Movement and differential consumption of short interfering RNA duplexes underlie mobile RNA interference

Abstract

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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Fig. 1: Identical signals account for long-distance and cell-to-cell movement of SS RNAi.
Fig. 2: Long-distance and cell-to-cell movement of RNAi triggered by endogenous inverted repeats.
Fig. 3: Cell-to-cell movement of RNAi triggered by TuYV.
Fig. 4: Consumption of siRNA during movement, diagnosed by concurrent 5′-nucleotide identity and AGO loading.
Fig. 5: A model for versatile non-autonomous RNAi based on free siRNA movement coupled with AGO-mediated consumption.

Data availability

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.

References

  1. 1.

    Bologna, N. G. & Voinnet, O. The diversity, biogenesis, and activities of endogenous silencing small RNAs in Arabidopsis. Annu. Rev. Plant Biol. 65, 473–503 (2014).

    CAS  PubMed  Google Scholar 

  2. 2.

    Voinnet, O. & Baulcombe, D. C. Systemic signalling in gene silencing. Nature 389, 553 (1997).

    CAS  PubMed  Google Scholar 

  3. 3.

    Palauqui, J. C., Elmayan, T., Pollien, J. M. & Vaucheret, H. Systemic acquired silencing: transgene-specific post-transcriptional silencing is transmitted by grafting from silenced stocks to non-silenced scions. EMBO J. 16, 4738–4745 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  4. 4.

    Palauqui, J. C. et al. Frequencies, timing, and spatial patterns of co-suppression of nitrate reductase and nitrite reductase in transgenic tobacco plants. Plant Physiol. 112, 1447–1456 (1996).

    CAS  PubMed  PubMed Central  Google Scholar 

  5. 5.

    Voinnet, O., Vain, P., Angell, S. & Baulcombe, D. C. Systemic spread of sequence-specific transgene RNA degradation in plants is initiated by localized introduction of ectopic promoterless DNA. Cell 95, 177–187 (1998).

    CAS  PubMed  Google Scholar 

  6. 6.

    Molnar, A. et al. Small silencing RNAs in plants are mobile and direct epigenetic modification in recipient cells. Science 328, 872–875 (2010).

    CAS  PubMed  Google Scholar 

  7. 7.

    Brosnan, C. A. et al. Nuclear gene silencing directs reception of long-distance mRNA silencing in Arabidopsis. Proc. Natl Acad. Sci. USA 104, 14741–14746 (2007).

    CAS  PubMed  Google Scholar 

  8. 8.

    Kobayashi, K., Otegui, M. S., Krishnakumar, S., Mindrinos, M. & Zambryski, P. INCREASED SIZE EXCLUSION LIMIT 2 encodes a putative DEVH box RNA helicase involved in plasmodesmata function during Arabidopsis embryogenesis. Plant Cell 19, 1885–1897 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  9. 9.

    Rosas-Diaz, T. et al. A virus-targeted plant receptor-like kinase promotes cell-to-cell spread of RNAi. Proc. Natl Acad. Sci. USA 115, 1388–1393 (2018).

    CAS  PubMed  Google Scholar 

  10. 10.

    Vaistij, F. E., Jones, L. & Baulcombe, D. C. Spreading of RNA targeting and DNA methylation in RNA silencing requires transcription of the target gene and a putative RNA-dependent RNA polymerase. Plant Cell 14, 857–867 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  11. 11.

    Himber, C., Dunoyer, P., Moissiard, G., Ritzenthaler, C. & Voinnet, O. Transitivity-dependent and -independent cell-to-cell movement of RNA silencing. EMBO J. 22, 4523–4533 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  12. 12.

    Liu, L. & Chen, X. Intercellular and systemic trafficking of RNAs in plants. Nat. Plants 4, 869–878 (2018).

    PubMed  PubMed Central  Google Scholar 

  13. 13.

    Brosnan, C. A. & Voinnet, O. Cell-to-cell and long-distance siRNA movement in plants: mechanisms and biological implications. Curr. Opin. Plant Biol. 14, 580–587 (2011).

    CAS  PubMed  Google Scholar 

  14. 14.

    Zhang, X. et al. Mini review: revisiting mobile RNA silencing in plants. Plant Sci. 278, 113–117 (2019).

    CAS  PubMed  Google Scholar 

  15. 15.

    Pyott, D. E. & Molnar, A. Going mobile: non-cell-autonomous small RNAs shape the genetic landscape of plants. Plant Biotechnol. J. 13, 306–318 (2015).

    CAS  PubMed  Google Scholar 

  16. 16.

    Imlau, A., Truernit, E. & Sauer, N. Cell-to-cell and long-distance trafficking of the green fluorescent protein in the phloem and symplastic unloading of the protein into sink tissues. Plant Cell 11, 309–322 (1999).

    CAS  PubMed  PubMed Central  Google Scholar 

  17. 17.

    Stadler, R. & Sauer, N. The Arabidopsis thaliana AtSUC2 gene is specifically expressed in companion cells. Bot. Acta 109, 299–306 (1996).

    CAS  Google Scholar 

  18. 18.

    Stadler, R. et al. Expression of GFP-fusions in Arabidopsis companion cells reveals non-specific protein trafficking into sieve elements and identifies a novel post-phloem domain in roots. Plant J. 41, 319–331 (2005).

    CAS  PubMed  Google Scholar 

  19. 19.

    Vargason, J. M., Szittya, G., Burgyan, J. & Hall, T. M. Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115, 799–811 (2003).

    CAS  PubMed  Google Scholar 

  20. 20.

    Papp, I. et al. Evidence for nuclear processing of plant micro RNA and short interfering RNA precursors. Plant Physiol. 132, 1382–1390 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  21. 21.

    Brosnan, C. A. et al. Genome-scale, single-cell-type resolution of microRNA activities within a whole plant organ. EMBO J. 38, e100754 (2019).

    PubMed  PubMed Central  Google Scholar 

  22. 22.

    Breakfield, N. W. et al. High-resolution experimental and computational profiling of tissue-specific known and novel miRNAs in Arabidopsis. Genome Res. 22, 163–176 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

  23. 23.

    Svozil, J., Gruissem, W. & Baerenfaller, K. Meselect—a rapid and effective method for the separation of the main leaf tissue types. Front Plant Sci. 7, 1701 (2016).

    PubMed  PubMed Central  Google Scholar 

  24. 24.

    Dengler, N. & Kang, J. Vascular patterning and leaf shape. Curr. Opin. Plant Biol. 4, 50–56 (2001).

    CAS  PubMed  Google Scholar 

  25. 25.

    Henderson, I. R. et al. Dissecting Arabidopsis thaliana DICER function in small RNA processing, gene silencing and DNA methylation patterning. Nat. Genet. 38, 721–725 (2006).

    CAS  PubMed  Google Scholar 

  26. 26.

    Boissinot, S. et al. Systemic propagation of a fluorescent infectious clone of a polerovirus following inoculation by agrobacteria and aphids. Viruses 9, 166 (2017).

    PubMed Central  Google Scholar 

  27. 27.

    Mutterer, J. D. et al. Role of the beet western yellows virus readthrough protein in virus movement in Nicotiana clevelandii. J. Gen. Virol. 80, 2771–2778 (1999).

    CAS  PubMed  Google Scholar 

  28. 28.

    Brodersen, P. et al. Widespread translational inhibition by plant miRNAs and siRNAs. Science 320, 1185–1190 (2008).

    CAS  PubMed  Google Scholar 

  29. 29.

    Pazhouhandeh, M. et al. F-box-like domain in the polerovirus protein P0 is required for silencing suppressor function. Proc. Natl Acad. Sci. USA 103, 1994–1999 (2006).

    CAS  PubMed  Google Scholar 

  30. 30.

    Dunoyer, P., Himber, C., Ruiz-Ferrer, V., Alioua, A. & Voinnet, O. Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways. Nat. Genet. 39, 848–856 (2007).

    CAS  PubMed  Google Scholar 

  31. 31.

    Lakatos, L., Szittya, G., Silhavy, D. & Burgyan, J. Molecular mechanism of RNA silencing suppression mediated by p19 protein of tombusviruses. EMBO J. 23, 876–884 (2004).

    CAS  PubMed  PubMed Central  Google Scholar 

  32. 32.

    Mi, S. et al. Sorting of small RNAs into Arabidopsis argonaute complexes is directed by the 5′ terminal nucleotide. Cell 133, 116–127 (2008).

    CAS  PubMed  PubMed Central  Google Scholar 

  33. 33.

    Morel, J. B. et al. Fertile hypomorphic ARGONAUTE (ago1) mutants impaired in post-transcriptional gene silencing and virus resistance. Plant Cell 14, 629–639 (2002).

    CAS  PubMed  PubMed Central  Google Scholar 

  34. 34.

    Harvey, J. J. et al. An antiviral defense role of AGO2 in plants. PLoS ONE 6, e14639 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  35. 35.

    Sorin, C. et al. Auxin and light control of adventitious rooting in Arabidopsis require ARGONAUTE1. Plant Cell 17, 1343–1359 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  36. 36.

    Poulsen, C., Vaucheret, H. & Brodersen, P. Lessons on RNA silencing mechanisms in plants from eukaryotic argonaute structures. Plant Cell 25, 22–37 (2013).

    CAS  PubMed  PubMed Central  Google Scholar 

  37. 37.

    Derrien, B. et al. A suppressor screen for AGO1 degradation by the viral F-box P0 protein uncovers a role for AGO DUF1785 in sRNA duplex unwinding. Plant Cell 30, 1353–1374 (2018).

    CAS  PubMed  PubMed Central  Google Scholar 

  38. 38.

    Bohmert, K. et al. AGO1 defines a novel locus of Arabidopsis controlling leaf development. EMBO J. 17, 170–180 (1998).

    CAS  PubMed  PubMed Central  Google Scholar 

  39. 39.

    Allen, E., Xie, Z., Gustafson, A. M. & Carrington, J. C. MicroRNA-directed phasing during trans-acting siRNA biogenesis in plants. Cell 121, 207–221 (2005).

    CAS  PubMed  Google Scholar 

  40. 40.

    Branscheid, A. et al. SKI2 mediates degradation of RISC 5′-cleavage fragments and prevents secondary siRNA production from miRNA targets in Arabidopsis. Nucleic Acids Res. 43, 10975–10988 (2015).

    CAS  PubMed  PubMed Central  Google Scholar 

  41. 41.

    Zhang, X. et al. Plant biology. Suppression of endogenous gene silencing by bidirectional cytoplasmic RNA decay in Arabidopsis. Science 348, 120–123 (2015).

    CAS  PubMed  Google Scholar 

  42. 42.

    Axtell, M. J., Jan, C., Rajagopalan, R. & Bartel, D. P. A two-hit trigger for siRNA biogenesis in plants. Cell 127, 565–577 (2006).

    CAS  PubMed  Google Scholar 

  43. 43.

    Zhai, J. et al. MicroRNAs as master regulators of the plant NB-LRR defense gene family via the production of phased, trans-acting siRNAs. Genes Dev. 25, 2540–2553 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  44. 44.

    Yoshikawa, M., Peragine, A., Park, M. Y. & Poethig, R. S. A pathway for the biogenesis of trans-acting siRNAs in Arabidopsis. Genes Dev. 19, 2164–2175 (2005).

    CAS  PubMed  PubMed Central  Google Scholar 

  45. 45.

    Boccara, M. et al. The Arabidopsis miR472–RDR6 silencing pathway modulates PAMP- and effector-triggered immunity through the post-transcriptional control of disease resistance genes. PLoS Pathog. 10, e1003883 (2014).

    PubMed  PubMed Central  Google Scholar 

  46. 46.

    Zhang, J. F. et al. The disturbance of small RNA pathways enhanced abscisic acid response and multiple stress responses in Arabidopsis. Plant Cell Environ. 31, 562–574 (2008).

    CAS  PubMed  Google Scholar 

  47. 47.

    Deleris, A. et al. Hierarchical action and inhibition of plant Dicer-like proteins in antiviral defense. Science 313, 68–71 (2006).

    CAS  PubMed  Google Scholar 

  48. 48.

    Xie, Z. et al. Genetic and functional diversification of small RNA pathways in plants. PLoS Biol. 2, E104 (2004).

    PubMed  PubMed Central  Google Scholar 

  49. 49.

    Blevins, T. et al. Massive production of small RNAs from a non-coding region of Cauliflower mosaic virus in plant defense and viral counter-defense. Nucleic Acids Res. 39, 5003–5014 (2011).

    CAS  PubMed  PubMed Central  Google Scholar 

  50. 50.

    Curtis, M. D. & Grossniklaus, U. A gateway cloning vector set for high-throughput functional analysis of genes in planta. Plant Physiol. 133, 462–469 (2003).

    CAS  PubMed  PubMed Central  Google Scholar 

  51. 51.

    Gibeaut, D. M., Hulett, J., Cramer, G. R. & Seemann, J. R. Maximal biomass of Arabidopsis thaliana using a simple, low-maintenance hydroponic method and favorable environmental conditions. Plant Physiol. 115, 317–319 (1997).

    CAS  PubMed  PubMed Central  Google Scholar 

  52. 52.

    Tocquin, P. et al. A novel high efficiency, low maintenance, hydroponic system for synchronous growth and flowering of Arabidopsis thaliana. BMC Plant Biol. 3, 2 (2003).

    PubMed  PubMed Central  Google Scholar 

  53. 53.

    Turnbull, C. G., Booker, J. P. & Leyser, H. M. Micrografting techniques for testing long-distance signalling in Arabidopsis. Plant J. 32, 255–262 (2002).

    CAS  PubMed  Google Scholar 

  54. 54.

    Pumplin, N. et al. DNA methylation influences the expression of DICER-LIKE4 isoforms, which encode proteins of alternative localization and function. Plant Cell 28, 2786–2804 (2016).

    CAS  PubMed  PubMed Central  Google Scholar 

  55. 55.

    Bologna, N. G. et al. Nucleo-cytosolic shuttling of ARGONAUTE1 prompts a revised model of the plant microRNA pathway. Mol. Cell 69, 709–719 (2018).

    CAS  PubMed  Google Scholar 

  56. 56.

    Karimi, M., Depicker, A. & Hilson, P. Recombinational cloning with plant gateway vectors. Plant Physiol. 145, 1144–1154 (2007).

    CAS  PubMed  PubMed Central  Google Scholar 

  57. 57.

    Clough, S. J. & Bent, A. F. Floral dip: a simplified method for Agrobacterium-mediated transformation of Arabidopsis thaliana. Plant J. 16, 735–743 (1998).

    CAS  PubMed  Google Scholar 

  58. 58.

    De Felippes, F. F., Ott, F. & Weigel, D. Comparative analysis of non-autonomous effects of tasiRNAs and miRNAs in Arabidopsis thaliana. Nucleic Acids Res. 39, 2880–2889 (2011).

    PubMed  Google Scholar 

  59. 59.

    Garcia, D. et al. Ago hook and RNA helicase motifs underpin dual roles for SDE3 in antiviral defense and silencing of nonconserved intergenic regions. Mol. Cell 48, 109–120 (2012).

    CAS  PubMed  Google Scholar 

  60. 60.

    Pall, G. S., Codony-Servat, C., Byrne, J., Ritchie, L. & Hamilton, A. Carbodiimide-mediated cross-linking of RNA to nylon membranes improves the detection of siRNA, miRNA and piRNA by northern blot. Nucleic Acids Res. 35, e60 (2007).

    PubMed  PubMed Central  Google Scholar 

  61. 61.

    Chen, C. J. ncPRO-seq: a tool for annotation and profiling of ncRNAs in sRNA-seq data. Bioinformatics 28, 3147–3149 (2012).

    CAS  PubMed  Google Scholar 

  62. 62.

    Langmead, B. & Salzberg, S. L. Fast gapped-read alignment with Bowtie 2. Nat. Methods 9, 357–359 (2012).

    CAS  PubMed  PubMed Central  Google Scholar 

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Acknowledgements

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.

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Contributions

E.A.D., C.A.B. and O.V. designed the project and all experiments. E.A.D. conducted the experiments on SS and TuYV and characterized the sRNA binding affinity of the ago1-18 and ago1-42 mutant alleles. C.A.B. conducted the experiments on dcl234 grafts and IR71. A.S. conducted all of the bioinformatics. D.A. contributed all of the plant material and RNA concerning TuYV infections of dcl234 and rdr126. A.C.A. conducted the Meselect procedure on the wild-type plants, and on the ago1-18 and ago1-42 mutant alleles. F.B. contributed all data related to amiRSUL. P.L. performed the cloning. G.S. and P.E.J. contributed the SS-graft deep-sequencing data. E.A.D., C.A.B., A.S. and O.V. analysed the data. E.A.D. and C.A.B. prepared all the figures and, together with O.V., wrote the manuscript. All authors read and approved the manuscript.

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Correspondence to Olivier Voinnet.

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Supplementary Figs. 1–15, discussion and Tables 1 and 2.

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Supplementary Data 1

Full-length, unprocessed blots for Supplementary Fig. 1.

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Full-length, unprocessed blots for Supplementary Fig. 10.

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Full-length, unprocessed blots.

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Full-length, unprocessed blots.

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Full-length, unprocessed blots.

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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

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