Use of intron-based RNA systems to study nuclear and cytoplasmic RNA interference processes in plants

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We have previously shown that the transcripts of an intronic hairpin RNA (int-hpRNA) transgene construct 1) were retained in the nucleus, 2) were inefficiently processed into small RNAs (sRNAs), 3) efficiently triggered RNA-directed DNA methylation (RdDM) of a transgenic target-promoter but 4) failed to trigger post-transcriptional gene silencing of a transgenic sensor mRNA. In the current proposal, we present how we aim to continue our research on intron-based RNA interference (RNAi) systems. Our studies will be focused on the elucidation of basic aspects of int-hpRNA-mediated gene silencing. However, we will also evaluate the potential of int-hpRNA constructs as a tool for biotechnological applications. By using conventional (non-intronic) hairpins, endogenous promoters have been shown to be poor targets for RdDM and transcriptional gene silencing (TGS). Based on the remarkable precision and efficiency of methylation induced by the int-hpRNA, it is tempting to speculate that int-hpRNA would efficiently initiate RdDM and TGS of endogenous promoters. If so, the intron-based RNAi approach would provide a powerful tool for heritable epigenetic gene silencing. In addition, we aim to take advantage of the intron-based RNAi system to investigate the ambiguous accessibility of introns against RNA-mediated silencing. Finally, the debatable nature of the primary RdDM-trigger (sRNAs or longer RNAs) will be examined by analysing the capacity of the non-small interfering RNA-producing int-hpRNA to trigger RdDM in Dicer-like-deficient plants.