Designer Endonuclease-mediated Gene Targeting in Barley

Transcription activator-like (TAL) effectors from pathogenic bacteria of the genus Xanthomonas are excreted into infected plant cells and bind to specific DNA motifs to manipulate host gene expression. The DNA-binding domain of TAL effectors consists of basically four types of repeats, each having specific affinity to one of the four nucleotides. This principle allows us to design sequences to code for DNA-binding domains with specificity to genomic target sequences of choice. In TAL effector nucleases (TALENs), such designer DNA-binding domain is coupled to a FokI nuclease which facilitates the creation of double strand breaks (DSBs) at a user-defined genomic position. DSBs are then processed by the cell's DNA repair machinery, which is error-prone to some extent and thus causes mutated target sites. To establish gene targeting technology in a cereal crop, a gfp specific TALEN pair was designed and used for gene transfer in barley embryogenic pollen cultures made from gfp lines. In this setup, a deleterious mutation of just one gfp allele is effective due to the haploid nature of the pollen-derived target cells, which could be converted into homozygous plants upon genome duplication. Screening for loss of fluorescence and sequencing the gfp gene of TALEN transgenics resulted in the detection of gfp knock-out mutants that were then analysed for genetic homogeneity and generative transmission.