title: A simple test for the cleavage activity of customized endonucleases in plants.
abstract: BACKGROUND
  Although customized endonucleases [transcription activator-like effector nucleases (TALENs) and RNA-guided endonucleases (RGENs)] are known to be effective agents of mutagenesis in various host plants, newly designed endonuclease constructs require some pre-validation with respect to functionality before investing in the creation of stable transgenic plants.
  
  
  RESULTS
  A simple, biolistics-based leaf epidermis transient expression test has been developed, based on reconstituting the translational reading frame of a mutated, non-functional yfp reporter gene. Quantification of mutation efficacy was made possible by co-bombarding the explant with a constitutive mCherry expression cassette, thereby allowing the ratio between the number of red and yellow fluorescing cells to serve as a metric for mutation efficiency. Challenging either stable mutant alleles of a compromised version of gfp in tobacco and barley or the barley MLO gene with TALENs/RGENs confirmed the capacity to induce site-directed mutations.
  
  
  CONCLUSIONS
  A convenient procedure to assay the cleavage activity of customized endonucleases has been established. The system is independent of the endonuclease platform and operates in both di- and monocotyledonous hosts. It not only enables the validation of a TALEN/RGEN's functionality prior to the creation of stable mutants, but also serves as a suitable tool to optimize the design of endonuclease constructs.
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Nagaveni
authors.initials: N
authors.lastname: Budhagatapalli
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Sindy
authors.initials: S
authors.lastname: Schedel
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Maia
authors.initials: M
authors.lastname: Gurushidze
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Stadt Seeland/OT Gatersleben, Germany.|Chair of Plant Breeding, Martin Luther University, Betty-Heimann-Str. 3, 06120 Halle (Saale), Germany.
authors.forename: Stefanie
authors.initials: S
authors.lastname: Pencs
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Hiekel
authors.affiliation: Structural Cell Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Twan
authors.initials: T
authors.lastname: Rutten
authors.affiliation: Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen University, Worringerweg 1, 52056 Aachen, Germany.
authors.forename: Stefan
authors.initials: S
authors.lastname: Kusch
authors.affiliation: ZMBP-General Genetics, University of Tübingen, Auf der Morgenstelle 32, 72076 Tübingen, Germany.
authors.forename: Robert
authors.initials: R
authors.lastname: Morbitzer
authors.affiliation: ZMBP-General Genetics, University of Tübingen, Auf der Morgenstelle 32, 72076 Tübingen, Germany.
authors.forename: Thomas
authors.initials: T
authors.lastname: Lahaye
authors.affiliation: Unit of Plant Molecular Cell Biology, Institute for Biology I, RWTH Aachen University, Worringerweg 1, 52056 Aachen, Germany.
authors.forename: Ralph
authors.initials: R
authors.lastname: Panstruga
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Jochen
authors.initials: J
authors.lastname: Kumlehn
authors.affiliation: Plant Reproductive Biology, Leibniz Institute of Plant Genetics and Crop Plant Research (IPK), Corrensstr. 3, 06466 Stadt Seeland/OT Gatersleben, Germany.
authors.forename: Goetz
authors.initials: G
authors.lastname: Hensel
keywords: Biolistic gene transfer; RNA-guided endonucleases; Site-directed mutagenesis; Transcription activator-like effector nucleases; Transient expression