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Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway

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Abstract

Previously, we had shown that stable expression of a hairpin RNA sharing homology with the coat protein (CP) of the Cucumber mosaic virus (CMV) (hpRNACMV) produced CMV resistant Nicotiana tabacum plants. However, only 17% of the hpRNACMV-expressing plants generated substantial amounts of siRNAs that mediated CMV resistance (siRNAsCMV). Here, we demonstrate that the transcription of a hpRNACMV per se is not sufficient to trigger cytoplasmic and nuclear RNAi. A multiple-transgene copy line showed a strong resistance phenotype. Segregation of individual copies revealed that in one locus, the transgene-produced hpRNACMV transcript was processed into 21-nt and 24-nt siRNAsCMV and lines containing this locus were resistant. At a second locus, where the transgene was shown to be transcribed, no siRNAsCMV were produced and lines harbouring only this locus were susceptible. In addition, the second locus failed to trigger de novo RNA-directed DNA methylation (RdDM) in cis, of its cognate sequence. However, after being induced in trans, methylation in the transcribed region of the transgene was maintained in both CG and CHG residues. Sequence-specific maintenance of methylation in transcribed regions, as well as diverse RNA degradation pathways in plants are discussed in view of our observations.

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Acknowledgments

We are thankful to Dr. Joszef Burgyan for providing the Reo-sigma 3 protein and Sergia Tzortzakaki for technical assistance. This work was supported by the grants of the Sixth Research Framework Programs of the European Union, Project LSHG-CT-2006-037900 (SIROCCO) and LSHG-CT-2004-005120 (FOSRAK).

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Authors and Affiliations

  1. RLP AgroScience GmbH, AlPlanta-Institute for Plant Research, 67435, Neustadt, Germany

    Athanasios Dalakouras & Michael Wassenegger

  2. Heidelberg Institute for Plant Sciences, University of Heidelberg, 69120, Heidelberg, Germany

    Athanasios Dalakouras & Michael Wassenegger

  3. Department of Biology, University of Crete, 71409, Heraklion, Greece

    Athanasios Dalakouras, Maria Tzanopoulou, Mina Tsagris & Kriton Kalantidis

  4. Institute of Molecular Biology and Biotechnology, Foundation for Research and Technology, 71110, Heraklion, Greece

    Athanasios Dalakouras, Maria Tzanopoulou & Kriton Kalantidis

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  1. Athanasios Dalakouras
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  2. Maria Tzanopoulou
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  3. Mina Tsagris
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  4. Michael Wassenegger
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  5. Kriton Kalantidis
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Correspondence to Kriton Kalantidis.

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Dalakouras, A., Tzanopoulou, M., Tsagris, M. et al. Hairpin transcription does not necessarily lead to efficient triggering of the RNAi pathway. Transgenic Res 20, 293–304 (2011). https://doi.org/10.1007/s11248-010-9416-3

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  • DOI: https://doi.org/10.1007/s11248-010-9416-3

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