Distinct extremely abundant siRNAs associated with cosuppression in petunia

Abstract

Cosuppression is a classical form of eukaryotic post-transcriptional gene silencing. It was first reported in transgenic petunia, where a sense transgene meant to overexpress the host Chalcone Synthase-A (CHS-A) gene caused the degradation of the homologous transcripts and the loss of flower pigmentation. In this work, we used deep sequencing technology to characterize in detail the small RNA population generated from the CHS-A sequence in cosuppressed transgenic petunia. Unexpectedly, two distinct small interfering RNAs (siRNAs) were found to vastly predominate. Our demonstration that they guide prominent cleavage events in CHS-A mRNA provides compelling and previously lacking evidence of a causative association between induction of individual siRNAs and an example of cosuppression. The preferential accumulation of these siRNAs provides new insights about sense cosuppression that may apply to other natural and engineered RNA silencing events.

Distinctive profiles of small RNA couple inverted repeat-induced post-transcriptional gene silencing with endogenous RNA silencing pathways in Arabidopsis

“…5). Because the full-length CHS-A mRNA had no strong detectable sequence complementarities near the major peak for sRNA accumulation (data not shown), the peak's position appears more consistent with the elevated frequency of rare codons than with complementarity in the predicted mRNA secondary structure as suggested previously (De Paoli et al 2009;Kasai et al 2013). …”

“…In addition, the five major peaks corresponding to the At-CHS gene fragment were distributed in approximate 79-nt intervals (Fig. 3A,B), which does not fit with the 21-nt phasing observed after sequential DCL4-mediated cleavage (Napoli et al 1990;Howell et al 2007;De Paoli et al 2009;Kasai et al 2013). In fact, the positions of the peaks corresponding to the At-CHS fragment used to generate ihpRNA were the same as those of the peaks produced by sRNAs associated with uncapped RNA decay of At-CHS mRNA in the ein5/abh1-1 mutant (Gregory et al 2009).…”

“…S2). RNAi-mediated RNA decay and transitivity both involve RDR-mediated synthesis of dsRNA from single-stranded (ss) RNA templates that are likely perceived in the cell as aberrant (De Paoli et al 2009;Gregory et al 2009). Perhaps preferential accumulation of sRNA corresponding to ORFs in such aberrant mRNA ( Fig.…”

“…Therefore, we explored characteristics of the trigger sequence that might correlate with the observed peaks of sRNA accumulation. Previous reports (De Paoli et al 2009;Kasai et al 2013) suggested that secondary structures in CHALCONE SYNTHASE A (CHS-A) mRNA may trigger production of both endogenous and transgene-derived sRNAs during sense-mediated PTGS (S-PTGS) in petunia (Napoli et al 1990). Consistent with this proposal, one of the five major peaks (P2) (Fig.…”

MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs

“…The eight Arabidopsis ''TAS'' genes generate miRNA-triggered secondary siRNAs in a 21-nt ''phased'' pattern (Howell et al 2007). We previously refined and applied a computational approach to evaluate the phasing pattern of small RNAs (De Paoli et al 2009). The small RNAs identified from this algorithm are phased but do not necessarily function in trans (or even in cis); therefore, we call these phased siRNAs ''phasiRNAs.''…”