DNA damage response (DDR) is an essential mechanism by which living organisms maintain their genomic stability. In plants, DDR is important also for normal growth and yield. Here, we explored the DDR of a temperate model crop barley (Hordeum vulgare) at the phenotypic, physiological, and transcriptomic levels. By a series of in vitro DNA damage assays using the DNA strand break (DNA-SB) inducing agent zeocin, we showed reduced root growth and expansion of the differentiated zone to the root tip. Genome-wide transcriptional profiling of barley wild-type and plants mutated in DDR signaling kinase ATAXIA TELANGIECTASIA MUTATED AND RAD3-RELATED (hvatr.g) revealed zeocin-dependent, ATR-dependent, and zeocin-dependent/ATR-independent transcriptional responses. Transcriptional changes were scored also using the newly developed catalog of 421 barley DDR genes with the phylogenetically-resolved relationships of barley SUPRESSOR OF GAMMA 1 (SOG1) and SOG1-LIKE (SGL) genes. Zeocin caused up-regulation of specific DDR factors and down-regulation of cell cycle and histone genes, mostly in an ATR-independent manner. The ATR dependency was obvious for some factors associated with DDR during DNA replication and for many genes without an obvious connection to DDR. This provided molecular insight into the response to DNA-SB induction in the large and complex barley genome.

• MeSH
• ATM protein genetika   metabolismus   MeSH
• bleomycin * MeSH
• DNA MeSH
• ječmen (rod) * genetika   metabolismus   MeSH
• oprava DNA MeSH
• poškození DNA MeSH
• Publikační typ
• časopisecké články MeSH
• Názvy látek
• ATM protein MeSH
• bleomycin * MeSH
• DNA MeSH
• Zeocin MeSH Prohlížeč

Gene targeting is becoming an important tool for precision genome engineering in plants. During gene replacement, a variant of gene targeting, transformed DNA integrates into the genome by homologous recombination (HR) to replace resident sequences. We have analysed gene targeting in barley (Hordeum vulgare) using a model system based on double-strand break (DSB) induction by the meganuclease I-SceI and a transgenic, artificial target locus. In the plants we obtained, the donor construct was inserted at the target locus by homology-directed DNA integration in at least two transformants obtained in a single experiment and was stably inherited as a single Mendelian trait. Both events were produced by one-sided integration. Our data suggest that gene replacement can be achieved in barley with a frequency suitable for routine application. The use of a codon-optimized nuclease and co-transfer of the nuclease gene together with the donor construct are probably the components important for efficient gene targeting. Such an approach, employing the recently developed synthetic nucleases/nickases that allow DSB induction at almost any sequence of a genome of interest, sets the stage for precision genome engineering as a routine tool even for important crops such as barley.

• Klíčová slova
• Barley, Hordeum vulgare, double-strand break induction, gene replacement, gene targeting, homology-directed DNA integration, precision genome engineering.,
• MeSH
• dvouřetězcové zlomy DNA * MeSH
• genetické lokusy MeSH
• geneticky modifikované rostliny MeSH
• genový targeting metody   MeSH
• ječmen (rod) genetika   MeSH
• kvantitativní polymerázová řetězová reakce MeSH
• modely genetické MeSH
• reprodukovatelnost výsledků MeSH
• rostlinné geny MeSH
• transformace genetická MeSH
• typy dědičnosti genetika   MeSH
• Publikační typ
• časopisecké články MeSH
• práce podpořená grantem MeSH