Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways

Abstract

In RNA interference (RNAi), double-stranded RNA (dsRNA) is processed into short interfering RNA (siRNA) to mediate sequence-specific gene knockdown. The genetics of plant RNAi is not understood, nor are the bases for its spreading between cells. Here, we unravel the requirements for biogenesis and action of siRNAs directing RNAi in Arabidopsis thaliana and show how alternative routes redundantly mediate this process under extreme dsRNA dosages. We found that SMD1 and SMD2, required for intercellular but not intracellular RNAi, are allelic to RDR2 and NRPD1a, respectively, previously implicated in siRNA-directed heterochromatin formation through the action of DCL3 and AGO4. However, neither DCL3 nor AGO4 is required for non-cell autonomous RNAi, uncovering a new pathway for RNAi spreading or detection in recipient cells. Finally, we show that the genetics of RNAi is distinct from that of antiviral silencing and propose that this experimental silencing pathway has a direct endogenous plant counterpart.

The Persistent Contributions of RNA to Eukaryotic Gen(om)e Architecture and Cellular Function

“…The development of deep and ultradeep sequencing methods for cellular, organ-specific, and whole transcriptomes of organisms greatly accelerated the deluge of data (Wang et al 2009). The past 15 years have surprised most of the scientific community with the incessant discovery of thousands of novel RNAs, some extending RNA species from already known classes such as snoRNAs, some establishing novel RNA classes (e.g., miRNAs and siRNAs), and some unclassified (Fire et al 1998 (Benner 1988;Wu et al 2002;Dunoyer et al 2007;Leslie 2013a). Major RNA classes are summarized in Table 1.…”

Epigenetics: heterochromatin meets RNAi

“…Likewise, in C. elegans, overexpression of Argonaute proteins predicted to be catalytically inactive are reported to enhance silencing [47]. The four Dicer isoforms of A. thaliana have been, when exposed to high doses of siRNA, reported to partially substitute for each other [73]. Hence, there is some functional redundancy between members of the same protein family.…”

“…The genome of A. thaliana has four Dicer genes, ten genes encoding Argonaute proteins and six paralogues for RNA-dependent RNA polymerases (Table 1) [47,98]. The various RNAi pathways in plants are interrelated and partially overlapping ( [62] and references therein), and accumulation of double stranded precursor RNA above a threshold value can result in Dicer enzymes from parallel pathways substituting for each other [73]. Although plants were among the earliest systems investigated for RNA-mediated silencing of transcription, the large number of RNAi enzymes combined with the high level of interconnection between different RNAi pathways have made it difficult to produce a comprehensive picture of the phenomenon.…”

“…Still a large number of enzymes involved in RNAi in plants have been identified and the complexity of the system is beginning to be dissected [73]. A common consequence of all plant RNAi pathways is RNA-dependent DNA methylation of cytosines and thereby silencing of genes at the transcriptional level [99].…”

La bataille du silence

“…Ces résultats suggèrent donc que P19 bloque très certainement la synthèse ou le mouvement du signal du silencing antiviral qui permet la destruction des ARN viraux en aval du front d'infection. En accord avec cette hypothèse, il est à noter que P19 fixe spécifiquement les sARN de 21nt produits par DCL4 qui ont été identifiés comme étant le principal, si ce n'est exclusif, signal du mouvement de cellule à cellule du RNA silencing [49,50] (Figure 3B). …”