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Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections

A Corrigendum to this article was published on 26 June 2015

Abstract

Regulation of gene expression through microRNAs (miRNAs) and antiviral defense through small interfering RNAs (siRNAs) are aspects of RNA silencing1, a process originally discovered as an unintended consequence of plant transformation by disarmed Agrobacterium tumefaciens strains2. Although RNA silencing protects cells against foreign genetic elements3, its defensive role against virulent, tumor-inducing bacteria has remained unexplored. Here, we show that siRNAs corresponding to transferred-DNA oncogenes initially accumulate in virulent A. tumefaciens–infected tissues and that RNA interference–deficient plants are hypersusceptible to the pathogen. Successful infection relies on a potent antisilencing state established in tumors whereby siRNA synthesis is specifically inhibited. This inhibition has only modest side effects on the miRNA pathway, shown here to be essential for disease development. The similarities and specificities of the A. tumefaciens RNA silencing interaction are discussed and contrasted with the situation encountered with plant viruses.

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Figure 1: Induction and suppression of RNA silencing in A. tumefaciens-infected tissues.
Figure 2: A. thaliana mutants with compromised miRNA accumulation show reduced susceptibility to A. tumefaciens.
Figure 3: Suppression of RNA silencing in tumors and calli in N. benthamiana.
Figure 4: Genetic dissection of RNAi suppression in tumors.
Figure 5: RNA blot analysis of endogenous small RNAs in leaves, stems and tumors of A. thaliana.

References

  1. Baulcombe, D. RNA silencing in plants. Nature 431, 356–363 (2004).

    CAS  Article  Google Scholar 

  2. Napoli, C., Lemieux, C. & Jorgensen, R.A. Introduction of a chimeric chalcone synthase gene into Petunia results in reversible co-suppression of homologous genes in trans. Plant Cell 2, 279–289 (1990).

    CAS  Article  Google Scholar 

  3. Voinnet, O. Induction and suppression of RNA silencing: insights from viral infections. Nat. Rev. Genet. 6, 206–220 (2005).

    CAS  Article  Google Scholar 

  4. Tzfira, T., Li, J., Lacroix, B. & Citovsky, V. Agrobacterium T-DNA integration: molecules and models. Trends Genet. 20, 375–383 (2004).

    CAS  Article  Google Scholar 

  5. Escobar, M.A. & Dandekar, A.M. Agrobacterium tumefaciens as an agent of disease. Trends Plant Sci. 8, 380–386 (2003).

    CAS  Article  Google Scholar 

  6. Gelvin, S.B. Agricultural biotechnology: gene exchange by design. Nature 433, 583–584 (2005).

    CAS  Article  Google Scholar 

  7. Bernstein, E., Caudy, A.A., Hammond, S.M. & Hannon, G.J. Role for a bidentate ribonuclease in the initiation step of RNA interference. Nature 409, 363–366 (2001).

    CAS  Article  Google Scholar 

  8. Hammond, S.M., Bernstein, E., Beach, D. & Hannon, G. An RNA-directed nuclease mediates post-transcriptional gene silencing in Drosophila cell extracts. Nature 404, 293–296 (2000).

    CAS  Article  Google Scholar 

  9. Ekwall, K. The RITS complex– a direct link between small RNA and heterochromatin. Mol. Cell 13, 304–305 (2004).

    CAS  Article  Google Scholar 

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

    Article  Google Scholar 

  11. Jacobsen, S.E., Running, M.P. & Meyerowitz, E.M. Disruption of an RNA helicase/RNAse III gene in Arabidopsis causes unregulated cell division in floral meristems. Development 126, 5231–5243 (1999).

    CAS  PubMed  Google Scholar 

  12. Yu, B. et al. Methylation as a crucial step in plant microRNA biogenesis. Science 307, 932–935 (2005).

    CAS  Article  Google Scholar 

  13. Bartel, B. & Bartel, D.P. MicroRNAs: at the root of plant development? Plant Physiol. 132, 709–717 (2003).

    CAS  Article  Google Scholar 

  14. Gasciolli, V., Mallory, A.C., Bartel, D.P. & Vaucheret, H. Partially redundant functions of Arabidopsis DICER-like enzymes and a role for DCL4 in producing trans-acting siRNAs. Curr. Biol. 15, 1494–1500 (2005).

    CAS  Article  Google Scholar 

  15. 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  Article  Google Scholar 

  16. Dalmay, T., Hamilton, A.J., Rudd, S., Angell, S. & Baulcombe, D.C. An RNA-dependent RNA polymerase gene in Arabidopsis is required for posttranscriptional gene silencing mediated by a transgene but not by a virus. Cell 101, 543–553 (2000).

    CAS  Article  Google Scholar 

  17. Chapman, E.J., Prokhnevsky, A.I., Gopinath, K., Dolja, V.V. & Carrington, J.C. Viral RNA silencing suppressors inhibit the microRNA pathway at an intermediate step. Genes Dev. 18, 1179–1186 (2004).

    CAS  Article  Google Scholar 

  18. Dunoyer, P., Lecellier, C.H., Parizotto, E.A., Himber, C. & Voinnet, O. Probing the microRNA and small interfering RNA pathways with virus-encoded suppressors of RNA silencing. Plant Cell 16, 1235–1250 (2004).

    CAS  Article  Google Scholar 

  19. Voinnet, O., Rivas, S., Mestre, P. & Baulcombe, D. An enhanced transient expression system in plants based on suppression of gene silencing by the p19 protein of tomato bushy stunt virus. Plant J. 33, 949–956 (2003).

    CAS  Article  Google Scholar 

  20. Hamilton, A.J., Voinnet, O., Chappell, L. & Baulcombe, D.C. Two classes of short interfering RNA in RNA silencing. EMBO J. 21, 4671–4679 (2002).

    CAS  Article  Google Scholar 

  21. 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  Article  Google Scholar 

  22. Nam, J., Matthysse, A.G. & Gelvin, S.B. Differences in susceptibility of Arabidopsis ecotypes to crown gall disease may result from a deficiency in T-DNA integration. Plant Cell 9, 317–333 (1997).

    CAS  Article  Google Scholar 

  23. Dalmay, T., Hamilton, A.J., Mueller, E. & Baulcombe, D.C. Potato virus X amplicons in arabidopsis mediate genetic and epigenetic gene silencing. Plant Cell 12, 369–379 (2000).

    CAS  Article  Google Scholar 

  24. Parizotto, E.A., Dunoyer, P., Rahm, N., Himber, C. & Voinnet, O. 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 (2004).

    CAS  Article  Google Scholar 

  25. Cluster, P.D., O'Dell, M., Metzlaff, M. & Flavell, R.B. Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression. Plant Mol. Biol. 32, 1197–1203 (1996).

    CAS  Article  Google Scholar 

  26. Gazzani, S., Lawerson, T., Woodward, D., Headon, R. & Sablowski, R. A link between mRNA turnover and RNA interference in Arabidopsis. Science 306, 1046–1048 (2004).

    CAS  Article  Google Scholar 

  27. Ooms, G. et al. T-DNA organization in homogeneous and heterogeneous octopine-type crown gall tissues of Nicotiana tabacum. Cell 30, 589–597 (1982).

    CAS  Article  Google Scholar 

  28. Chateau, S., Sangwan, R.S. & Sangwan-Norreel, B.S. Competence of Arabidopsis thaliana genotypes and mutants for Agrobacterium tumefaciens-mediated gene transfer: role of phytohormones. J. Exp. Bot. 51, 1961–1968 (2000).

    CAS  Article  Google Scholar 

  29. Jones-Rhoades, M.W. & Bartel, D.P. Computational identification of plant microRNAs and their targets, including a stress-induced miRNA. Mol. Cell 14, 787–799 (2004).

    CAS  Article  Google Scholar 

  30. Hood, E.E., Helmer, G.L., Fraley, R.T. & Chilton, M.D. The hypervirulence of Agrobacterium tumefaciens A281 is encoded in a region of pTiBo542 outside of T-DNA. J. Bacteriol. 168, 1291–1301 (1986).

    CAS  Article  Google Scholar 

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Acknowledgements

We thank P. Zambryski and M. Matzke for critical reading of the manuscript, S. Gelvin for providing strains and the root inoculation protocol, members of the Voinnet lab for discussions and R. Wagner's team for plant care. This work was supported by an Action thématique et incitative sur programme Jeune Chercheur award from the Centre National de la Recherche Scientifique and a postdoctoral fellowship to P.D. from the Federation of European Biochemical Societies. O.V. is recipient of a Young Investigator Award from the European Molecular Biology Organization.

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Correspondence to Olivier Voinnet.

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Supplementary information

Supplementary Fig. 1

A. thaliana plants expressing the P19 and P1-HcPro viral silencing suppressors show decreased susceptibility to A. tumefaciens. (PDF 456 kb)

Supplementary Fig. 2

Absence of secondary siRNAs in stems and tumors from GF-RNAi plants with the rdr6 mutation. (PDF 331 kb)

Supplementary Fig. 3

Optimal virus accumulation in stems of DAN2 plants carrying the rdr6 mutation, compared with tumors developing on similar DAN2 tissues. (PDF 185 kb)

Supplementary Fig. 4

Specific transcriptional repression of miR393 in tumors. (PDF 239 kb)

Supplementary Table 1

A. tumefaciens susceptibility assays in roots of the dcl2-1, dcl3-1 and rdr2-1 mutants. (PDF 807 kb)

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Dunoyer, P., Himber, C. & Voinnet, O. Induction, suppression and requirement of RNA silencing pathways in virulent Agrobacterium tumefaciens infections. Nat Genet 38, 258–263 (2006). https://doi.org/10.1038/ng1722

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