DICER-LIKE2 Plays a Primary Role in Transitive Silencing of Transgenes in Arabidopsis

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.

Developmental Defects Mediated by the P1/HC-Pro Potyviral Silencing Suppressor Are Not Due to Misregulation of AUXIN RESPONSE FACTOR 8

“…To try to reproduce that result, we used the same arf8-6 SALK line as in that study and crossed it to our established P1/HC-Pro transgenic Arabidopsis line (Mlotshwa et al, 2005(Mlotshwa et al, , 2008Endres et al, 2010). The P1/HC-Pro line is hemizygous for the turnip mosaic virus coding region, expresses high levels of the P1/HC-Pro mRNA, and has a severe developmental phenotype (Mlotshwa et al, 2005; Fig.…”

“…The paradoxical result was that the gel blot analysis in Figure 5E showed a total absence of CHS siRNAs in those plants, although Figure 5E also presented evidence that P1/HC-Pro was suppressing the hairpin transgene-induced posttranscriptional silencing. It has been well established, however, that primary siRNAs, such as those that would derive from the stem of a hairpin transgene, are not eliminated in P1/HC-Pro suppression of silencing (Johansen and Carrington, 2001;Mlotshwa et al, 2008;Zhang et al, 2008;Endres et al, 2010). Therefore, CHS siRNAs should have been present if P1/HC-Pro suppression of silencing was truly occurring, and their absence strongly suggested that some confounding factor had affected the Jay et al (2011) P1/HC-Pro studies.…”

“…On their own, however, these two transgenes do not induce transcriptional silencing (Béclin et al, 2002;Mlotshwa et al, 2008).…”

“…For the purpose of the molecular analysis, the P1/HC-Pro line we used for the cross contained two additional transgenes that provide an assay for silencing suppression (Mlotshwa et al, 2008). These additional loci are a sense uidA transgene (GUS) encoding b-glucuronidase and a hairpin transgene (hpGUS) that posttranscriptionally silences the GUS locus (Béclin et al, 2002).…”

“…2, compare lanes 1-6 with lanes 15-19). The GUS locus gives rise to a transcript that is slightly longer than the full-length hpGUS transcript; however, a prominent high M r hpGUS RNA species that also accumulates corresponds to the loop of the hairpin (Mlotshwa et al, 2008). With respect to low M r RNA species, the fully P1/HC-Pro phenotypic arf8-6 plants and the P1/HC-Pro parent show the same increased accumulation of miRNAs and of primary siRNAs, which are the siRNAs from the stem of the hpGUS transcript (Fig.…”