1887

Abstract

The cysteine-rich 16K protein of tobacco rattle virus (TRV), the type member of the genus is known to suppress RNA silencing. However, the mechanism of action of the 16K suppressor is not well understood. In this study, we used a GFP-based sensor strategy and an -mediated transient assay in to show that 16K was unable to inhibit the activity of existing small interfering RNA (siRNA)- and microRNA (miRNA)-programmed RNA-induced silencing effector complexes (RISCs). In contrast, 16K efficiently interfered with formation of miRNA- and siRNA-guided RISCs, thus preventing cleavage of target RNA. Interestingly, we found that transiently expressed endogenous miR399 and miR172 directed sequence-specific silencing of complementary sequences of viral origin. 16K failed to bind small RNAs, although it interacted with ARGONAUTE 4, as revealed by bimolecular fluorescence complementation and immunoprecipitation assays. Site-directed mutagenesis demonstrated that highly conserved cysteine residues within the N-terminal and central regions of the 16K protein are required for protein stability and/or RNA silencing suppression.

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2016-01-01
2019-12-06
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References

  1. Andika I. B., Kondo H., Nishiguchi M., Tamada T.. ( 2012;). The cysteine-rich proteins of beet necrotic yellow vein virus and tobacco rattle virus contribute to efficient suppression of silencing in roots. J Gen Virol 93: 1841–1850 [CrossRef] [PubMed].
    [Google Scholar]
  2. Azevedo J., Garcia D., Pontier D., Ohnesorge S., Yu A., Garcia S., Braun L., Bergdoll M., Hakimi M. A., other authors. ( 2010;). Argonaute quenching and global changes in Dicer homeostasis caused by a pathogen-encoded GW repeat protein. Genes Dev 24: 904–915 [CrossRef] [PubMed].
    [Google Scholar]
  3. Bhattacharjee S., Zamora A., Azhar M. T., Sacco M. A., Lambert L. H., Moffett P.. ( 2009;). Virus resistance induced by NB-LRR proteins involves Argonaute4-dependent translational control. Plant J 58: 940–951 [CrossRef] [PubMed].
    [Google Scholar]
  4. Brodersen P., Voinnet O.. ( 2006;). The diversity of RNA silencing pathways in plants. Trends Genet 22: 268–280 [CrossRef] [PubMed].
    [Google Scholar]
  5. Brodersen P., Voinnet O.. ( 2009;). Revisiting the principles of microRNA target recognition and mode of action. Nat Rev Mol Cell Biol 10: 141–148 [CrossRef] [PubMed].
    [Google Scholar]
  6. Burgyán J., Havelda Z.. ( 2011;). Viral suppressors of RNA silencing. Trends Plant Sci 16: 265–272 [CrossRef] [PubMed].
    [Google Scholar]
  7. Canto T., Uhrig J. F., Swanson M., Wright K. M., MacFarlane S. A.. ( 2006;). Translocation of Tomato bushy stunt virus P19 protein into the nucleus by ALY proteins compromises its silencing suppressor activity. J Virol 80: 9064–9072 [CrossRef] [PubMed].
    [Google Scholar]
  8. Carbonell A., Carrington J. C.. ( 2015;). Antiviral roles of plant ARGONAUTES. Curr Opin Plant Biol 27: 111–117 [CrossRef] [PubMed].
    [Google Scholar]
  9. Chapman E. J., Prokhnevsky A. I., Gopinath K., Dolja V. V., Carrington J. C.. ( 2004;). Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev 18: 1179–1186 [CrossRef] [PubMed].
    [Google Scholar]
  10. Chen X.. ( 2004;). A microRNA as a translational repressor of APETALA2 in Arabidopsis flower development. Science 303: 2022–2025 [CrossRef] [PubMed].
    [Google Scholar]
  11. Ciomperlik J. J., Omarov R. T., Scholthof H. B.. ( 2011;). An antiviral RISC isolated from Tobacco rattle virus-infected plants. Virology 412: 117–124 [CrossRef] [PubMed].
    [Google Scholar]
  12. Csorba T., Burgyán J.. ( 2011;). Gel mobility shift assays for RNA binding viral RNAi suppressors. Methods Mol Biol 721: 245–252 [CrossRef] [PubMed].
    [Google Scholar]
  13. Deng X., Kelloniemi J., Haikonen T., Vuorinen A. L., Elomaa P., Teeri T. H., Valkonen J. P.. ( 2013;). Modification of Tobacco rattle virus RNA1 to serve as a VIGS vector reveals that the 29K movement protein is an RNA silencing suppressor of the virus. Mol Plant Microbe Interact 26: 503–514 [CrossRef] [PubMed].
    [Google Scholar]
  14. Diao A., Chen J., Ye R., Zheng T., Yu S., Antoniw J. F., Adams M. J.. ( 1999;). Complete sequence and genome properties of Chinese wheat mosaic virus, a new furovirus from China. J Gen Virol 80: 1141–1145 [CrossRef] [PubMed].
    [Google Scholar]
  15. Donaire L., Barajas D., Martínez-García B., Martínez-Priego L., Pagán I., Llave C.. ( 2008;). Structural and genetic requirements for the biogenesis of tobacco rattle virus-derived small interfering RNAs. J Virol 82: 5167–5177 [CrossRef] [PubMed].
    [Google Scholar]
  16. Donald R. G., Jackson A. O.. ( 1994;). The barley stripe mosaic virus γb gene encodes a multifunctional cysteine-rich protein that affects pathogenesis. Plant Cell 6: 1593–1606 [PubMed].
    [Google Scholar]
  17. Dunoyer P., Pfeffer S., Fritsch C., Hemmer O., Voinnet O., Richards K. E.. ( 2002;). Identification, subcellular localization and some properties of a cysteine-rich suppressor of gene silencing encoded by peanut clump virus. Plant J 29: 555–567 [CrossRef] [PubMed].
    [Google Scholar]
  18. Fernández-Calvino L., Osorio S., Hernández M. L., Hamada I. B., del Toro F. J., Donaire L., Yu A., Bustos R., Fernie A. R., other authors. ( 2014;). Virus-induced alterations in primary metabolism modulate susceptibility to Tobacco rattle virus in Arabidopsis. Plant Physiol 166: 1821–1838 [CrossRef] [PubMed].
    [Google Scholar]
  19. Fernández-Calvino L., Guzmán-Benito I., Del Toro F. J., Donaire L., Castro-Sanz A. B., Ruíz-Ferrer V., Llave C.. ( 2015;). Activation of senescence-associated Dark-inducible (DIN) genes during infection contributes to enhanced susceptibility to plant viruses. Mol Plant Pathol [CrossRef].
    [Google Scholar]
  20. Garcia-Ruiz H., Carbonell A., Hoyer J. S., Fahlgren N., Gilbert K. B., Takeda A., Giampetruzzi A., Garcia Ruiz M. T., McGinn M. G., other authors. ( 2015;). Roles and programming of Arabidopsis ARGONAUTE proteins during Turnip mosaic virus infection. PLoS Pathog 11: e1004755 [CrossRef] [PubMed].
    [Google Scholar]
  21. Ghazala W., Waltermann A., Pilot R., Winter S., Varrelmann M.. ( 2008;). Functional characterization and subcellular localization of the 16K cysteine-rich suppressor of gene silencing protein of tobacco rattle virus. J Gen Virol 89: 1748–1758 [CrossRef] [PubMed].
    [Google Scholar]
  22. Giner A., Lakatos L., García-Chapa M., López-Moya J. J., Burgyán J.. ( 2010;). Viral protein inhibits RISC activity by argonaute binding through conserved WG/GW motifs. PLoS Pathog 6: e1000996 [CrossRef] [PubMed].
    [Google Scholar]
  23. González I., Martínez L., Rakitina D. V., Lewsey M. G., Atencio F. A., Llave C., Kalinina N. O., Carr J. P., Palukaitis P., Canto T.. ( 2010;). Cucumber mosaic virus 2b protein subcellular targets and interactions: their significance to RNA silencing suppressor activity. Mol Plant Microbe Interact 23: 294–303 [CrossRef] [PubMed].
    [Google Scholar]
  24. Hamera S., Song X., Su L., Chen X., Fang R.. ( 2012;). Cucumber mosaic virus suppressor 2b binds to AGO4-related small RNAs and impairs AGO4 activities. Plant J 69: 104–115 [CrossRef] [PubMed].
    [Google Scholar]
  25. Johansen L. K., Carrington J. C.. ( 2001;). Silencing on the spot. Induction and suppression of RNA silencing in the Agrobacterium-mediated transient expression system. Plant Physiol 126: 930–938 [CrossRef] [PubMed].
    [Google Scholar]
  26. Jones-Rhoades M. W., Bartel D. P.. ( 2004;). Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol Cell 14: 787–799 [CrossRef] [PubMed].
    [Google Scholar]
  27. Karlowski W. M., Zielezinski A., Carrère J., Pontier D., Lagrange T., Cooke R.. ( 2010;). Genome-wide computational identification of WG/GW Argonaute-binding proteins in Arabidopsis. Nucleic Acids Res 38: 4231–4245 [CrossRef] [PubMed].
    [Google Scholar]
  28. Kasschau K. D., Xie Z., Allen E., Llave C., Chapman E. J., Krizan K. A., Carrington J. C.. ( 2003;). P1/HC-Pro, a viral suppressor of RNA silencing, interferes with Arabidopsis development and miRNA unction. Dev Cell 4: 205–217 [CrossRef] [PubMed].
    [Google Scholar]
  29. Lakatos L., Csorba T., Pantaleo V., Chapman E. J., Carrington J. C., Liu Y. P., Dolja V. V., Calvino L. F., López-Moya J. J., Burgyán J.. ( 2006;). Small RNA binding is a common strategy to suppress RNA silencing by several viral suppressors. EMBO J 25: 2768–2780 [CrossRef] [PubMed].
    [Google Scholar]
  30. Li J., Reichel M., Millar A. A.. ( 2014;). Determinants beyond both complementarity and cleavage govern microR159 efficacy in Arabidopsis. PLoS Genet 10: e1004232 [CrossRef] [PubMed].
    [Google Scholar]
  31. Lin S. S., Wu H. W., Elena S. F., Chen K. C., Niu Q. W., Yeh S. D., Chen C. C., Chua N. H.. ( 2009;). Molecular evolution of a viral non-coding sequence under the selective pressure of amiRNA-mediated silencing. PLoS Pathog 5: e1000312 [CrossRef] [PubMed].
    [Google Scholar]
  32. Liu D. H., Robinson D. J., Duncan G. H., Harrison B. D.. ( 1991;). Nuclear location of the 16K non-structural protein of tobacco rattle virus. J Gen Virol 72: 1811–1817 [CrossRef] [PubMed].
    [Google Scholar]
  33. Liu H., Reavy B., Swanson M., MacFarlane S. A.. ( 2002;). Functional replacement of the tobacco rattle virus cysteine-rich protein by pathogenicity proteins from unrelated plant viruses. Virology 298: 232–239 [CrossRef] [PubMed].
    [Google Scholar]
  34. Llave C.. ( 2004;). MicroRNAs: more than a role in plant development?. Mol Plant Pathol 5: 361–366 [CrossRef] [PubMed].
    [Google Scholar]
  35. Llave C., Xie Z., Kasschau K. D., Carrington J. C.. ( 2002;). Cleavage of Scarecrow-like mRNA targets directed by a class of Arabidopsis miRNA. Science 297: 2053–2056 [CrossRef] [PubMed].
    [Google Scholar]
  36. Ma X., Nicole M. C., Meteignier L. V., Hong N., Wang G., Moffett P.. ( 2015;). Different roles for RNA silencing and RNA processing components in virus recovery and virus-induced gene silencing in plants. J Exp Bot 66: 919–932 [CrossRef] [PubMed].
    [Google Scholar]
  37. Macfarlane S. A.. ( 2010;). Tobraviruses–plant pathogens and tools for biotechnology. Mol Plant Pathol 11: 577–583 [CrossRef] [PubMed].
    [Google Scholar]
  38. Martín-Hernández A. M., Baulcombe D. C.. ( 2008;). Tobacco rattle virus 16-kilodalton protein encodes a suppressor of RNA silencing that allows transient viral entry in meristems. J Virol 82: 4064–4071 [CrossRef] [PubMed].
    [Google Scholar]
  39. Martínez-Priego L., Donaire L., Barajas D., Llave C.. ( 2008;). Silencing suppressor activity of the Tobacco rattle virus-encoded 16-kDa protein and interference with endogenous small RNA-guided regulatory pathways. Virology 376: 346–356 [CrossRef] [PubMed].
    [Google Scholar]
  40. Niu Q. W., Lin S. S., Reyes J. L., Chen K. C., Wu H. W., Yeh S. D., Chua N. H.. ( 2006;). Expression of artificial microRNAs in transgenic Arabidopsis thaliana confers virus resistance. Nat Biotechnol 24: 1420–1428 [CrossRef] [PubMed].
    [Google Scholar]
  41. Palatnik J. F., Allen E., Wu X., Schommer C., Schwab R., Carrington J. C., Weigel D.. ( 2003;). Control of leaf morphogenesis by microRNAs. Nature 425: 257–263 [CrossRef] [PubMed].
    [Google Scholar]
  42. Pantaleo V., Szittya G., Burgyán J.. ( 2007;). Molecular bases of viral RNA targeting by viral small interfering RNA-programmed RISC. J Virol 81: 3797–3806 [CrossRef] [PubMed].
    [Google Scholar]
  43. Parizotto E. A., Dunoyer P., Rahm N., Himber C., Voinnet O.. ( 2004;). In vivo investigation of the transcription, processing, endonucleolytic activity, and functional relevance of the spatial distribution of a plant miRNA. Genes Dev 18: 2237–2242 [CrossRef] [PubMed].
    [Google Scholar]
  44. Pérez-Cañamás M., Hernández C.. ( 2015;). Key importance of small RNA binding for the activity of a glycine-tryptophan (GW) motif-containing viral suppressor of RNA silencing. J Biol Chem 290: 3106–3120 [CrossRef] [PubMed].
    [Google Scholar]
  45. Qi Y., He X., Wang X. J., Kohany O., Jurka J., Hannon G. J.. ( 2006;). Distinct catalytic and non-catalytic roles of ARGONAUTE4 in RNA-directed DNA methylation. Nature 443: 1008–1012 [CrossRef] [PubMed].
    [Google Scholar]
  46. Qu F., Ye X., Morris T. J.. ( 2008;). Arabidopsis DRB4, AGO1, AGO7, and RDR6 participate in a DCL4-initiated antiviral RNA silencing pathway negatively regulated by DCL1. Proc Natl Acad Sci U S A 105: 14732–14737 [CrossRef] [PubMed].
    [Google Scholar]
  47. Schott G., Mari-Ordonez A., Himber C., Alioua A., Voinnet O., Dunoyer P.. ( 2012;). Differential effects of viral silencing suppressors on siRNA and miRNA loading support the existence of two distinct cellular pools of ARGONAUTE1. EMBO J 31: 2553–2565 [CrossRef] [PubMed].
    [Google Scholar]
  48. Schwab R., Palatnik J. F., Riester M., Schommer C., Schmid M., Weigel D.. ( 2005;). Specific effects of microRNAs on the plant transcriptome. Dev Cell 8: 517–527 [CrossRef] [PubMed].
    [Google Scholar]
  49. Silhavy D., Molnár A., Lucioli A., Szittya G., Hornyik C., Tavazza M., Burgyán J.. ( 2002;). A viral protein suppresses RNA silencing and binds silencing-generated, 21- to 25-nucleotide double-stranded RNAs. EMBO J 21: 3070–3080 [CrossRef] [PubMed].
    [Google Scholar]
  50. Simón-Mateo C., García J. A.. ( 2006;). MicroRNA-guided processing impairs Plum pox virus replication, but the virus readily evolves to escape this silencing mechanism. J Virol 80: 2429–2436 [CrossRef] [PubMed].
    [Google Scholar]
  51. Szittya G., Molnár A., Silhavy D., Hornyik C., Burgyán J.. ( 2002;). Short defective interfering RNAs of tombusviruses are not targeted but trigger post-transcriptional gene silencing against their helper virus. Plant Cell 14: 359–372 [CrossRef] [PubMed].
    [Google Scholar]
  52. Te J., Melcher U., Howard A., Verchot-Lubicz J.. ( 2005;). Soilborne wheat mosaic virus (SBWMV) 19K protein belongs to a class of cysteine rich proteins that suppress RNA silencing. Virol J 2: 18 [CrossRef] [PubMed].
    [Google Scholar]
  53. Todesco M., Rubio-Somoza I., Paz-Ares J., Weigel D.. ( 2010;). A collection of target mimics for comprehensive analysis of microRNA function in Arabidopsis thaliana. PLoS Genet 6: e1001031 [CrossRef] [PubMed].
    [Google Scholar]
  54. Vaucheret H.. ( 2008;). Plant ARGONAUTES. Trends Plant Sci 13: 350–358 [CrossRef] [PubMed].
    [Google Scholar]
  55. Yelina N. E., Savenkov E. I., Solovyev A. G., Morozov S. Y., Valkonen J. P.. ( 2002;). 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 [CrossRef] [PubMed].
    [Google Scholar]
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