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Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA

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Abstract

Gene silencing is a natural defense response of plants against invading RNA and DNA viruses. The RNA post-transcriptional silencing system has been commonly utilized to generate transgenic crop plants that are “immune” to plant virus infection. Here, we applied this approach against the devastating DNA virus tomato yellow leaf curl virus (TYLCV) in its host tomato (Solanum lycopersicum L.). To generate broad resistance to a number of different TYLCV viruses, three conserved sequences (the intergenic region [NCR], V1-V2 and C1-C2 genes) from the genome of the severe virus (TYLCV) were synthesized as a single insert and cloned into a hairpin configuration in a binary vector, which was used to transform TYLCV-susceptible tomato plants. Eight of 28 independent transgenic tomato lines exhibited immunity to TYLCV-Is and to TYLCV-Mld, but not to tomato yellow leaf curl Sardinia virus, which shares relatively low sequence homology with the transgene. In addition, a marker-free (nptII-deleted) transgenic tomato line was generated for the first time by Agrobacterium-mediated transformation without antibiotic selection, followed by screening of 1180 regenerated shoots by whitefly-mediated TYLCV inoculation. Resistant lines showed a high level of transgene-siRNA (t-siRNA) accumulation (22 % of total small RNA) with dominant sizes of 21 nt (73 %) and 22 nt (22 %). The t-siRNA displayed hot-spot distribution (“peaks”) along the transgene, with different distribution patterns than the viral-siRNA peaks observed in TYLCV-infected tomato. A grafting experiment demonstrated the mobility of 0.04 % of the t-siRNA from transgenic rootstock to non-transformed scion, even though scion resistance against TYLCV was not achieved.

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Acknowledgments

We acknowledge a contribution from the Agricultural Research Organization, The Volcani Center, Bet Dagan, Israel, No. 547/15. This work was supported by grants from Volcani Center ARO and Zeraim Gedera Seed Company (grant no.132-150110).

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    Authors and Affiliations

    1. Department of Plant Pathology and Weed Science, ARO, Volcani Center, 50250, Bet Dagan, Israel

      Diana Leibman, Shanmugam Prakash, Sabrina Haviv, Marina Brumin, Victor Gaba & Amit Gal-On

    2. Department of Vegetable Research, ARO, Volcani Center, 50250, Bet Dagan, Israel

      Dalia Wolf, Aaron Zelcer & Moshe Lapidot

    3. Department of Ornamental Horticulture, ARO, Volcani Center, 50250, Bet Dagan, Israel

      Tzahi Arazi

    4. Department of Biotechnology, Al-Balqa’ Applied University, Al-Salt, 19117, Jordan

      Ghandi Anfoka

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    1. Diana Leibman
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    Diana Leibman, Shanmugam Prakash and Dalia Wolf have contributed equally to this work.

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    Leibman, D., Prakash, S., Wolf, D. et al. Immunity to tomato yellow leaf curl virus in transgenic tomato is associated with accumulation of transgene small RNA. Arch Virol 160, 2727–2739 (2015). https://doi.org/10.1007/s00705-015-2551-7

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