Mismatch hpRNA prevents intrinsic self-silencing and enhances RNAi stability

ABSTRACT: Hairpin RNA (hpRNA) transgenes, with a perfect inverted-repeat (IR) DNA, have been the most successful RNA interference (RNAi) method in plants. Here we show that hpRNA transgenes were invariably methylated in the IR DNA and the adjacent promoter, causing transcriptional self-silencing and preventing the full potential of RNAi. Nucleotide substitutions in the sense sequence, which disrupts the perfect IR DNA structure, were sufficient to prevent the intrinsic DNA methylation resulting in more uniform and persistent RNAi. Substituting all cytosine (C) with thymine (T) nucleotides, in a G:U hpRNA design, prevented DNA methylation and self-silencing but still allowed for the formation of perfect 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. Furthermore, while a traditional hpRNA transgene showed increasing DNA methylation and self-silencing from cotyledons to true leaves, the G:U transgenes avoided this developmental progression of self-silencing and induced RNAi throughout plant growth. The G:U and traditional hpRNA transgenes generated small interfering RNA (siRNA) with different 5’ phosphorylation, which resembled the endogenous tasiRNA and miRNA, respectively. Furthermore, our results suggest that siRNAs from the two transgene designs function differently to induce target DNA methylation, one (from traditional hpRNA) through the canonical RdDM pathway and the other (G:U hpRNA) a non-canonical pathway. Our study not only revealed a methylation-resistant RNAi transgene design but also provided new mechanistic insights into small RNA biogenesis and function in plants

ORGANISM(S): Arabidopsis thaliana

PROVIDER: GSE178565 | GEO | 2022/03/31

REPOSITORIES: GEO