title: A novel arrangement of zinc finger nuclease system for in vivo targeted genome engineering: the tomato LEC1-LIKE4 gene case.
abstract: A selection-free, highly efficient targeted mutagenesis approach based on a novel ZFN monomer arrangement for genome engineering in tomato reveals plant trait modifications. 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.
authors.affiliation: Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thermi, Thessaloniki, 57001, Greece. zhilioti@certh.gr.
authors.forename: Zoe
authors.initials: Z
authors.lastname: Hilioti
authors.affiliation: Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thermi, Thessaloniki, 57001, Greece.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Ganopoulos
authors.affiliation: Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thermi, Thessaloniki, 57001, Greece.
authors.forename: Sabna
authors.initials: S
authors.lastname: Ajith
authors.affiliation: Animal Science Department, Agricultural University of Athens, Athens, 11855, Greece.
authors.forename: Ioannis
authors.initials: I
authors.lastname: Bossis
authors.affiliation: Institute of Applied Biosciences, Centre for Research and Technology Hellas, Thermi, Thessaloniki, 57001, Greece.
authors.forename: Athanasios
authors.initials: A
authors.lastname: Tsaftaris
mesh_terms: D001483:Base Sequence; D004720:Endonucleases / Q000378:metabolism*; D005638:Fruit / Q000033:anatomy & histology; D018506:Gene Expression Regulation, Plant; D018390:Gene Targeting / Q000379:methods*; D017343:Genes, Plant; D005818:Genetic Engineering / Q000379:methods*; D005820:Genetic Testing; D018551:Solanum lycopersicum / Q000201:enzymology* / Q000235:genetics* / Q000254:growth & development; D009154:Mutation / Q000235:genetics; D010641:Phenotype; D030821:Plants, Genetically Modified; D016335:Zinc Fingers / Q000235:genetics*
chemical_list: D004720:Endonucleases
keywords: Development; Fruit; Heterochronic; Tomato; Transcription factor; Zinc finger nuclease technology