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Plant Genome Modification by Homologous Recombination 157 Plant genome modification by homologous recombination Moez Haninà and Jerzy Paszkowskiy The mechanisms and frequencies of various types of progeny. For example, a somatic rearrangement at the homologous recombination (HR) have been studied in plants for zein locus of maize reached the gametes and was trans- several years. However, the application of techniques involving mitted meiotically to the next generation [1]. HR for precise genome modification is still not routine. The low frequency of HR remains the major obstacle but recent progress DSBs can also be repaired by non-homologous end join- in gene targeting in Arabidopsis and rice, as well as accumulating ing (NHEJ), which requires no or very limited sequence knowledge on the regulation of recombination levels, is an homology. However, NHEJ often results in sequence encouraging sign of the further development of HR-based alteration at the restored junctions [2]. Although the approaches for genome engineering in plants. mechanisms and molecular components of DSB repair seem to be evolutionarily conserved, the usage of these Addresses alternative repair pathways varies between organisms. ÃInstitut Supe´ rieur de Biotechnologie de Sfax, route M’harza, HR is prevalent in prokaryotes and yeast, whereas NHEJ 3018 Sfax., Tunisia is predominant in multicellular eukaryotes [3]. This dif- yFriedrich Miescher Institute for Biomedical Research, Maulbeerstrasse 66, CH-4058 Basel, Switzerland ference is reflected in the dissimilar outcomes of yeast e-mail: [email protected] transformation experiments compared to those in plant and animal cells. In yeast, even short stretches (approxi- mately 50 bp) of homology to chromosomal DNA within Current Opinion in Plant Biology 2003, 6:157–162 transformation vectors are able to direct the integration of This review comes from a themed issue on transforming DNA by homologous recombination. In Plant biotechnology contrast, transforming DNA is integrated into plant and Edited by Wolf B Frommer and Roger Beachy mammalian cells at random positions by the action of the 1369-5266/03/$ – see front matter NHEJ pathway, regardless of the presence of stretches in ß 2003 Elsevier Science Ltd. All rights reserved. the transforming DNA that are homologous to the host chromosomes. Random DNA integration by NHEJ DOI 10.1016/S1369-5266(03)00016-5 occurs even when the number of random DSBs is artifi- cially increased by DNA-damaging treatments such as Abbreviations X-ray irradiation [4]. Such natural preference for NHEJ DSB double-strand breaks and the low frequency of HR are the main obstacles to the ECR extrachromosomal recombination development of efficient gene-targeting (GT) technology EGT ectopic targeting GT gene-targeting for use in angiosperms. In contrast, GT is an established HR homologous recombination tool for engineering the mouse genome [5] and has been ICR intrachromosomal recombination successful in the moss Physcomitrella patens [6 ]. Its poten- NHEJ non-homologous end joining tial has also been demonstrated in Drosophila [7,8], sheep PPO PROTOPORPHYRINOGEN OXIDASE [9] and human somatic cells [10]. SDSA synthesis-dependent strand-annealing In this review, we briefly depict the most likely fate of Introduction foreign DNA before and after chromosomal integration During meiosis, homologous recombination (HR) guides in plant cells. We review how current strategies that exchanges between chromosomes that produce novel involve HR may influence this fate by targeting DNA combinations of alleles and is essential for proper chro- to a specific chromosomal position and how superfluous mosome segregation. In somatic cells, HR allows the DNA may be eliminated by intrachromosomal recombi- precise repair of DNA double-strand breaks (DSBs) by nation (ICR) events. the copying of homologous templates. Consequently, HR affects genome structure in more than one way. HR not Fate of foreign DNA in the plant cell only ensures genome integrity (through DSB-repair) but Extrachromosomal phase also contributes to genome evolution by the creation of Transforming DNA is usually introduced into a plant cell new alleles or new allelic combinations through meiotic in one of two forms, either as a purified chemical molecule and possibly somatic recombination events. Somatic (naked DNA) or as a T-DNA complex that is transferred recombination may be of particular importance in plants from Agrobacterium tumefaciens. Interestingly, these two as the plant germ line, in contrast to that of animals, forms of DNA are almost equally prone to efficient differentiates late in development. Thus, genetic changes extrachromosomal recombination (ECR) after uptake that are generated in somatic cells can be transmitted to [11,12]. ECR has been investigated extensively during www.current-opinion.com Current Opinion in Plant Biology 2003, 6:157–162 158 Plant biotechnology the past decade in plant species such as tobacco, Arabi- tion, less-defined transposon-specific effects may also dopsis, maize and petunia. In all of these species, it occurs contribute to these unusually high induction levels with efficiencies ranging from 10À1 to 10À3, as measured [26]. Other stress factors that are not directly linked to by a comparison of the transformation frequencies with the formation of recombinogenic DSBs have been impli- two non-overlapping deletions of a selectable marker cated in the stimulation of ICR. For example, pathogen gene to transformation frequencies with the intact gene. attack stimulates ICR [27], providing a compelling link to Linear molecules are more prone to ECR than circular the proposed involvement of HR in the rapid evolution of DNAs, suggesting the involvement of single-strand resistance gene clusters [28]. On the other hand, the annealing (SSA) [13] or synthesis-dependent strand- potential instability of loci with sequence redundancies annealing (SDSA) processes in ECR [14]. Both SSA can be induced by adverse stress conditions. This poten- and SDSA processes involve strand invasion but the latter tial source of instability must be considered when design- is also supported by DNA synthesis [15]. Co-transforma- ing transgenic constructs, selecting reliable transgenic tion by Agrobacterium strains harboring two different strains or devising strategies for further processing of T-DNAs or by two different purified plasmids has facili- transgenic loci by ICR. tated the analysis of ECR [16,17]. Junctions between DNA molecules that are involved in recombination can Altering the fate of foreign DNA by be associated with deletions or insertions of filler DNA homologous recombination that originates from either transforming or host chromo- The most straightforward and widely explored way to somal sequences. Regardless of the detailed mechanism, reduce or prevent the disadvantageous integration of ECR results in head-to-tail linked arrays of transgenes. multicopy DNAs is to select single copy inserts, assuming Although ECR is thought to occur before chromosomal that single-copy DNAs have an enhanced genetic (phy- integration, it is still possible that the concatamerization sical) and epigenetic (functional) stability. In general, also takes place during or even after integration through transformation followed by the selection of single copy some kind of ‘homing mechanism’ [16–19]. Clearly, a inserts has been successful. As the transformed DNAs are certain proportion of transgenic DNA integrates without integrated into random positions, however, this approach ECR, resulting in a single-copy locus or several unlinked requires that several transgenic lines are characterized in loci that contain single copies of foreign DNA that can be detail to identify a line with a single functional copy of the separated by genetic segregation. inserted DNA. Site-specific recombination systems for the integration of DNA into a predetermined chromoso- Intrachromosomal phase mal position would appear to be an attractive way to Linked multicopy transgenes are prone to epigenetic and eliminate position effects. Surprisingly, however, this is genetic instability governed by gene silencing or by ICR, not the case; the integration of identical transgenic inserts respectively. The frequency of ICR in plants has been into the same genomic location by site-specific integra- determined using various recombination substrates that tion resulted in variable levels of expression among the were inserted into the genome as direct or inverted transgenic events studied [29]. The ultimate goal of plant repeats [20–22]. Recombination frequencies have usually genome modification is not only the controlled addition of been measured by the frequency with which gene func- foreign genes with regard to position, orientation and tion is restored after ICR. This system only reveals copy number, but also the in situ modification of endo- precise recombination events, however, and probably genous genes. Here, the technologies of choice are cer- overlooks inaccurate rearrangements. As in mammals, tainly the various GT strategies (Figure 1). the basal frequency of ICR in plants is low, varying between 10À5 and 10À6 of cells containing a recombina- Gene targeting tion cassette. This can be increased by up to 9-fold by The first reports of successful gene targeting in mouse genotoxic stress factors such as irradiation (e.g. ultra- and plant cells appeared within the space of three years violet light or X-rays) or radio-mimicking chemicals [30,31]. By 2001, the functions of more than 7000 chro- (e.g. methyl methanesulfonate [MMS]) [23,24]. mosomal loci
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