves & Steve M. Swain & Ren Zhang & Ming-Bo Wang, 2022. "Nucleotide mismatches prevent intrinsic self-silencing of hpRNA transgenes to enhance RNAi stability in plants," Nature Communications, Nature, vol. 13(1), pages 1-16, December.
    • Handle: RePEc:nat:natcom:v:13:y:2022:i:1:d:10.1038_s41467-022-31641-5
    DOI: 10.1038/s41467-022-31641-5
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    References listed on IDEAS

    as
    1. David Baulcombe, 2004. "RNA silencing in plants," Nature, Nature, vol. 431(7006), pages 356-363, September.
    2. Neil A. Smith & Surinder P. Singh & Ming-Bo Wang & Peter A. Stoutjesdijk & Allan G. Green & Peter M. Waterhouse, 2000. "Total silencing by intron-spliced hairpin RNAs," Nature, Nature, vol. 407(6802), pages 319-320, September.
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