Letter

Neutralization of mobile antiviral small RNA through peroxisomal import

  • Nature Plants 3, Article number: 17094 (2017)
  • doi:10.1038/nplants.2017.94
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Abstract

In animals, certain viral proteins are targeted to peroxisomes to dampen the antiviral immune response mediated by these organelles1,​2,​3. In plants, RNA interference (RNAi) mediated by small interfering (si)RNA is the main antiviral defence mechanism. To protect themselves against the cell- and non-cell autonomous effects of RNAi, viruses produce viral suppressors of RNA silencing (VSR)4, whose study is crucial to properly understand the biological cycle of plant viruses and potentially find new solutions to control these pathogens. By combining biochemical approaches, cell-specific inhibition of RNAi movement and peroxisome isolation, we show here that one such VSR, the peanut clump virus (PCV)-encoded P15, isolates siRNA from the symplasm by delivering them into the peroxisomal matrix. Infection with PCV lacking this ability reveals that piggybacking of these VSR-bound nucleic acids into peroxisomes potentiates viral systemic movement by preventing the spread of antiviral siRNA. Collectively, these results highlight organellar confinement of antiviral molecules as a novel pathogenic strategy that may have its direct counterpart in other plant and animal viruses.

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References

  1. 1.

    et al. Activation of type I and III interferon response by mitochondrial and peroxisomal MAVS and inhibition by hepatitis C virus. PLoS Pathog. 11, e1005264 (2015).

  2. 2.

    et al. Hepatitis C virus NS3-4A inhibits the peroxisomal MAVS-dependent antiviral signalling response. J. Cell. Mol. Med. 20, 750–757 (2016).

  3. 3.

    et al. Peroxisomes are platforms for cytomegalovirus’ evasion from the cellular immune response. Sci. Rep. 6, 26028 (2016).

  4. 4.

    & RNA silencing and its suppression novel insights from in planta analyses. Trends Plant Sci. 18, 382–392 (2013).

  5. 5.

    et al. Peroxisomes are signaling platforms for antiviral innate immunity. Cell 141, 668–681 (2010).

  6. 6.

    , & Identification of a type 1 peroxisomal targeting signal in a viral protein and demonstration of its targeting to the organelle. J. Virol. 76, 2543–2547 (2002).

  7. 7.

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

  8. 8.

    et al. The 21-nucleotide, but not 22-nucleotide, viral secondary small interfering RNAs direct potent antiviral defense by two cooperative Argonautes in Arabidopsis thaliana. Plant Cell 23, 1625–1638 (2011).

  9. 9.

    et al. Functional analysis of three Arabidopsis ARGONAUTES using slicer-defective mutants. Plant Cell 24, 3613–3629 (2012).

  10. 10.

    et al. Pattern formation via small RNA mobility. Genes Dev. 23, 549–554 (2009).

  11. 11.

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

  12. 12.

    , , & In situ characterization of Cymbidium ringspot tombusvirus infection-induced posttranscriptional gene silencing in Nicotiana benthamiana. J. Virol. 77, 6082–6086 (2003).

  13. 13.

    et al. Identification, subcellular localization and some properties of a cysteine-rich suppressor of gene silencing encoded by peanut clump virus. Plant J. 29, 555–567 (2002).

  14. 14.

    , , , & Intra- and intercellular RNA interference in Arabidopsis thaliana requires components of the microRNA and heterochromatic silencing pathways. Nat. Genet. 39, 848–856 (2007).

  15. 15.

    , , & Size selective recognition of siRNA by an RNA silencing suppressor. Cell 115, 799–811 (2003).

  16. 16.

    et al. Arabidopsis RNA-dependent RNA polymerases and Dicer-like proteins in antiviral defense and small interfering RNA biogenesis during Turnip Mosaic Virus infection. Plant Cell 22, 481–496 (2010).

  17. 17.

    et al. Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein. Genes Dev. 24, 904–915 (2010).

  18. 18.

    , & Uniqueness of the mechanism of protein import into the peroxisome matrix: transport of folded, co-factor-bound and oligomeric proteins by shuttling receptors. Biochim. Biophys. Acta 1763, 1552–1564 (2006).

  19. 19.

    et al. Roles and programming of Arabidopsis ARGONAUTE proteins during Turnip Mosaic Virus infection. PLoS Pathog. 11, e1004755 (2015).

  20. 20.

    , , , & Long-distance movement, virulence, and RNA silencing suppression controlled by a single protein in hordei- and potyviruses: complementary functions between virus families. J. Virol. 76, 12981–12991 (2002).

  21. 21.

    Viruses exploiting peroxisomes. Curr. Opin. Microbiol. 14, 458–469 (2011).

  22. 22.

    , , , & The influenza A virus NS1 protein binds small interfering RNAs and suppresses RNA silencing in plants. J. Gen. Virol. 85(Pt 4), 983–991 (2004).

  23. 23.

    et al. Interferon antagonist proteins of influenza and vaccinia viruses are suppressors of RNA silencing. Proc. Natl Acad. Sci. USA 101, 1350–1355 (2004).

  24. 24.

    , , & DICER-LIKE 4 functions in trans-acting small interfering RNA biogenesis and vegetative phase change in Arabidopsis thaliana. Proc. Natl Acad. Sci. USA 102, 12984–12989 (2005).

  25. 25.

    & In planta Agrobacterium-mediated transformation of adult Arabidopsis thaliana plants by vacuum infiltration. Methods Mol. Biol. 82, 259–266 (1998).

  26. 26.

    , , & Two classes of short interfering RNA in RNA silencing. EMBO J. 21, 4671–4679 (2002).

  27. 27.

    , & Virus-induced gene silencing in tomato. Plant J. 31, 777–786 (2002).

  28. 28.

    , & Nonsense-mediated decay serves as a general viral restriction mechanism in plants. Cell Host. Microbe. 16, 391–402 (2014).

  29. 29.

    & Solubilization of plant membrane proteins for analysis by two-dimensional gel electrophoresis. Plant Physiol. 81, 802–806 (1986).

  30. 30.

    , , & Higher plant mitochondria encode an homolog of the nuclear-coded 30 kDa subunit of bovine mitochondrial complex I. Eur. J. Biochem. 217, 831–838 (1993).

  31. 31.

    , & Biochemical specialization within Arabidopsis RNA silencing pathways. Mol. Cell 19, 421–428 (2005).

  32. 32.

    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).

  33. 33.

    & Isolation of leaf peroxisomes from Arabidopsis for organelle proteome analyses. Methods Mol. Biol. 1072, 541–552 (2014).

  34. 34.

    et al. A transmitting tissue- and pollen-expressed protein from sunflower with sequence similarity to the human RTP protein. Plant Sci. 129, 191–202 (1997).

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Acknowledgements

This work was supported by a research grant from Agence Nationale de la Recherche (ANR-14-CE19-0014-01). It was also performed under the framework of the LABEX: ANR-10-LABX-0036_NETRNA and benefits from a funding from the state managed by the French National Research Agency as part of the Investments for the future program. We deeply thank S. Reumann for advice on plant peroxisome isolation and S. Bouzoubaa for advice on PCV purification.

Author information

Affiliations

  1. Institut de Biologie Moléculaire des Plantes du CNRS, UPR2357, Université de Strasbourg, F-67000 Strasbourg, France

    • M. Incarbone
    • , A. Zimmermann
    • , M. Erhardt
    • , F. Michel
    •  & P. Dunoyer
  2. Institut de Biologie Moléculaire et Cellulaire du CNRS, Plateforme Protéomique Strasbourg – Esplanade, FRC1589, F-67000 Strasbourg, France

    • P. Hammann

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Contributions

M.I. and P.D. designed the experiments. M.I. performed transgene construction, plant transformation and manipulation, viral purification and infection, immunoprecipitation and peroxisome isolation. M.I. and A.Z. performed RNA and protein extraction, RNA and protein gel blot analysis with the assistance of F.M. P.H. performed MS-MS protein analyses, while M.I. and M.E. performed immunohistochemistry. P.D. and M.I. wrote the manuscript and prepared the figures.

Competing interests

The authors declare no competing financial interests.

Corresponding author

Correspondence to P. Dunoyer.

Supplementary information

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  1. 1.

    Supplementary Information

    Supplementary Methods, Supplementary References, Supplementary Figures 1–10, Supplementary Source Data, Supplementary Raw Data 1–10.

Excel files

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    Supplementary Table 1

    Materials and Methods.