Abstract
Dicer-like (DCL) enzymes play a pivotal role in RNA silencing in plants, processing the long double-stranded RNA (dsRNA) that triggers silencing into the primary short interfering RNAs (siRNAs) that mediate it. The siRNA population can be augmented and silencing amplified via transitivity, an RNA-dependent RNA polymerase (RDR)-dependent pathway that uses the target RNA as substrate to generate secondary siRNAs. Here we report that Arabidopsis DCL2–but not DCL4-is required for transitivity in cell-autonomous, post-transcriptional silencing of transgenes. An insertion mutation in DCL2 blocked sense transgene-induced silencing and eliminated accumulation of the associated RDR-dependent siRNAs. In hairpin transgene-induced silencing, the dcl2 mutation likewise eliminated accumulation of secondary siRNAs and blocked transitive silencing, but did not block silencing mediated by primary siRNAs. Strikingly, in all cases, the dcl2 mutation eliminated accumulation of all secondary siRNAs, including those generated by other DCL enzymes. In contrast, mutations in DCL4 promoted a dramatic shift to transitive silencing in the case of the hairpin transgene and enhanced silencing induced by the sense transgene. Suppression of hairpin and sense transgene silencing by the P1/HC-Pro and P38 viral suppressors was associated with elimination of secondary siRNA accumulation, but the suppressors did not block processing of the stem of the hairpin transcript into primary siRNAs. Thus, these viral suppressors resemble the dcl2 mutation in their effects on siRNA biogenesis. We conclude that DCL2 plays an essential, as opposed to redundant, role in transitive silencing of transgenes and may play a more important role in silencing of viruses than currently thought.
Highlights
RNA silencing is an ancient network of highly related pathways that repress gene expression in eukaryotic organisms by means of small regulatory RNAs [1,2,3,4,5]
To identify which DCL genes are required for sense transgeneinduced silencing, we crossed the L1 line with dcl mutant lines and examined the accumulation of GUS mRNA and short interfering RNAs (siRNAs) in F2 progeny that were homozygous for the dcl mutation and carried at least one copy of the L1 GUS transgene
This propensity of the L1 locus to become transcriptionally silenced in the presence of additional copies of the cauliflower mosaic virus (CaMV) promoter highlights the importance of examining siRNA accumulation before concluding that absence of GUS mRNA or activity indicates that the locus is posttranscriptionally silenced
Summary
RNA silencing is an ancient network of highly related pathways that repress gene expression in eukaryotic organisms by means of small regulatory RNAs [1,2,3,4,5]. The mechanism is triggered by dsRNA and targets any related RNA (post-transcriptional pathways) or DNA (transcriptional gene silencing). Key steps in the process are: 1) the dsRNA trigger is cut into 21–24 nucleotide (nt) short-interfering (si) RNA duplexes by a ribonuclease III-like enzyme termed Dicer, 2) one strand of the siRNA duplex associates with an argonaute-like protein to form the core of a silencing effector complex, and 3) the siRNA directs the complex to complementary genetic elements. C. elegans, and fungi amplify silencing via transitivity, a pathway that produces dsRNA and additional siRNAs from the targeted mRNA [6,7]. Transitivity increases the siRNA population, and degrades the target mRNA in the process of making secondary siRNAs, thereby amplifying silencing in two ways. Transitive silencing is important in plants, where it is essential for some types of transgene-induced silencing [11] and thought to play a key role in defense against viruses [12,13]
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