Targeted mutagenesis of the tomato PROCERA gene using transcription activator-like effector nucleases

Journal Article / 2014

Lor, Vai S.; Starker, Colby G.; Voytas, Daniel F.; Weiss, David; Olszewski, Neil E.

We report the successful use of transcription activatorlike effector nucleases (TALENs) under the control of an estrogen-inducible promoter for targeted mutagenesis in tomato (Solanum lycopersicum)ofthe negative regulator of GA signaling PROCERA (PRO). TALEN expression was induced and plants were regenerated from cotyledons of seedlings derived from stable transgenic lines. Six of 40 regenerated plants carried pro alleles, and the mutations in the two lines examined were heritable. Homozygous pro segregants exhibited phenotypes consistent with increased GA response. Tomato is an important agricultural crop in which significant resources are invested for breeding traits such as disease resistance and fruit shape and color (Foolad and Panthee, 2012). Mutations affecting these traits can be generated using random mutagens such as ethyl methanesulfonate and transfer DNA integration (Mathews et al., 2003; Menda et al., 2004); however, screening for the desired mutation is laborious and time consuming. A potentially more efficient method of gene disruption is targeted mutagenesis using sequence-specificnucleases, which create a double strand break in the target sequence. These breaks are then repaired either by the homologous recombination or nonhomologous end-joining pathway (Jasin and Rothstein, 2013). Nonhomologous end-joining is sometimes imprecise, resulting in deletions or insertions at the double strand break site.