Hohn, B.

Puchta, H.

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Green light for gene targeting in plantsHolger Puchta* and Barbara Hohn†‡*Botany II, University of Karlsruhe, D-76128 Karlsruhe, Germany; and †Friedrich Miescher Institute for Biomedical Research,Maulbeerstrasse 66, CH-4058 Basel, SwitzerlandPrecision engineering of eukary-otic organisms requires efficienttechniques for homology-basedreplacement of an endogenousgene by an introduced gene, a processtermed gene targeting (GT). Becauseforeign DNA is preferentially integratedby nonhomologous recombination inplants and animals, special strategies areneeded to increase the frequency andfacilitate the detection of GT. Develop-ing these strategies has proved particu-larly difficult in plants, for which GTefficiencies are on the order of 103 to104 targeted events per transformedplant (1, 2). This low frequency seemsto be the result of a natural barrier tointegration of homologous sequencesin higher plants. By contrast, efficientGT has been demonstrated in lowerplants, such as the moss Physcomitrellapatens (3).The time appears ripe for a break-through in this field, and one has nowbeen reported in this issue of PNAS.Avraham Levy and his group (4) de-scribe a jump of between one and twoorders of magnitude in GT frequency inArabidopsis thaliana plants overexpress-ing the yeast chromatin remodeling pro-tein Rad54. Their innovative approachwas facilitated by the development of aningenious assay that uses fluorescentseeds to identify targeted insertions.Targeting by BreakingPrevious attempts to improve GT effi-ciency include the introduction of dou-ble-stranded breaks (DSBs) at the targetsite and modification of proteins in-volved in homologous recombination(HR). DSBs are obligatory recombina-tion intermediates, and dedicated endo-nucleases are recruited to introducethem during meiosis. The introductionof a target site for a rare cutter restric-tion enzyme increased the targeting effi-ciency to such a site by two orders ofmagnitude in the somatic tissues inwhich GT normally takes place (5).However, the DSB has to be introducedat or close to the gene to be changed,which has been achieved in mammaliancells by using reprogrammed endonucle-ases whose catalytic domain was fusedto a sequence-specific DNA-bindingdomain (ref. 6 and references therein).A site-specific zinc-finger endonucleasehas been successfully employed to inducesite-specific mutations by nonhomolo-gous end-joining in Arabidopsis (7).Most important in the context of GT isthe recent achievement in tobacco of atargeting efficiency of 10% using a zinc-finger endonuclease to introduce breaksin a model target gene (D. Voytas, per-sonal communication).Targeting by Modifications of ProteinsInvolved in the Mechanics of HRThe molecular machinery involved inHR and nonhomologous recombinationis largely conserved in eukaryotes and,to some extent, in prokaryotes. An obvi-ous approach to improving the effi-ciency of GT in plants is to overexpressHR or heterologous recombination pro-teins. However, overexpression of nei-ther the bacterial RecA protein, themolecule central to strand exchange, norRuvC, the Holliday junction resolvase,improved GT efficiency (8, 9). Screen-ing of Arabidopsis mutants for plantswith increased levels of somatic HR orof radiation sensitivity for use in GTapplications remains an option (dis-cussed in ref. 10).Now Shaked et al. (4) report that theSaccharomyces cerevisiae Rad54 proteinsubstantially boosts GT frequency.These authors devised an ingenious GTassay by using a promoterless GFP geneinserted in an Arabidopsis cruciferingene. Cruciferin is a seed storage pro-tein; hence, integration of the GFP-containing gene into the one of thegenomic genes should (and does) pro-duce fluorescent seeds (Fig. 1). BecauseArabidopsis plants produce many thou-sands of seeds, this convenient and non-destructive assay allowed the authors toidentify, recover, and analyze many pu-tative GT events. Surprisingly, Shaked etal. report that the GFP–cruciferin con-struct was correctly integrated into aresident cruciferin gene in all of the 19plants grown from fluorescent seeds andsubjected to molecular analysis. Thisprecision is particularly remarkable be-cause expression of the GFP gene re-quired a single crossover to capture thecruciferin promoter, yet all analyzed in-sertions had the genomic sequencedownstream of the GFP gene as well.Ectopic targeting, or integration by acombination of HR and nonhomologousrecombination, had been reported inearlier GT experiments (11,12). More-over, the frequency of GT increasedfrom 0.1–1% in control plants to 3–17%in transgenic Rad54 plants, a frequencysufficiently high to form the basis of aroutine genetic modification procedure.Thus, Rad54 might not only enhancetargeting efficiency per se but might alsosuppress nonhomologous recombination.Either alternatively or in addition, therelatively long stretch of homology onthe 3 side of the GFP gene may lead topreferential use of the HR pathway,given that the assay does not select forrestoration of the sequence downstreamof the marker gene.GT by Chromatin Remodeling?HR, like many other DNA-related func-tions, depends on sequence recognition,necessitating the temporary removal ofnucleosomes. The Arabidopsis INO80protein is, like Rad54, a member of theSwi2Snf2 ATPase superfamily and hasbeen shown to be important for efficientsomatic HR (13). Its yeast ortholog ispart of a chromatin remodeling complex(14). Because Arabidopsis orthologs ofthe other components of the complexhave been identified in the plant ge-nome (O. Fritsch, personal communi-cation), INO80 may have a functionsimilar to that of the Rad54 protein.Rad54 orthologs in other organismshave also been implicated in GT. Aknockout of the chicken RAD54 or-tholog substantially decreased GT in thehighly recombinogenic chicken DT40cell line (15); likewise, disruption of theSee companion article on page 12265.‡To whom correspondence should be addressed. E-mail:barbara.hohn@fmi.ch.© 2005 by The National Academy of Sciences of the USAFig. 1. Glowing seeds: The cruciferin promoter–GFP-based GT technology. (Image courtesy ofA. Levy, Weizmann Institute, Rehovot, Israel.)www.pnas.orgcgidoi10.1073pnas.0505202102 PNAS  August 23, 2005  vol. 102  no. 34  11961–11962COMMENTARYmouse RAD54 ortholog suppresses GT(16). RAD54 proteins, especially thewell characterized yeast protein, havebeen shown to participate in severalaspects of HR (reviewed in ref. 17).Therefore, several scenarios can be en-visaged for how the yeast protein mightenhance GT in plants. Fig. 2 shows amodel based on the assumption thatthe yeast Rad54 protein interacts withthe Rad51 strand-exchange protein ofArabidopsis. An alternative explanationfavored by Shaked et al. (4) suggeststhat the chromatin remodeling activityof Rad54 is permitting invasion ofthe incoming DNA strand withoutinvolvement of other plant proteins.Analysis of Arabidopsis plants overex-pressing other Rad54 proteins, especiallyplant orthologs, will help explore thismechanism.Challenges for the FutureThe results reported by Shaked et al. (4)represent a major advance toward thegoal of precision gene modification inplants. If increased levels of either theyeast Rad54 protein or its plant or-thologs confer similar increases in GTfrequency in crop plants, it will becomefeasible to modify gene sequences invitro and replace the resident gene withthe modified gene, something that is notcurrently possible. However, there arelikely to be major hurdles ahead.Will the results reported with the Ara-bidopsis cruciferin gene be generalizableto other Arabidopsis genes? It is gener-ally believed, at least in animal systems,that the acceptor gene must be highlyexpressed at the moment of the target-ing event. Yet egg cells, the putativetargets of T-DNA-mediated transforma-tion (18), probably do not express theseed-specific cruciferin gene. Becausechromatin structures differ between ac-tive and inactive genes, it will be inter-esting to see whether GT efficienciesdepend on the gene expression levels ofRad54-overexpressing plants. Will suchtechniques work in crop plants whosegenomes are very much larger than thatof Arabidopsis? Comparisons of GT effi-ciencies obtained in previous work inArabidopsis and rice rather suggest thatgenome size is not limiting and there-fore that enhancing chromating remod-eling activity may work (1, 2).A more serious limitation may be thedifficulty of transforming crop plants.Arabidopsis plants are readily trans-formed by dipping or infiltrating plantswith Agrobacterium strains carrying thetransgene (19). This simple procedurehas so far not been successful in majorcrop species, and the heroic efforts re-quired to transform rice calli and screenthem for GT events are far from routine(2). The choice of GT vector may alsobe important. In the experiments de-scribed in this Commentary, the organ-ism used for transformation in mostcases was Agrobacterium tumefaciens, thetransforming entity of which is single-stranded DNA. Thus far, differenceshave not been observed in GT efficien-cies between experiments using T-DNAand those using DNA transformation.It is to be hoped that the techniquedescribed here will be independent ofthe transformation procedure used.Despite the many questions to be an-swered, the development of this new GTtechnology represents a crucial step inimproving GT for experimental pur-poses. Moreover, it has the potential toincrease public acceptance of plant genemodification by molecular techniques.1. Hanin, M., Volrath, S., Bogucki, A., Bricker, M.,Ward, E. & Paszkowski, J. (2001) Plant J. 28,671–677.2. Terada, R., Urawa, H., Inagaki, Y., Tsugane, K. &Iida, S. (2002) Nat. Biotechnol. 20, 1030–1034.3. Schaefer, D. G. (2002) Annu. Rev. Plant Biol. 53,477–501.4. Shaked, H., Melamed-Bessudo, C. & Levy, A. A.(2005) Proc. Natl. Acad. Sci. USA 102, 12265–12269.5. Puchta, H., Dujon, B. & Hohn, B. (1996) Proc.Natl. Acad. Sci. USA 93, 5055–5060.6. Urnov, F. D., Miller, J. C., Lee, Y. L., Beausejour,C. M., Rock, J. M., Augustus, S., Jamieson, A. C.,Porteus, M. H., Gregory, P. D. & Holmes, M. C.(2005) Nature 435, 646–651.7. Lloyd, A., Plaisier, C. L., Carroll, D. & Drews,G. N. (2005) Proc. Natl. Acad. Sci. USA 102,2232–2237.8. Reiss, B., Schubert, I., Köpchen, K., Wendeler, E.,Schell, J. & Puchta, H. (2000) Proc. Natl. Acad.Sci. USA 96, 3358–3363.9. Shalev, G., Sitrit, Y., Avivi-Ragolski, N., Lichten-stein, C. & Levy, A. A. (1999) Proc. Natl. Acad. Sci.USA 96, 7398–7402.10. Schuermann, D., Molinier, J., Fritsch, O. & Hohn,B. (2005) Trends Genet. 21, 172–181.11. Puchta, H. (2002) Plant Mol. Biol. 48, 173–182.12. Hohn, B. & Puchta, H. (2003) Trends Plant Sci. 8,51–53.13. Fritsch, O., Benvenuto, G., Bowler, C., Molinier,J. & Hohn, B. (2004) Mol. Cell. 16, 479–485.14. Shen, X., Mizuguchi, G., Hamiche, A. & Wu, C.(2000) Nature 406, 541–544.15. Bezzubova, O., Silbergleit, A., Yamaguchi-Iwai,Y., Takeda, S. & Buerstedde, J. M. (1997) Cell 89,185–193.16. Essers, J., Hendriks, R. W., Swagemakers, S. M.,Troelstra, C., de Wit, J., Bootsma, D., Hoeijmak-ers, J. H. & Kanaar, R. (1997) Cell 89, 195–204.17. Tan, T. L. R., Kanaar, R. & Wyman, C. (2003)DNA Repair 2, 787–794.18. Bechtold, N., Jaudeau, B., Jolivet, S., Maba, B. G.,Vezon, D., Voisin, R. & Pelletier, G. (2000)Genetics 155, 1875–188719. Bechtold, N., Ellis, J. & Pelletier, G. (1993) C. R.Acad. Sci. Paris Life Sci. 316, 1194 –119920. Alexeev, A., Mazin, A. & Kowalczykowski, S. C.(2003) Nat. Struct. Biol. 10, 182–186.21. Jaskelioff, M., Van Komen, S., Krebs, J. E., Sung,P. & Peterson, C. L. (2003) J. Bio.Chem. 278,9212–9218.22. Solinger, J. A., Kiianitsa, K. & Heyer, W.-D.(2002) Mol. Cell 10, 1175–1188Fig. 2. Hypothetical model for Rad54-aided chromatin remodeling and GT. Shown is the chromatinremodeling activity of Rad54 stimulated by the presence of a filament consisting of single-stranded DNAand the single-stranded DNA-binding protein RAD51 (20, 21). In vitro data suggest the followinghypothetical scenario for GT in Arabidopsis: T-DNA, coated by the plant ortholog RAD51, recruits yeastRAD54, which, by its capacity to interact with nucleosomes, promotes remodeling of chromatin untilhomology is encountered. This homology would lead to strand invasion and subsequent formation andresolution of Holliday junctions. It is not known, however, whether the yeast protein would interact withthe endogenous RAD51 protein, and species specificity in this interaction has indeed been demonstrated(22). The activity of the yeast Rad54 protein in dislocating Rad51 from double-stranded DNA (22) may beessential for the later stages of the recombination reaction. (Image courtesy of T. Hohn, BotanicalInstitute, Basel.)11962  www.pnas.orgcgidoi10.1073pnas.0505202102 Puchta and Hohn

Green light for gene targeting in plants

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Green light for gene targeting in plants

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