Abstract
Increased tolerance to pathogens is an important goal in conventional and biotechnology-assisted grapevine breeding programs worldwide. Fungal and viral pathogens cause direct losses in berry production, but also affect the quality of the final products. Precision breeding strategies allow the introduction of resistance characters in elite cultivars, although the factors determining the plant’s overall performance are not fully characterized. Grapevine plants expressing defense proteins, from fungal or plant origins, or of the coat protein gene of grapevine leafroll-associated virus 3 (GLRaV-3) were generated by Agrobacterium-mediated transformation of somatic embryos and shoot apical meristems. The responses of the transformed lines to pathogen challenges were investigated by biochemical, phytopathological and molecular methods. The expression of a Metarhizium anisopliae chitinase gene delayed pathogenesis and disease progression against the necrotrophic pathogen Botrytis cinerea. Modified lines expressing a Solanum nigrum osmotin-like protein also exhibited slower disease progression, but to a smaller extent. Grapevine lines carrying two hairpin-inducing constructs had lower GLRaV-3 titers when challenged by grafting, although disease symptoms and viral multiplication were detected. The levels of global genome methylation were determined for the genetically engineered lines, and correlation analyses demonstrated the association between higher levels of methylated DNA and larger portions of virus-derived sequences. Resistance expression was also negatively correlated with the contents of introduced viral sequences and genome methylation, indicating that the effectiveness of resistance strategies employing sequences of viral origin is subject to epigenetic regulation in grapevine.
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Acknowledgements
The authors would like to thank the following for kindly sharing their expertise and excellent assistance: Renata Gava for fungus growth and aid with pathogenesis analyses, and Heitor Corbelini for grafting and conducting the plants in the greenhouse. The work was financed by Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) (Grant No. 381107/97-3)/BioEX and Sistema Embrapa de Gestão (SEG) (Grant No. 03.08.06.003.00.00) to PSR, TVMF, UAC and VQ.
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DDB, IS and VQ performed genetic transformation experiments, plant selection and culture, fungal pathogen challenges, and insertion/expression molecular analyses. PSR and UAC designed the strategies for fungal disease resistance, and selected the grapevine cultivars. RH and TVMF designed and constructed the virus hairpin-inducing constructs and performed viral pathogen challenges. PSR, UAC, TVMF and VQ conceived the work, analyzed the results and drafted the manuscript.
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Dal Bosco, D., Sinski, I., Ritschel, P.S. et al. Expression of disease resistance in genetically modified grapevines correlates with the contents of viral sequences in the T-DNA and global genome methylation. Transgenic Res 27, 379–396 (2018). https://doi.org/10.1007/s11248-018-0082-1
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DOI: https://doi.org/10.1007/s11248-018-0082-1