Localized egg‐cell expression of effector proteins for targeted modification of the Arabidopsis genome

Abstract

). † These authors contributed equally to this work. SUMMARYTargeted modification of the genome is an important genetic tool, which can be achieved via homologous, nonhomologous or site-specific recombination. Although numerous efforts have been made, such a tool does not exist for routine applications in plants. This work describes a simple and useful method for targeted mutagenesis or gene targeting, tailored to floral-dip transformation in Arabidopsis, by means of specific protein expression in the egg cell. Proteins stably or transiently expressed under the egg apparatus-specific enhancer (EASE) were successfully localized to the area of the egg cell. Moreover, a zinc-finger nuclease expressed under EASE induced targeted mutagenesis. Mutations obtained under EASE control corresponded to genetically independent events that took place specifically in the germline. In addition, RAD54 expression under EASE led to an approximately 10-fold increase in gene targeting efficiency, when compared with wild-type plants. EASE-controlled gene expression provides a method for the precise engineering of the Arabidopsis genome through temporally and spatially controlled protein expression. This system can be implemented as a useful method for basic research in Arabidopsis, as well as in the optimization of tools for targeted genetic modifications in crop plants.

Gene targeting in plants: 25 years later

“…As Cruciferin is a seed storage protein, integration of the GFPcontaining gene into the genomic target site produced fluorescent seeds, making it a convenient and non-destructive marker for GT (Shaked et al, 2005). The same authors expanded on their work in a later study with egg-cell specific expression of RAD54 to enhance GT using the Agrobacterium infiltration technique for transformation (Even-Faitelson et al, 2011).…”

“…One must bear in mind that if HR is more efficient, than enhanced recombination might occur not only between the targeting vector and the target locus but also between repetitive sequences all over the genome, leading to undesired and uncontrolled off-site effects. Attempts have been made to minimize the time window for GT using inducible or organ-specific promoters (Even-Faitelson et al, 2011), but even with these approaches, exclusive activation of the site of interest has not been possible.…”

Nonhomologous End Joining-Mediated Gene Replacement in Plant Cells

“…We used the QQR ZFN, which is a three-finger ZFN capable of binding to the sequence 59-GGGGAAGAA-39 (Smith et al, 2000). QQR ZFN has been successfully used for targeting experiments in Arabidopsis, tobacco, and petunia (Petunia hybrida) plants (Lloyd et al, 2005;Tovkach et al, 2009;Marton et al, 2010;Even-Faitelson et al, 2011). We selected gfp as the target DNA molecule to facilitate the phenotypical monitoring of putative targeting events by loss of function.…”

“…However, it is important to note that while ZFNinduced DSBs can indeed induce the HR repair machinery, most of these breaks are repaired by the cell's NHEJ DNA-repair machinery in plant and other species. Thus, ZFNs have also been used for the induction of site-specific mutagenesis in many eukaryotic cells, including plant cells (Lloyd et al, 2005;Tovkach et al, 2009;Marton et al, 2010;Osakabe et al, 2010;Zhang et al, 2010;Curtin et al, 2011;Even-Faitelson et al, 2011). More recently, ZFNs have been used for NHEJmediated targeted chromosomal deletions (Lee et al, 2010;Söllü et al, 2010), transgene removal (Petolino et al, 2010), and even NHEJ-mediated targeted DNA integration in mammalian genomes (Orlando et al, 2010).…”

Precision genetic modifications: a new era in molecular biology and crop improvement

“…however, ZFs may bind in a context-dependent and unpredictable fashion, requiring iterative selection to get ZF arrays with the desired specificity . nevertheless, many endogenous loci have been targeted at high frequencies using Zinc Finger nucleases (ZFns) in Arabidopsis (De Pater et al 2009;Even-Faitelson et al 2011;lloyd et al 2005), tobacco (Townsend et al 2009) and maize (shukla et al 2009). The reader may refer to recent reviews on this topic (Urnov et al 2010;Weinthal et al 2010).…”