In RNA interference (RNAi), the RNase III Dicer processes long double-stranded RNA (dsRNA) into short interfering RNA (siRNA), which, when loaded into ARGONAUTE (AGO) family proteins, execute gene silencing1. Remarkably, RNAi can act non-cell autonomously2,3: it is graft transmissible4,5,6,7, and plasmodesmata-associated proteins modulate its cell-to-cell spread8,9. Nonetheless, the molecular mechanisms involved remain ill defined, probably reflecting a disparity of experimental settings. Among other caveats, these almost invariably cause artificially enhanced movement via transitivity, whereby primary RNAi-target transcripts are converted into further dsRNA sources of secondary siRNA5,10,11. Whether siRNA mobility naturally requires transitivity and whether it entails the same or distinct signals for cell-to-cell versus long-distance movement remains unclear, as does the identity of the mobile signalling molecules themselves. Movement of long single-stranded RNA, dsRNA, free/AGO-bound secondary siRNA or primary siRNA have all been advocated12,13,14,15; however, an entity necessary and sufficient for all known manifestations of plant mobile RNAi remains to be ascertained. Here, we show that the same primary RNAi signal endows both vasculature-to-epidermis and long-distance silencing movement from three distinct RNAi sources. The mobile entities are AGO-free primary siRNA duplexes spreading length and sequence independently. However, their movement is accompanied by selective siRNA depletion reflecting the AGO repertoires of traversed cell types. Coupling movement with this AGO-mediated consumption process creates qualitatively distinct silencing territories, potentially enabling unlimited spatial gene regulation patterns well beyond those granted by mere gradients.
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All sequencing data files have been deposited onto the Gene Expression Omnibus under the accession numbers GSE112885, GSE112929, GSE113029 and GSE143746. Full-length, unprocessed blots were deposited at the Mendeley database accessible at https://doi.org/10.17632/s6fz43svsz.1. Source data are provided with this paper.
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We thank the Voinnet laboratory members for fruitful discussions, A. Imboden for plant care, V. Ziegler-Graff for providing the TuYV wild-type strain, V. Brault for providing the TuYV–GFP strain and comments on the manuscript, H. Vaucheret for providing seeds of ago1-18 and ago1-42, and the ETH ScopeM unit for providing the microscopy facility. This work was supported by a Marie Curie Intra-European Fellowship for career development (FP7-PEOPLE-IEF; number 623826) attributed to E.A.D., an EMBO Long-Term Fellowship (ALTF 728-2009) to C.A.B., and a European Research Council advanced grant (Frontiers of RNAi-II; number 323071) to O.V.
The authors declare no competing interests.
Publisher’s note Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary Figs. 1–15, discussion and Tables 1 and 2.
Full-length, unprocessed blots for Supplementary Fig. 1.
Full-length, unprocessed blots for Supplementary Fig. 3.
Full-length, unprocessed blots for Supplementary Fig. 4.
Full-length, unprocessed blots for Supplementary Fig. 8.
Full-length, unprocessed blots for Supplementary Fig. 10.
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Devers, E.A., Brosnan, C.A., Sarazin, A. et al. Movement and differential consumption of short interfering RNA duplexes underlie mobile RNA interference. Nat. Plants 6, 789–799 (2020). https://doi.org/10.1038/s41477-020-0687-2