A novel arrangement of zinc finger nuclease system for in vivo targeted genome engineering: the tomato LEC1-LIKE4 gene case

Abstract

Key message

A selection-free, highly efficient targeted mutagenesis approach based on a novel ZFN monomer arrangement for genome engineering in tomato reveals plant trait modifications.

Abstract

How to achieve precise gene targeting in plants and especially in crops remains a long-sought goal for elucidating gene function and advancing molecular breeding. To address this issue, zinc finger nuclease (ZFN)-based technology was developed for the Solanum lycopersicum seed system. A ZFN architecture design with an intronic sequence between the two DNA recognition sites was evaluated for its efficiency in targeted gene mutagenesis. Custom engineered ZFNs for the developmental regulator LEAFY-COTYLEDON1-LIKE4 (L1L4) coding for the β subunit of nuclear factor Y, when transiently expressed in tomato seeds, cleaved the target site and stimulated imperfect repair driven by nonhomologous end-joining, thus, introducing mutations into the endogenous target site. The successful in planta application of the ZFN platform resulted in L1L4 mutations which conferred heterochronic phenotypes during development. Our results revealed that sequence changes upstream of the DNA binding domain of L1L4 can lead to phenotypic diversity including fruit organ. These results underscore the utility of engineered ZFN approach in targeted mutagenesis of tomato plant which may accelerate translational research and tomato breeding.

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Acknowledgments

This work was benefited from the networking activities within the European funded COST ACTION FA1106 Quality Fruit. SA was supported by the European funded Erasmus Mundus Panacea Project. We are grateful to Prof. Petros Koidis, Fotini Dalipi, Theodoros Moysiadis and Dimitrios Valasiadis for valuable comments on the manuscript.

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Correspondence to Zoe Hilioti.

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Communicated by W. Harwood.

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Hilioti, Z., Ganopoulos, I., Ajith, S. et al. A novel arrangement of zinc finger nuclease system for in vivo targeted genome engineering: the tomato LEC1-LIKE4 gene case. Plant Cell Rep 35, 2241–2255 (2016). https://doi.org/10.1007/s00299-016-2031-x

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Keywords

  • Transcription factor
  • Zinc finger nuclease technology
  • Heterochronic
  • Development
  • Tomato
  • Fruit