Plant Molecular Biology 48: 173182, 2002. 2002 Kluwer Academic Publishers. Printed in the Netherlands. 173

Gene replacement by homologous recombination in plants

Holger PuchtaInstitut fr Pflanzengenetik und Kulturpflanzenforschung (IPK), Corrensstrasse 3, 06466 Gatersleben, Germany(e-mail puchta@ipk-gatersleben.de)

Key words: chimeric oligonucleotides, double-strand break repair, gene targeting, illegitimate recombination,Physcomitrella patens, site-specific integration

Abstract

After the elucidation of the sequence of the yeast genome a major effort was started to elucidate the biologicalfunction of all open reading frames of this organisms by targeted gene replacement via homologous recombination.The establishment of the complete sequence of the genome of Arabidopsis thaliana would principally allow asimilar approach. However, over the past dozen years all attempts to establish an efficient gene targeting techniquein flowering plants were in the end not successful. In contrast, in Physcomitrella patens an efficient gene targetingprocedure has been set up, making the moss a valuable model system for plant molecular biologists. But also forflowering plants recently several new approaches some of them based on the availability of the genomic sequenceof Arabidopsis were initiated that might finally result on the set up of a general applicable technique. Beside theproduction of hyper-recombinogenic plants either via expression or suppression of specific gene functions or viaundirected mutagenesis, the application of chimeric oligonucleotides might result in major progress.

Introduction

Two different ways for alteration of covalent link-age between DNA molecules is possible: either aregion of sequence identity between the partners isneeded (homologous recombination) or no sequence-specific requirements have to be fulfilled (illegitimaterecombination also referred to as non-homologous endjoining). For the controlled manipulation of genomeshomologous recombination is of special interest as theproduct of the reaction is predictable. In transforma-tion experiments in prokaryotes and lower eukaryotessuch as yeast homologous recombination is predomi-nating. In yeast, for instance, an external piece of DNAharbouring homologous sequences to the genome willalmost exclusively integrate into the correspondinggenomic position, a phenomenon called gene tar-geting. Transformation with sequences that carry nohomology to the yeast genome is hardly possible.At present reverse genetics is used to elucidate therole of specific proteins in an organism. Using in-formation obtained from various genome sequencing

programs the gene coding for the protein of interestcan be modified to render it non-functional. The trans-formed organism carrying a loss-of-function mutationcan then be analysed for its phenotype. Gene target-ing is thus a powerful tool for directed knockout ofgenes. Furthermore, using this technique it is possibleto perform subtle changes such as the modificationof single nucleotides in a gene in situ, an achieve-ment that cannot be obtained by random insertionmutagenesis. In higher eukaryotes including plantsDNA integrates mainly via illegitimate recombina-tion in an undirected, sequence-independent mannerin the genome. Tremendous efforts have been un-dertaken to improve gene targeting in animals andplants and the development of a feasible techniquefor mouse embryonic stem cells has revolutionizedbasic as well as applied research in this area. Unfortu-nately, similar improvements have not been achievedfor higher plants. However, a number of approacheswere developed recently that might indeed lead to afeasible gene targeting technique in the near future.The main emphasis of this review will lie on these

174

new developments; mainly gene targeting experimentsperformed in plants will be discussed. Results fromother organisms or basic aspects of recombination inplants will only be discussed were it is necessaryfor understanding. To obtain a more detailed pictureof specific aspects of homologous recombination andgene targeting in plants the reader is recommended toconsult a row of recently published reviews (Puchta,1998a; Gorbunova and Levy, 1999; Hohn and Puchta,1999; Mengiste and Paszkowski, 1999; Vergunst andHooykaas, 1999; Oh and May, 2001; Schaefer, 2001).

Attemps to improve gene targeting via thetargeting vector

Mammals as guiding light

In mammals gene targeting has become a routine tech-nique for knocking out gene functions. Since the pio-neering work of the groups of Smithies (Doetschmanet al., 1987) and Capecci (Thomas and Capecci, 1987)thousands of loss-of-function mutations of mousehave been generated by gene targeting in embryonicstem (ES) cells. Numerous experiments indicate genetargeting frequencies of 102 (one homologous inte-gration event per hundred random integration events)or higher in ES cells (for review see Jasin et al., 1996).Unfortunately, similar frequencies have not been ob-tained in gene targeting experiments in plants. Sincethe pioneering first report on the targeting of a trans-gene locus in plants (Paszkowski et al., 1988) noconvincing enhancement of the targeting frequencycould be achieved within the next dozen of years.In principle, there are two ways to produce trans-genic plants. Either the DNA is directly transferredinto plants cells by PEG transformation or electropo-ration of protoplasts or by the bombardment of var-ious plant tissues (for details of plant transformationsee Potrykus and Spangenberg, 1996). Alternatively,Agrobacterium tumefaciens is used as a vector forplant transformation (for recent revieuws see Rossieet al., 1996; Tinland, 1996; Hansen and Chilton,1999; Zupan et al., 2000). Both direct gene trans-fer (Paszkowski et al., 1988; Halfter et al., 1992)and Agrobacterium-mediated T-DNA transformation(Offringa et al., 1990; Lee et al., 1990) were ap-plied in the initial studies in which the restorationof selectable marker genes (confering kanamycin orhygromycin resitance) was used to estimate the fre-quency of gene targeting in plants. In all cases the

observed gene targeting frequencies were low, 104to 105, independent of the plant species (tobaccoor Arabidopsis) and the transformation method. Con-sidering the factors that improved gene targeting inmouse, various attempts have been undertaken in thefollowing years to improve the ratio of homologous toillegitimate recombination. However, neither extend-ing the length of homology in the transferred DNAto up to 22 kb (Thykjaer et al., 1997) nor includingnegative selectable markers outside of the homologyof the targeting vector to be able to select against ran-dom integration (Risseeuw et al., 1997; Gallego et al.,1999; for a detailed discussion on negative selectablemarkers, see Vergunst and Hooykaas, 1999) did resultin a significantly higher frequency of gene targeting inplants.

Nevertheless, it is well possible to enhance ho-mologous recombination in plant cells by activatingthe target site in the genome by the induction ofa double-strand break (DSB). Recombination is in-ducible by DSBs and all current recombination modelsare based on the repair of such breaks (for review seePasques and Haber, 1999). Via transient expressionof the restriction enzyme I-SceI a DSB was intro-duced at a transgenic restriction site within the tobaccogenome in vivo resulting in a homologous integrationfrequency of up to 102 at this site (Puchta et al.,1996). However, this strategy is not applicable fortargeting genes at all, as such a break can only beinduced at the specific recognation sites of the rare-cutting restriction enzyme. Therefore this techniquecan only be used for the integration of transgenes ata specific predetermined site in the genome and is assuch an alternative to site-specific integration medi-ated by sequence-specific recombinases as describedin the accompanying review by David Ow (Ow, 2001).

The targeting of natural genes in Arabidopsisthaliana

Two cases have been reported over the years in whichnatural genes (the loss of function cannot be selectedfor) were knocked out in Arabidopsis thaliana bygene targeting after Agrobacterium-mediated transfor-mation. Eric Lams group was able to isolate oneArabidopsis callus out of 2580 tissue culture transfor-mants in which targeting of the TGA3 locus had oc-curred. However, the respective callus was a chimerafor the targeted locus and could not be regenerated(Mia and Lam, 1995). Two years later Martin Yanof-skys group in collaboration with Eric Lam was finally

175

able to obtain a mutant Arabidopsis plant by knockingout the AGL5 MADS-box gene. Using vacuum infil-tration of bolting plants, they were able to recover onetargeted event in 750 transformations (Kempin et al.,1997). This relatively high frequency induced hopesthat the application of vacuum infiltration might havehelped to transform especially active in homologousrecombination, reminiscent of mouse ES cells (Esserset al., 2000) and that indeed gene targeting wouldbecome feasible for Arabidopsis. However, no statis-tically sound conclusion as to the frequencies of tar-geting could be drawn from the reported single eventand the results were discussed controversially (Puchta1998a; Liljegren and Yanofsky, 1998). Unfortunately,for the following years no further successful genetargeting experiments with vacuum infiltration werereported, so that the initial hopes were not fulfilled.

Ectopic recombination: a not wanted by-product ofgene targeting

Beside the low frequencies of gene targeting anothercause of disillusionment for several researchers wasthe fact that although the respective marker gene wasrestored via homologous recombination, in some casesthe target locus was not changed in the expected way(Offringa et al., 1993; Risseeuw et al., 1995; Reisset al., 2000). The classical double-strand break repair(DSBR) model of recombination (Szostak et al., 1983)predicts that integration of both ends of the extrachro-mosomal piece of DNA into the target locus shouldoccur via homologous recombination. However genetargeting experiments in tobacco revealed two furtherclasses of homologous recombinants. Firstly, inte-gration of the extrachromosomal DNA in the targetlocus by a combination of homologous and illegiti-mate recombination was detected. Secondly, sequencehomology can also be copied from the target locus toextrachromosomal DNA. This modified sequence maythen integrate elsewhere in the genome (ectopic tar-geting). Recent studies in somatic plant cells indicatethat these findings are not suprising because the mainmechanism of recombination is described best bya synthesis-dependent strand annealing (SDSA)-likemodel of recombination (Rubin et al., 1997; Puchta1998b; for review see Gorbunova and Levy, 1999)rather then by the DSBR model. It could be demon-strated that DNA ends can react independently and oneend might be repaired by homologous and the other byillegitimate recombination. Therefore, gene targetingexperiments in higher plants have to include a thor-

ough Southern analysis of recombinants to identify thedesired knock-out, in which both ends of the vectorsequence have been integrated via homology in the tar-get locus. But why is the SDSA mechanism operatingin somatic plant cells? It seems to be a prerequisite forthe stability of the highly repetitive plant genome. Ho-mologous interactions between ectopic sequences can,according to the DSBR model, result in chromosomalrearrangements as translocations whereas according tothe SDSA model translocations are avoided (for de-tailed discussion on DSB repair, see Puchta, 1999;Gorbunova and Levy, 1999).

Production of a targeting vector in vivo: a solution tothe problem?

An apparently highly efficient variant of gene tar-geting has been developed recently for Drosophilamelanogaster by the group of Kent Golic (Rong anGolic, 2000, 2001). In this method the contruct for tar-geting is integrated in the host genome flanked by tworecognitions sites of a site-specific recombinase andincludes a site for a rare-cutting restriction endonu-clease. By induced expression of the site-specific re-combinase a DNA circle is excised from the genome.This circle is then linearized after the restriction en-zyme (in this case I-SceI) has been expressed resultingin an activated DNA molecule with both ends ho-mologous to the target sequence. In the female germline of Drosophila gene targeting occurred in aboutone out of 500 cases. Although questions were raisedabout the general applicability of the technique (En-gels, 2000) and certain product classes obtained arehard to explain by a simple mechanism, it seemsworthwhile to test such a strategy in plants, too (fordiscussion see also Kumar and Fladung, 2001). On theone hand, the set-up of the system seems to be verycomplex, as beside constructions of a donor sequencewith sites for recombinase and restriction enzyme, ex-pression cassettes both enzymes have to be includedinto the transgene construct or supplied in trans. Onthe other hand, if the reaction occurs in an efficientway in planta, every single seedling should repre-sent an excision event. Thus, by the use of respectivemarker genes large numbers of plants can be producedand easily screened.

176

Productions op plants with a hyper-recombinationphenotype

Over-expression of heterologous genesIf homologous recombination is not working effi-ciently in plants why not try to transfer the enzymemachinery from an organism in which gene targetingis working well to plants? This idea is followed sincea row of years in the field, and pioneering work wasdone by the group of Bernd Reiss. Transgenic tobaccoplants were produced which expressed the key ac-tor of homologous recombination in Escherichia coli,RecA. As expected, the rate of intrachromosomal ho-mologous recombination was enhanced by one orderor magnitude (Reiss et al., 1996). Hopes were fly-ing high that also gene targeting frequencies would beenhanced by RecA, however in the respective exper-iments for two different transgene loci no significantof the gene targeting frequency could be achievedwith Agrobacterium-mediated transformation (Reisset al., 2000). Although one can speculate that the T-DNA complexed with bacterial proteins might havehindered homologous recombination, experiments inyeast demonstrate that in this organism T-DNA isperfectly integrated via homology (Bundock et al.,1996). In a different approach the group of Avi Levyover-expressed the RuvC gene in tobacco. RuvC isresponsible for the resolution of homologous recom-bination intermediates (Holliday junctions) in E. coli.They were able to demonstrate that frequencies ofextrachromosomal, intrachromosomal as well as inter-chromosomal recombination were enhanced up to twoorders of magnitude, although no conclusive explana-tion for this phenomenon could be supplied (Shalevet al., 1999). It will be interesting to see whether a sim-ilar effect can also be obtained with gene targeting. Alesson learnt from these experiments is, however, thatit is very hard to predict the effects of the expressionof a heterologous protein in plants on recombination.Due to complex interactions with different host pro-teins it might be possible to influence some or evendifferent but not all of the reactions in which therespective protein is participating in its natural host.

Modulating the function of plant genesBasic features of the enzyme machinery involved inrecombination are conserved between yeast mammalsand plants. Thus, factors of putatively similar func-tion can be identified by sequence homologies (Ver-gunst and Hooykaas, 1999; Bhatt et al., 2001). With

the complete sequence of the Arabidopsis genomeat hand, multiple open reading frames with similar-ities to known genes of other organisms involted inrepair and recombination can be identified (see thecompilation of homologous to known repair and re-combination genes of other organisms; ArabidopsisGenome Initiative, 2000). Moreover, it has been re-ported for vertebrates that mutations in certain geneslead to enhanced homologous recombination frequen-cies. For example, the knockout of the Mre 11 geneof chicken leads to increased gene targeting frequen-cies (Yamaguchi-Iwai et al., 1999). Mre 11 togetherwith RAD50 is in yeast part of a multifunctional pro-tein complex that can act as nuclease and is involvedin certain reactions of homologous as well as in ille-gitimate recombination (for review see Haber, 1998).Homologous factors exist in Aribidopsis (Hartung andPuchta, 1999; Gallego et al., 2001) and the groupof Charles White could indeed demonstrate that aRAD50 T-DNA insertion mutant showed an increaseof intrachromosomal homologous recombination byone order of magnitude (Gherbi et al., 2001). It willbe interesting to see whether such a similar increasecan also be detected for gene targeting.

There are other genes the mutation or suppressionof which might lead to higher targeting frequencies.In chicken enhanced target integration of DNA wasfound for a mutant of the gene responsible for Bloomssyndrome (BLM) (Wang et al., 2000). BLM is a mem-ber of the RecQ helicase family involved in pro- andeukaryotes in DNA replication and recombination (forreview see Karow et al., 2000). Recently several RecQhomologues have been characterized in Arabidopsis(Hartung et al., 2000).

Blocking illegitimate recombination should in-crease the relation of homologous to random integra-tion events. Two components of this pathway, DNALigase IV and XRCC4, have already been character-ized from Arabidopsis (West et al., 2000) and morewill follow. Without question, in the next few yearsa series of mutants with insertions within differentgenes involved in recombination processes will be pro-duced and characterized. Analysis of their effects onrecombination will lead to identification of plants withenhanced gene targeting frequencies. Alternatively,also over-expression of Arabidopsis genes involvedin homologous recombination or its regulation mighthelp to increase gene targeting efficiency. For theMIM gene (Hannin et al., see below) it has alreadybeen recently demonstrated that over-expression is

177

correlated with increased homologous recombinationfrequencies.

Undirected mutagenesis can result in the productionof hyper-recombinogenic plantsThe classical genetic method to use undirected muta-genesis to isolate a phenotype of interest can provideimportant new insights because, in contrast to geneisolation by homology search, no assumptions needto be made about which genes might be involved inthe phenomenon of interest. This strategy was fol-lowed by the group of Jurek Paszkowski. As defectsin recombination should be correlated with an im-paired ability to repair DSBs resulting in an increasedsensitivity against X-rays, screening of Arabidopsispopulations, mutagenized by T-DNA insertions, forradiation-sensitive mutants should lead to the identi-fication of genes involved in this process. Indeed, aninsertion mutant into a gene (MIM) closely relatedto the SMC (structural maintenance of chromosomes)gene family of other eukaryotes was correlated withan X-ray-sensitive phenotype. The mutant showeda decreased frequency of intrachromosomal homol-ogous recombination (Mengiste et al., 1999). Over-expression of the MIM gene in wild-type Arabidopsisplants, on the other hand, did indeed increase thefrequency of homologous intrachromosomal recom-bination, although the effect was too small to expecta decisive improvement of gene targeting frequencies(Hannin et al., 2000).

A more direct strategy would be to screen forhyper-recombination mutants. For this purpose thegroup of Avi Levy used a sulfur mutation of tabacco.In this mutant somatic cross-overs between homo-logues can be detected in planta as yellow/green twinsectors, although the molecular nature of the processis not characterized in detail (Carlson, 1974). Usinga transposon as insertion mutagen, they were able toobtain a plant in which the frequency of interchro-mosomal recombination was enhanced by three ordersof magnitude. Moreover, the mutant proved to be re-sistant to elevated doses of radiation. Interestingly,although the frequency of homologous recombina-tion between extrachromosomal substrates was alsoincreased, no enhancement could be detected with anintrochromosomal substrate (Gorbunova et al., 2001).It will be very interesting to test this mutant for genetargeting, especially as an enhancement of severalorders of magnitude would indeed be a major break-through. Even if this will not be the case for this

mutant, the approach taken seems to be promising.There is no question that further mutant screens, forexample also with intrachromosomal recombinationsubstrates and also with Arabidopsis (e.g. Swobodaet al., 1994), should lead to the identification of morehyperrecombining plant mutants making this strategya very promising path that indeed might lead to theset-up of a feasible gene targeting technique in plants.

Chimeraplasty

A technique for targeted genome mutationsBased on findings that transcription enhances RecA-mediated homologous pairing of DNA in vitro, EricKmiec and his group came to the conclusion that pair-ing can be enhanced in the presence of complementaryRNA. Therefore they constructed self-complementarychimeric oligonucleotides that consist of a combina-tion of DNA and RNA sequences (for reviews see Yeet al., 1998; Rice et al., 2001). Chimeric oligonu-cleotides consist of a DNA mutator region of 5 or6 nucleotides complementary to the target including amutation which should be introduced into the genome,framed by two 2 O-methyl RNA bridges of 812nucleotides that are also complementary to the tar-get locus (O-methylation increases the stability of theRNA against degradation within cells). These regionsin turn are flanked by hairpin loops of 3 or 4 T nu-cleotides each. The hairpins are connected to a DNAregion of some 30 nucleotides, fully complementaryto the upper RNA-DNA-RNA strand. The lowerstrand contains a strand break that seems to be nec-essary for the topological interwinding of the chimerainto the target DNA. That chimeric oligonucleotidescan be used to induce mutations in a mammalian ge-nomic target sequence in vivo was first demonstratedby using chimeric oligonucleotides with a single-basemismatch corresponding to the wild-type sequencewithin the -globin gene. This construct was usedfor repairing the sickle allele, resulting in a chro-mosomal reversion to the wild-type -globulin. Anastonishingly high reversion frequency of 20% wasreported (Cole-Strauss et al., 1996). Many reportshave been published since then about the success-ful use of chimeric oligonucleotides in mammaliancells, demonstrating the usefulnesss of the technique.However, the mutation frequencies reported differeddramatically within and between experiments (e.g. vander Steege et al., 2001).

178

Targeted mutations in plants

Facing the gene targeting dilemma in plants it wastherefore obvious to use the technique for the tar-geted mutation of plant genomes (for review see Ohand May, 2001). In 1999, in parallel studies in to-bacco (Beetham et al., 1999) and maize (Zhu et al.,1999), an endogenous gene and a transgene were mu-tated by chimeric oligonucleotides. In both studies thefirst enzyme of the biosynthetic pathway of branchedchain amino acids was chosen as endogenous gene.In tobacco, the gene is referred to as acetolacetatesynthase (ALS) and in maize as acetohydroxy acidsynthase (AHAS). The mutation of certain aminoacids in the protein results in a resistant phenotype,which can be selected for by application of imidazo-line and sulfonylurea herbicides. In contrast to ani-mals cells, where chimeric oligonucleotides are nor-mally delivered via cationic liposomes, the chimericoligonucleotides were delivered into the plant cellsby biolistics. The strategy chosen in the experimentswas to obtain a selectable, dominant mutation in onegene, as both plant species harbour gene familiesof ALS or AHAS. Two different chimeric oligonu-cleotides were applied in both studies and mutationfrequencies were assessed to be arround 104 (Zhuet al., 1999; Hohn and Puchta, 1999). Surprisingly,the specificity of the reaction was not as precise as ex-pected. In tobacco none of the isolated resistant plantcell lines proved to harbour the expected target muta-tion. In all cases one nucleotide 5 to the mismatchednucleotide was changed, thus, strictly speaking, no tar-geted but only semi-targeted events were obtained.As controls indicate that chimeric oligoncleotides donot enhance mutation rates all over the genome, thechimeric oligonucleotides at least in plants seemsto induce mutation not only at the specific mismatchbut within the DNA stretch complementary to the tar-get locus. In maize the predicted target mutations wereindeed recovered, although semi-targeted mutationswere also found (Zhu et al., 1999, 2000). In bothstudies transgenic model systems using the green flu-orescent protein (GFP) as marker gene were set up.Plants containing an inactive GFP gene with eithera frame shift mutation (Beetham et al., 1999) or aninternal termination codon (Zhu et al., 1999) werebombarded with the respective COs to reverse the mu-tations. Putative revertants that are able to express afunctional GFP can easily be detected by fluorescencemicroscopy. In tobacco as well as in maize GFP-positive cells were found at frequencies at least as

high as reported for chimeric oligonucleotide-inducedmutations in the ALS or AHAS genes. The molecu-lar analysis of one mutant line in maize indicated thatthe induced mutation occurred indeed at the mismatchposition of the chimeric oligonucleotide, but insteadof the intended T a C was detected. The mutatedmaize line was regenerated and it could be domon-strated that the mutation was transferred faithfully tothe progeny (Zhu et al., 1999). Similarly stable propa-gation of the desired targeted mutations in the AHASgene could be demonstrated recently for maize (Zhuet al., 2000). Taking advantage of the ease of the GFPassay system the establishment of the mutated linesin tobacco and in maize will difinitely help to furtheroptimize the efficiency of chimeric oligonucleotideinduced mutations in plants.

In vitro assays and a bunch of open questionsExperiments were also performed using chimericoligonucleotides in vitro with extracts of mammalianand, more recently, plant cells. It could be demon-strated with human cell extracts that the repair re-action requires the presence of the mismatch repairprotein Msh2. In extracts of a cell line devoid ofMsh2 and wild-type cell extracts depleted of Msh2by a specific antibody, a reduced efficiency of re-pair was measured (Cole-Strauss et al., 1999). Thus,whereas gene targeting relies exclusively on fac-tors involved in homologous recombination, chimericoligonucleotide-directed repair seems to require fac-tors involved in mismatch reapair. In line with thisargument is the finding that via the use of chimericoligonucleotides only base substitutions and, withless efficiency, deletions or insertions of one or twobases on the target locus can be achieved (Ye et al.,1998), whereas via homologous recombination ge-nomic sequences of tens of kilobases can be changed,inserted or deleted. In vitro experiments were alsoperformed with nuclear extracts of different plantspecies, leading to the discovery that in extractsof tobacco the repair was in some cases less pre-cise then in the extracts of other plant species (Riceet al., 2000). This finding is in accordance with thedata obtained in vivo (Beetham et al., 1999). How-ever, recent result of Eric Kmiecs group indicatethat with single-stranded DNA oligonucleotides withvarying numbers of phosophorothiote linkages at the3 and 5 ends (to block exonucleolitic degradation)higher repair efficiencies can be achieved than withchimeric oligonucleotides in animal as well as in plant

179

extracts (Gamper et al., 2000). Moreover, the in-volvement of Msh2 in the repair process could notbe sustained, when the phosphorothioate-containingoligonucleotides were used. Thus, at present severalquestions remain to be answered, the most importantof which are whether phosphorothioate-containingoligonucleotides can also be used to mutate genesin vivo and whether the low frequencies obtained forgenomic repair in plants can be drastically increased infuture. The situation is somehow similar to that afterthe first report on gene targeting in plants (Paszkowskiet al., 1988). It has been unambigously demonstratedthat a basic principle works in plants, and hopes areflying high that a feasible technique will be avail-able soon. Hopefully, the oligionucleotide-mediatedDNA repair will leads us in the next years up thehills of glory and not into a similar valley of tears asgene targeting, when we where trying to optimize thetechnique.

Physcomitrella patens : an alternative to be takenseriously

Gene targeting in moss

In contrast to flowering plants, the moss Physcomitrellapatens is able to integrate DNA efficiently by homolo-gous recombination (for a recent review see Schaefer,2001). This finding, first reported by Didier Schae-fer and Jean-Pierre Zryd (Schaefer an Zryd, 1997),marked a major breakthrough for plant molecular biol-ogy. Soon afterwards it could be demonstrated by RalfReskis group that the technique can indeed be usedefficiently for the knock-out of gene functions (Streppet al., 1998). The moss has turned out to be a veryvaluable system for investigation of basic processesof plant biology. Mosses share with other plants thesame kind of hormones, high similarities in light andstress signalling response systems, as well as featuresof cell differentation and nucleus organelle interac-tions. This is also demonstrated by the fact that mostof the ESTs of Physecomitrella patens show stronghomology to genes of other plants (e.g. Reski et al.,1998). Thus, it seems likely that in many cases wherethe biological function of a specific gene is under in-vestigation in a higher plant a homologue might befound in Physcomitrella. A gene targeting experimentin Physcomitrella might therefore help to elucidate itsgeneral function. Although this strategy was under de-bate in the beginning (Puchta, 1998; Reski, 1998a;

Liljegren and Yanofsky, 1998), the reports of suc-cessful knockouts of moss are starting to accumulate(e.g. Hofmann et al., 1999; Girod et al., 1999) andthe technique is in the process of becoming a popu-lar tool in plant biology. Also from a practical pointof view experiments with Physcomitrella are attrac-tive. The life cycle takes 23 months per generationand consists mainly of the gametophyte stage. Thus,in contrast to diploid organisms, recessive phenotypescan be scored immediately after a single gene target-ing reaction. After fertilization multiple spores can beharvested easily for analysis (for recent reviews onthe biology of Physcomitrella patens see Cove et al.,1997; Reski, 1998b, 1999).

Why is gene targeting so efficient in moss?An extremely important question that remains to

be answered especially as it might help to establishgene targeting in higher plants is why homolo-gous integration takes place so efficiently in moss?Many possible explanations can be brought forward.Is the enzyme machinery different or the regulationof its expression? Apparently PEG-mediated trans-formation is especially efficient for obtaining genetargeting in moss (Schaefer, 2001) but not in higherplants (Mengiste and Paszkowski, 1999). Transfor-mation apparently takes place during a specific phaseof the cell cycle, at the G2/M transition, and it canbe speculated that homologous recombination is es-pecially efficient at this time (Reski, 1998a). Indeed,indirect evidence exists that in mammals homologousrecombination is more efficient during S and G2 phasethan within G1 (Dronkert et al., 2000). However, itremains to be shown whether a putative moss-specificcell cycle block is indeed responsible for the dramaticenhancement in homologous recombination. It waspostulated that the haploid state per se is a prereq-uisite for the efficiency of gene targeting (Schaefferand Zryd, 1998); however, in higher plants transfor-mation experiments with haploid tissue did not resultin higher gene targeting frequencies (Mengiste andPaszkowski, 1999). An intriguing finding is the factthat in many cases several copies of the plasmid DNAwere integrated per target locus (Schaeffer and Zryd,1997) and that extrachromosomal DNA can persistin Physcomitrella over longer periods (Ashton et al.,2000). This hints to the possibility that incommingplasmid DNA might become more activated for ho-mologous interactions. Only further studies about therecombination mechanisms in Physcomitrella patensand the factors involved will give us a deeper un-

180

derstanding of the recombination behaviour of thisorganism.

Conclusions

A major goal of plant biotechnology is to develop newand improve existing elite cultivars. To meet this goal,it will be necessary to both improve existing and de-velop novel strategies for plant genome manipulation.The ability to modify a resident gene in situ or to in-tegrate a transgene at a specific genomic position in acontrolled way is a central issue in this context. Genetargeting will permit validation of gene function inplants through knockout of target genes. This reversegenetics approach is expected to become increasinglyimportant in the post-genomic era in order to demon-strate gene function thus permitting protein engineer-ing and overcoming problems of gene silencing andposition effects so often encountered in transgenicplants. Time will tell when and by which approachgene targeting in higher plants will be achieved in afeasible way. That it will be achieved in the long runseems to me to be out of question, but is is hard toguess when it will be the case. As we have variousapproaches at hand, we should not be too pessimisticabout time frames. In general, two different strate-gies have been developed to improve gene targetingfrequencies in plants. The first one aims at achievinggene targeting in a wild-type genetic background andincludes the production of a targeting vector in vivo,chimeraplasty and the moss Physcomitrella patens inwhich gene targeting is naturally efficient. The secondstrategy aims at improving gene targeting efficien-cies in a mutated genetic background and includes theover-expression of heterologous recombination genes,the modulation of the expression of endogenous plantgenes involved in recombination and the identifica-tion of hyper-recombinogenic mutants. Wheres thefirst strategy will result in mutants with a stable ge-netic background, further offorts have to be taken toavoid unwanted secondary effects due to a mutatedgenetic background obtained with the second strategy.All these approaches are followed up in an active com-munity by a growing number of researchers and wehave to wait and see what their results will be.

Acknowledgements

I thank Ingo Schubert for critical comments on themanuscript and all my colleagues in the field, espe-

cially my partners within the framework IV and Vprogrammes of the EU, for many fruitful interactionsover the past dozen years. The work in my labora-tory is funded by grants of the Deutche Forschungs-gemeinschaft, the Kultusministerium of the LandSachsen-Anhalt, the Bundesministerium fr Bildingund Forschung and the EU.

References

Arabidopsis Genome Initiative. 2000. Analysis of the genome se-quence of the flowering plant Arabidopsis thaliana. Nature 408:796815.

Ashton, N.W., Champagne, C.E.M., Weiler, T. and Verkoczy, L.K.2000. The bryophyte Physcomitrella patens replicates extrachro-mosomal transgenic elements. New Phytol. 146: 391402.

Beetham, P.R., Kipp, P.B., Sawycky, X.L., Arntzen, C.J. andMay, G.D. 1999. A tool for functional plant genomics: chimericRNA/DNA oligonucleotides cause in vivo genespecific muta-tions. Proc. Natl. Acad. Sci. USA 96: 87748778.

Bhatt, A.M., Canales, C. and Dickinson, H.G. 2001. Plant meiosis:the means to 1N. Trends Plant Sci. 6: 114121.

Bundock, P., den Dulk-Ras, A., Beijersbergen, A. and Hooykaas,P.J. 1995. Trans-kingdom T-DNA transfer from Agrobacteriumtumefaciens to Saccharomyces cerevisiae. EMBO J. 14: 32063214.

Carlson, P.S. 1974. Mitotic crossing over in a higher plant. Genet.Res. Camb. 24: 109112.

Cole-Strauss, A., Gamper, H., Holloman, W.K., Munoz, M., Cheng,N. and Kmiec, E.B. 1999. Targeted gene repair directed by thechimeric RNA/DNA oligonucleotide in a mammalian cell-freeextract. Nucl. Acids Res. 27: 13231330.

Cole-Strauss, A., Yoon, K., Xiang, Y., Byrne, B.C., Rice, M.C.,Gryn, J., Holloman, W.K. and Kmiec, E.B. 1996. Correction ofthe mutation responsible for sickle cell anemia by an RNA-DNAoligonucleotide. Science 273: 13861389.

Cove, D.J., Knight, C.D. and Lamparter, T. 1997. Mosses as modelsystems. Trends Plant Sci. 2: 99105.

Doetschman, T., Gregg, R.G., Maeda, N., Hooper, M.L., Melton,D.W., Thompson, S. and Smithies, O. 1987. Targeted correctionof a mutant HPRT gene in mouse embryonic stem cells. Nature330: 576578.

Dronkert, M.L., Beverloo, H.B., Johnson, R.D., Hoeijmakers, J.H.,Jasin, M. and Kanaar, R. 2000. Mouse RAD54 affects DNAdouble-strand break repair and sister chromatid exchange. Mol.Cell Biol. 20: 31473156.

Engels, W.R. 2000. Reversal of fortune for Drosophila geneticists?Science 288: 19731975.

Essers, J., van Steeg, H., de Wit, J., Swagemakers, S.M., Ver-meij, M., Hoeijmakers. J.H. and Kanaar, R. 2000. Homologousand non-homologous recombination differentially affect DNAdamage repair in mice. EMBO J. 19: 17031710.

Gallego, M.E., Jeanneau, M., Granier, F., Bouchez, D., Bechtold, N.and White I. 2001. Disruption of the Arabidopsis RAD50 geneleads to plant sterility and MMS sensitivity. Plant J. 25: 3141.

Gallego, M.E., Sirand-Pugnet, P. and White, C.I. 1999. Positive-negative selection and T-DNA stability in Arabidopsis transfor-mation. Plant Mol. Biol. 39: 8393.

Gamper, H.B., Parekh, H., Rice, M.C., Bruner, M., Youkey, H.and kmiec, EB. 2000. The DNA strand of chimeric RNA/DNAoigonucleotides can direct gene repair/conversion activity in

181

mammalian and plant cell-free extracts. Nucl. Acids Res. 28:43324339.

Gherbi, H., Gallego, M.E., Jalut, N., Lucht, J.M., Hohn, B.and White, C.I. 2001. Homologous recombination in planta isstimulated in the absence of Rad50. EMBO Rep. 2: 287291.

Girke, T., Schmidt, H., Zahringer, U., Reski, R. and Heinz, E. 1998.Identification of a novel 6-acyl-group desaturase by targetedgene disruption in Physcomitrella patens. Plant J. 15: 3948.

Girod, P.A., Fu, H., Zryd, J.P. and Vierstra, R.D. 1999. Multiubiqui-tin chain binding subinit MCB1 (RPN10) of the 26S proteasomeis essential for developmental progression in Physcomitrellapatens. Plant Cell 11: 14571472.

Gorbunova, V., Avivi-Ragolski, N., Shalev, G., Kovalchuk, I., Abbo,S., Hohn, B. and Levy, A.A. 2000. A new hyperrecombinogenicmutant of Nicotiana tabacum. Plant J. 24: 601611.

Gorbunova, V. and Levy, A.A. 1999. How plants make ends meet:DNA double-strand break repair. Trends Plant Sci. 4: 263269.

Haber, J.E. 1998. The many interfaces of Mre11. Cell 95: 583586.Halfter, U., Morris, P.C., and Willmitzer, L. 1992. Gene targeting in

Arabidopsis thaliana. Mol. Genet. 231: 186193.Hanin, M., Mengiste, T., Bogucki, A. and Paszkowski, J. 2000.

Elevated levels of intrachromosomal homologous recombinationin Arabidopsis overexpressing the MIM gene. Plant J. 24: 183189.

Hansen, G. and Chilton, M.D. 1999. Lessons in gene transfer toplants by a gifted microbe. Curr. Top. Microbiol. Immunol. 240:2157.

Hartung, F., Plchova, H. and Puchta, H. 2000. Molecular characteri-zation of RecQ homologues in Arabidopsis thaliana. Nucl. AcidsRes. 28: 42754282.

Hartung, F. and Puchta, H. 1999. Isolation of the complete cDNA ofthe Mre11 homologue of Arabidopsis (Accession No. AJ243822)indicates conservation of DNA recombination mechanisms be-tween plants and other eucaryotes (PGR 99132). Plant Physiol.121: 311.

Hofman, A.H., Codon, A.C., Ivascu, C., Russo, V.E., Knight, C.,Cove, D., Schaefer, D.G., Chakhparoian, M. and Zryd, J.P. 1999.A specific member of the Cab multigene family can be efficientlytargeted and disrupted in the moss Physcomitrella patens. Mol.Gen. Genet. 261: 9299.

Hohn, B. and Puchta, H. 1999. Gene therapy in plants. Proc. Natl.Acad. Sci. USA 96: 83218323.

Jasin, M., Moynaham, M.E. and Richardson, C. 1996. Targetedtransgenesis. Proc. Natl. Acad. Sci. USA 93: 88048808.

Karow, J.K., Wu, L. and Hickson, I.D. 2000. RecQ family helicases:roles in cancer and ageing. Curr. Opin. Genet. Dev. 10: 3238.

Kempin, S.A., Liljegren, S.J., Block, L.M., Rounsley, S.D., Yanof-sky, M.F. and Lam E. 1997. Targeted disruption in Arabidopsis.Nature 389: 802803.

Kumar, S. and Fladung, M. 2001. Controlling trangene integrationin plants. Trends Plant Sci. 6: 155159.

Lee, K.Y., Lund, P., Lowe, K. and Dunsmuir, P. 1990. Homolo-gous recombination in plant cells after Agrobacterium-mediatedtransformation. Plant Cell 2: 415425.

Liljegren, S.J and Yanofsky, M.F. 1998. Towards targeted transfor-mation in plants. Response: targeting Arabidopsis. Trends PlantSci. 3: 7980.

Mengiste, T. and Paszkowski, J. 1999. Prospects for the precise en-gineering of plant genomes by homologous recombination. Biol.Chem. 380: 749758.

Mengiste, T., Revenkova, E., Bechtold, N. and Paszkowski, J.1999. An SMC-like protein is required for efficient homologousrecombination in Arabidopsis. EMBO J. 18: 45054512.

Miao, Z.-H. and Lam, E. 1995. Targeted disruption of the TGA3locus in Arabidopsis thaliana. Plant J. 7: 359365.

Offringa, R., de Groot, M.J.A., Haagsman, H.J., Does, M.P.,van den Elzen, P.J.M. and Hooykaas, P.J.J. 1990. Extrachro-mosomal homologous recombination and gene targeting in plantcells after Agrobacterium-mediated transformation. EMBO J. 9:30773084.

Offringa, R., Franke-van Dijk, M.E.I., de Groot, M.J.A., van denElzen, P.J.M. and Hooykaas, P.J.J. 1993. Nonreciprocal homol-ogous recombination between Agrobacterium transferred DNAand a plant chromosomal locus. Proc. Natl. Acad. Sci. USA 90:73467350.

Oh, T.J. and May, G.D. 2001. Oligonucleotide-directed plant genetargeting. Curr. Opin. Biotechnol. 12: 169172.

Ow, D. 2001. Applications of site-specific recombination. PlantMol. Biol., this issue.

Paques, F. and Haber, J.E. 1999. Multiple pathways of recom-bination induced by double-strand breaks in Saccharomycescerevisiae. Microbiol. Mol. Biol. Rev. 63: 349404.

Paszkowski, J., Baur, M., Bogucki, A. and Potrykus I. 1988. Genetargeting in plants. EMBO J. 7: 40214026.

Potrykus, I. and Spangenberg, G. 1995. Gene Transfer to Plants.Springer-Verlag, Berlin.

Puchta, H. 1998a. Towards targeted transformation in plants. TrendsPlant Sci. 3: 7778.

Puchta, H. 1998b. Repair of genomic double-strand breaks insomatic plant cells by one-sided invasion of homologous se-quences. Plant J. 13: 331339.

Puchta, H. 1999. DSB-induced recombination between ectopichomologous sequences in somatic plant cells. Genetics 152:11731181.

Puchta, H., Dujon, B. and Hohn, B. 1996. Two different but relatedmechanisms are used in plants for the repair of genomic double-strand breaks by homologous recombination. Proc. Natl. Acad.Sci. USA 93: 50555060.

Reiss, B., Klemm, M., Kosak, H. and Schell, J. 1996. RecA pro-tein stimulates homologous recombination in plants. Proc. Natl.Acad. Sci. USA 93: 30943098.

Reiss, B. Schubert, I., Kpchen, K., Wendeler, E., Schell, J. andPuchta, H. 2000. RecA stimulates sister chromatid exchange andthe fidelity of double-strand break repair, but not gene targeting,in plants transformed by Agrobacterium. Proc. Natl. Acad. Sci.USA 97: 33583363.

Reski, R. 1998a. Physcomitrella and Arabidopsis: the David andGoliath of reverse genetics. Trends Plant Sci. 3: 209210.

Reski, R. 1998b. Development, genetics and molecular biology ofmosses. Bot. Acta 111: 115.

Reski, R. 1999. Molecular genetics of Physcomitrella. Planta 208:301309.

Reski, R., Reynolds, S., Wehe, M., Kleber-Janke, T. and Kruse,S. 1998. Moss (Physcomitrella patens) expressed sequence tagsinclude several sequences which are novel for plants. Bot. Acta111: 145151.

Rice, M.C., May, G.D., Kipp, P.B., Parekh, H. and Kmiec, E.B.2000. Genetic repair of mutations in plant cell-free extracts di-rected by specific chimeric oligonucleotides. Plant Physiol. 123:427438.

Rice, M.C., Czymmek, K. and Kmiec, E.B. 2001. The potential ofnucleic acid repair in functional genomics. Nature Biotechnol.19: 321326.

Risseeuw, E., Offringa, R., Franke-van Dijk, M. E. and Hooykaas,P.J. 1995. Targeted recombination in plants using Agrobacteriumcoincides with additional rearrangements at the target locus.Plant J. 7: 109119.

182

Risseeuw, E., Franke-van Dijk, M.E. and Hooykaas, P.J. 1997. Genetargeting and instability of Agrobacterium T-DNA loci in theplant genome. Plant J. 11: 717728.

Rong, Y.S. and Golic, K.G. 2000. Gene targeting by homologousrecombination in Drosophila. Science 288: 20132018.

Rong, Y.S. and Golic, K.G. 2001. A targeted gene knockout inDrosophila. Genetics 157: 13071312.

Rossi, L., Tinland, B. and Hohn, B. 1996. Role of the virulenceprotein of Agrobacterium tumefaciens in the plant cell. In: H.Spaink, P. Hooykaas and A. Kondorosi (Eds.) The Rhizobiaceae.Kluwer Academic Publishers, Dordrecht, Netherlands, pp. 303320.

Rubin, E. and Levy, A.A. 1997. Abortive gap repair: the underlyingmechanism for Ds elements formation. Mol. Cell Biol. 17: 62946302.

Schaefer, D.G. 2001. Gene targeting in Physcomitrella patens. Curr.Opin. Plant Biol. 4: 143150.

Schaefer, D.G. and Zryd, J.P. 1997. Efficient gene targeting in themoss Physcomitrella patens. Plant J. 11: 11951206.

Shalev, G., Sitrit, Y., Avivi-Ragolski, N., Lichenstein, C. and LevyA.A. 1999. Stimulation of homologous recombination in plantsby expression of the bacterial resolvase RuvC. Proc. Natl. Acad.Sci. USA 96: 73987402.

Strepp, R., Scholz., S., Kruse, S., Speth, V. and Reski R. 1998. Plantnuclear gene knockout reveals a role in plastid division for thehomolog of the bacterial cell division protein FtsZ, an ancestraltubulin. Proc. Natl. Acad. Sci. USA 95: 43684373.

Swoboda, P., Gal, S., Hohn, B. and Puchta, H. 1994. Intrachromo-somal homologous recombination in whole plants. EMBO J. 13:484489.

Szostak, J.W., Orr-Weaver, T.L., Rothstein, R.J. and Stahl, F.W.1983. The double-strand break repair model of recombination.Cell 33: 2535.

Thomas, K.R. and Capecchi, M.R. 1987. Site-directed mutagenesisby gene targeting in mouse embryo-derived stem cells. Cell 51:503512.

Thykjaer, T., Finnemann, J., Schausser, L., Christensen, L., Poulsen,C. and Stougaard, J. 1997. Gene targeting approaches using

positive-negative selection and large flanking regions. Plant Mol.Biol. 35: 523530.

Tinland, B. 1996. The integration of T-DNA into plant genomes.Trends Plant Sci. 1: 178184.

van der Steege, G., Schuilenga-Hut, P.H., Buys, C.H., Scheffer, H.,Pas, H.H. and Jonkman M.F. 2001. Persistent failures in generepair. Nature Biotechnol. 19: 305306.

Vergunst, A.C. and Hooykaas, P.J.J. 1999. Recombination in theplant genome and its application in biotechnology. Crit. Rev.Plant. Sci. 18: 131.

Wang, W., Seki, M., Narita, Y., Sonoda. E., Takeda, S., Yamada,K., Masuko, T., Katada. T. and Enomoto, T. 2000. Possible asso-ciation of BLM in decreasing DNA double strand breaks duringDNA replication. EMBO J. 19: 34283435.

West, C.E., Waterworth, W.M., Jiang, Q. and Bray C.M. 2000. Ara-bidopsis DNA ligase IV is induced by gamma-irradiation andinteracts with an Arabidopsis homologue of the double standbreak repair protein XRCC4. Plant J. 24: 6778.

Yamaguchi-Iwai, Y., Sonoda, E., Sasaki, M.S., Morrison, C.,Haraguchi, T., Hiraoka, Y., Yamashita, Y.M., Yagi, T., Takata,M., Price, C., Kakazu, N. and Takeda, S. 1999. Mre11 is essentialfor the maintenance of chromosomal DNA in vertebrate cells.EMBO J. 18: 66196629.

Ye, S., Cole-Strauss, A.C., Frank, B. and Kmiec, E.B. 1998. Tar-geted gene correction: a new strategy for molecular medecine.Mol. Med. Today 4: 431437.

Zhu, T., Peterson, D.J., Tagliani, L., St. Clair, G., Baszczynski,C. and Bowen B. 1999. Targeted manipulation of maize genesin vivo using chimeric RNA/DNA oligonucleotides. Proc. Natl.Acad. Sci. USA 96: 87688773.

Zhu, T., Mettenburg, K., Peterson, D.J., Tagliana, L. and Baszczyn-ski, C.L. 2000. Engineering herbicide-resistant maize usingchimeric RNA/DNA oligonucleotides. Nature Biotechnol. 18:555558.

Zupan, J., Muth, T.R., Draper, O. and Zambryski, P. 2000. Thetransfer of DNA from Agrobacterium tumefaciens into plants: afeast of fudamental insights. Plant J. 23: 1128.