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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.

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Figure 1: Effects of key components of the miRNA pathway on SUL silencing.
Figure 2: Distinct dcl4 mutations uncouple production of hairpin-derived and viral-derived 21-nt siRNAs.
Figure 3: Effects of the rdr2 and nrpd1a mutations on SUL silencing.
Figure 4: The drb4 mutation uncovers alternative pathways for SUL silencing.
Figure 5: RNAi-competent siRNAs are loaded into AGO1.
Figure 6: A speculative model for intra- and intercellular RNAi in SUC-SUL plants.

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Acknowledgements

The SUC-GFP line was a gift of N. Sauer (University of Erlangen). TRV-PDS was provided by S. Macfarlane (Scottish Research Crop Institute). Antiserum to GFP was a gift of D. Gilmer (Institut de Biologie Moléculaire des Plantes-Centre National de la Recherche Scientifique). We thank members of the Voinnet laboratory for critical reading of the manuscript and R. Wagner's team for plant care. This work was supported by the European Molecular Biology Organization Young Investigator Grant to O.V. and by a Marie Curie Intra-European Fellowship (number 041419) to V.R-F.

Author information

Authors and Affiliations

Authors

Contributions

O.V. and P.D. designed this study; P.D., C.H. and V.R.F. performed the experiments; M.A. carried out DNA sequencing and O.V. and P.D. wrote the paper.

Corresponding author

Correspondence to Olivier Voinnet.

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The authors declare no competing financial interests.

Supplementary information

Supplementary Fig. 1

Splicing defects in the dcl4-9 allele. (PDF 146 kb)

Supplementary Fig. 2

The effects of dcl2-dcl3 and dcl1-dcl2-dcl3-dcl4 combination mutations on SUL silencing. (PDF 270 kb)

Supplementary Fig. 3

Accumulation of the hairpin section of the SUL transgene in triple and quadruple dcl mutant backgrounds (PDF 210 kb)

Supplementary Fig. 4

Side-by-side comparison of siRNA levels produced during RNAi of a GFP transgene in Arabidopsis, as triggered by the depicted GF-FG IR construct, driven either by the strong 35S promoter (35S transgenic line) or by the SUC promoter (S1 and S2 independent transgenic lines). (PDF 181 kb)

Supplementary Fig. 5

VIGS and SUL silencing phenotypes, and accumulation of viral and endogenous siRNAs in various RNA silencing mutants of Arabidopsis. (PDF 519 kb)

Supplementary Fig. 6

Lack of detectable secondary siRNA production from the 5′ and 3′ portions of the endogenous SUL transcript (At4g18480). (PDF 131 kb)

Supplementary Table 1

Primer sequences used for RDR2 and NRPD1α amplification. (PDF 21 kb)

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Dunoyer, P., Himber, C., Ruiz-Ferrer, V. et al. Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways. Nat Genet 39, 848–856 (2007). https://doi.org/10.1038/ng2081

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