Plant Molecular Biology 51: 263–279, 2003.© 2003 Kluwer Academic Publishers. Printed in the Netherlands.

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Cre recombinase expression can result in phenotypic aberrations in plants

Eric R. Coppoolse1,∗, Marianne J. de Vroomen, Dick Roelofs, Jaap Smit, Femke van Gennip,Bart J.M. Hersmus, H. John J. Nijkamp and Mark J.J. van Haaren2

Department of Genetics, Institute for Molecular Biological Sciences, BioCentrum Amsterdam, Vrije Univer-siteit, De Boelelaan 1087, 1081 HV Amsterdam, Netherlands; present addresses: 1Plant Research International,Droevendaalsesteeg 1, P.O. Box 16, 6700 AA Wageningen, Netherlands (∗author for correspondence; e-mail:e.r.coppoolse@plant.wag-ur.nl; 2KeyGene N.V., AgroBusiness Park 90, P.O. Box 216, 6700 AE Wageningen,Netherlands

Received 18 July 2000; accepted in revised form 29 May 2002

Key words: Cre recombinase, lox P, petunia, site-specific recombination, tobacco, tomato, toxicity

Abstract

The cre recombinase gene was stably introduced and expressed in tomato, petunia and Nicotiana tabacum. Someplants expressing the cre gene driven by a CaMV 35S promoter displayed growth retardation and a distinct patternof chlorosis in their leaves. Although no direct relation can be proven between the phenotype and cre expression,aberrant phenotypes always co-segregate with the transgene, which strongly suggests a correlation. The severityof the phenotype does not correlate with the level of steady-state mRNA in mature leaves, but with the timing ofcre expression during organogenesis. The early onset of cre expression in tomato is correlated with a more severephenotype and with higher germinal transmission frequencies of site-specific deletions. No aberrant phenotype wasobserved when a tissue-specific phaseolin promoter was used to drive the cre gene. The data suggest that for theapplication of recombinases in plants, expression is best limited to specific tissues and a short time frame.

Abbreviations: bar, the phosphinotricin acetyltransferase gene; CAM, chloramphenicol resistance gene; Ds 5′ &Ds 3′, borders of the Ds transposable element from maize forming a functional transposable element that embodiesthe interjacent DNA; gus, the β-glucoronidase gene; gus-int, the gus gene interrupted by a plant intron; hpt,the hygromycin phosphotransferase gene; nptII, the neomycin phosphotransferase gene; ORI, bacterial origin forplasmid replication in Escherichia coli of plasmid p15A

Introduction

Site-specific recombinases of bacteriophages andyeasts have become popular tools in heterologous sys-tems. In particular the Cre/lox system has been usedin many organisms including plants and animals, re-viewed by Ow and Medberry (1995). The popularityof the Cre/lox system is based on its simplicity: be-sides the 38 kDa recombinase subunits and the 34 bplox sites, no accessory factors are required (Sternberget al., 1986). The exact determination of the 3D struc-ture of the synaptic complex of four Cre subunits andtwo lox sites (Guo et al., 1997) makes the Cre recom-

binase one of the better defined molecular biologicaltools.

In plants, the Cre/lox system has been used for avariety of purposes including chromosomal translo-cations (Qin et al., 1994), chromosomal inversions(Medberry et al., 1995; Osborne et al., 1995; Stuur-man et al., 1996), excision of marker genes (Dale andOw, 1991; Russell et al., 1992), tissue-specific activa-tion of a reporter gene (Odell et al., 1994), site-specificintegration (Albert et al., 1995, reviewed by Vergunstand Hooijkaas, 1999) and the resolution of complextransgene integration patterns (Srivastava et al., 1999).

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To achieve these goals, the cre gene was mostlyexpressed from a 35S promoter in Nicotiana tabacum(Odell and Russell, 1994; Ow and Medberry, 1995),Arabidopsis thaliana (Russell et al., 1992; Osborneet al., 1995), and petunia (Que et al., 1998). Asidefrom one report on Petunia hybrida (Que et al., 1998),no anomalies were reported as a result of the intro-duction of cre in plants. In Drosophila melanogaster,however, the chronic expression of Cre recombinaseis reported to be toxic to proliferating cells leadingto clear phenotypic aberrations in the insects (Heid-mann and Lehner, 2001). In transgenic mice, Schmidtet al. (2000) showed that the over-expression of crein the spermatids causes chromosome scrambling af-ter meiosis II, leading to complete male sterility. Inmammalian cell cultures that continuously express thecre gene decreased growth, cytopathic effects andchromosomal aberrations are reported (Silver and Liv-ingston, 2001). Loonstra et al. (2001) show that thetoxicity of Cre in mamelian cells depends on the strandexchange activity and the dose of the active protein.Their cytological analyses indicate that Cre expressionin mouse embryonic fibroblast cell cultures induceschromosome abnormalities and increased frequenciesof sister chromatid exchange.

In this report we show that cre expression canlead to distinct phenotypes in tomato (Lycopersiconesculentum). Similar phenotypes were observed inN. tabacum, petunia and A. thaliana after transforma-tion with cre gene-containing constructs.

Materials and methods

DNA constructs

Escherichia coli strains DH5α and JM101 were usedas a host for recombinant DNA constructions. Stan-dard laboratory procedures were used as described bySambrook et al. (1989)

Construction of the Cre vectors

pMH303 was constructed by sub-cloning a 3.4 kbSacI/HindIII fragment of pED23, containing thecauliflower mosaic virus (CaMV) 35S-cre-nos 3′fusion (Dale and Ow, 1990), into SacI/HindIII-cut pIC19R (Marsh, 1984), From pMH303, a3.4 kb HindIII/XhoI fragment was cloned into theHindIII/SalI-cut pBIN19 binary vector (Bevan, 1984)to obtain the binary vector plasmid pMH2626. Bi-nary vector plasmid pMM222 was constructed by

cloning the 3.2 kb BglII fragment of pMH303, con-taining most of the CaMV 35S promoter, fused to thecre gene and the nos terminator into the BamHI siteof pMOG222 (MOGEN, Netherlands). Binary vectorplasmid pJB30 was constructed by cloning the 3.2 kbBglII fragment of pMH303, containing most of theCaMV 35S promoter, fused to the cre gene and the nosterminator into the BamHI site of pVdH212 (van derHave, Netherlands). pVdH212 contained between theT-DNA borders a GUS gene interrupted with an intronand a CaMV 35S promoter-neomycin phosphotrans-ferase (nptII)-CaMV 35S terminator fusion, for selec-tion of transformants. Binary vector plasmid pJB40was constructed by cloning the HindIII fragment ofp108-cre described by Odell, et al. (1994), contain-ing the French Bean (Phaseolus vulgaris) β-phaseolinpromoter, fused to the cre gene and the phaseolin ter-minator into the HindIII site of pCGN1548 (McBrideand Summerfelt, 1990). Binary vector pJB110 wasconstructed by inserting a 1.2 kb BglII/SalI linker-PCR fragment of pED23 (Dale and Ow, 1990), con-taining the cre coding region, into the BamHI/SalIsites of pBIN19 derivative pVDH275 (van der Have).pVDH275 contains a 284 bp plastocyanin (petE) pro-moter fragment of pea (Pisum sativum) (Pwee andGray, 1993), fused to a nopaline synthase termi-nator (nos) sequence. Besides this plastocyanin ex-pression cassette, pVDH275 contains a CaMV 35Spromoter-nptII-CaMV 35S terminator fusion betweenthe T-DNA borders, for selection of transformants.

Construction of the pMV933 and pD3 constructs

In the binary vector pCGN1548 (McBride andSummerfelt, 1990), a lox-P sequence for site-specific recombination was introduced by cloninga 40 bp double-stranded oligonucleotide (BamHI-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-BamHI) in the BamHI site to produce pMH1241.To produce pMH55, a new polylinker (EcoRI-GGTACCCTCGAGGCTGCAGGGCATGCGGGTA-CC-HindIII) was inserted as a double-strandedoligonucleotide in EcoRI/HindIII-cut pUC19. Toclone the tmsII gene of the Ti plasmid of A. tume-faciens, a 3.5 kb SphI fragment from pTT218 (Har-ing et al., 1989) was cloned into the SphI site ofpMH55, to produce pMH787. In the SphI site ofpMH787 downstream of tmsII, a 2.2 kb SphI frag-ment of pTT283 (Rommens et al., 1992) was inserted,containing the hygromycin phosphotransferase (hpt)gene between a CaMV 35S promoter and the nopaline

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synthase gene (nos) terminator from A. tumefaciens.The resulting plasmid, pMH2057, has a BamHI site inbetween the hpt gene and its 35S promoter.

The construction of the pMV933 T-DNA

For the construction of pMV933, a lox-P sequencewas introduced in the HindIII site of pTT281 (Rom-mens et al., 1992) as a double stranded oligonucleotide(HindIII-GCGGCCGCATAACTTCGTATAATGTAT-GCTATACGAAGTTAT-HindIII). The plasmid pMH-1725 obtained in this way harbours a single BglII sitebetween the inverted repeat termini of the Ds borders,by which this construct can be made linear and clonedas a functional transposon. After BglII digestion ofpMH1725, the linear 7.8 kb fragment was introducedinto the BamHI site between the 35S promoter andthe hpt gene in pMH2057 to obtain pMV904. FrompMV904, a 13.7 kb KpnI fragment is cloned into theKpnI site of pMH1241 to produce pMV933.

The construction of the pD3 T-DNA

The Ds borders of the activator transposable ele-ment from maize were sub-cloned as a single 1.1kb SphI-NruI fragment from pTT280 (Rommenset al., 1992) in SphI/NruI-cut pACYC184 (Changand Cohen, 1978) to produce pACYC-Ds. FrompGSFR280 (De Block et al., 1987) the 35S-bar-nos cassette was cloned as a 1.9 kb EcoRI-HindIIIfragment into the HindIII/AseI-cut pACYC-Ds vec-tor to produce pCTP103. Also, a lox-P sequencewas introduced in pCTP103 by cloning a dou-ble stranded oligonucleotide (HindIII-GCGGCCGC-ATAACTTCGTATAATGTATGCTATACGAAGTTAT-HindIII) in the HindIII site. The plasmid pMH1737obtained this way harbours a single BglII site in be-tween the inverted repeat termini of the Ds borders, bywhich this construct can be made linear and clonedas a functional transposon. After BglII digestion ofpMH1737, the linear 6.8 kb fragment was introducedin the BamHI site between 35S promoter and hpt genein pMH2057 to obtain pMH3030. From pMH3030, a12.7 kb KpnI fragment is cloned into the KpnI site ofpMH1241 to produce pMH3524.

Transformation of tomato

Binary vector plasmids were transferred to A. tume-faciens strain LBA4404 (Hoekema et al., 1983, forpMV933 and pD3), EHA105 (Hood et al., 1993, for97 of 222 pMH2626 explants) or MOG301 (Hood

et al., 1993, for pJB40, pMM222 and for 125 of222 pMH2626 explants) using a cold shock (Chenet al., 1994). Explants (ca. 8 mm × 8 mm) were cutfrom cotyledons of in vitro grown L. esculentum cv.Moneymaker (for pMH2626, pMM222 and pJB40) orUC82B (for pD3 and pMV933). The explants weredipped in an overnight A. tumefaciens culture. In-fected explants were kept on feeder layers of Petunia‘Albino Comanche’ cells for two days. Transformedcalluses were selected on Murashige and Skoog (1962)medium containing 30 g/l sucrose (MS 30), 7.5 g/lagarose, 250 mg/l carbenicillin, 2 mg/l zeatin and40 mg/l kanamycin sulfate according to Horsch et al.(1985). Shoots were rooted on MS 15 medium, con-taining 4% Gelrite, 250 mg/l carbenicillin and 40 mg/lkanamycin sulfate. The putative transformants weretransferred to soil in the greenhouse. Ploidy levelwas estimated by counting the number of chloro-plasts in the stomata (Koornneef et al., 1989); onlydiploid plants were kept for further analysis by PCR(described below). PCR-positive plants were desig-nated primary transformants and, after out-crossingthe primary transformants, with Moneymaker as therecipient parent, F1 plants containing the cre constructwere selected by antibiotic assays as described below.Only resistant plants were used for further analysis.

Transformation of Petunia hybrida

Binary vector plasmids were transferred to A. tumefa-ciens strain LBA4404 (Hoekema et al., 1983) using acold shock (Chen et al., 1994). Leaf discs from youngleaves of in vitro grown Petunia hybrida line V26 weretransformed and shoots were regenerated on shootinduction medium (containing 250 mg/l kanamycin;Duchefa, Netherlands) according to Horsch et al.(1985). In the greenhouse, primary transformants wereselected by Southern and northern analysis.

Transformation of N. tabacum

Binary vector plasmids were transferred to A. tumefa-ciens strain LBA4404 (Hoekema et al., 1983) using acold shock (Chen et al., 1994). Explants (ca. 4 mm ×6 mm) were cut from young leaves of in vitro grownN. tabacum cv. Samsun NN. Explants were pre cul-tured for 48 h at 25 ◦C in liquid MS 20 containing1 mg/l BA and co cultivated for 20 h at 25 ◦C withan overnight A. tumefaciens culture (added to the pre-culture up to OD600 = 0.05). Infected explants wererinsed in liquid pre-culture medium for 20 min andtransferred to MS 20 plates, containing 8 g/l agarose,

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200 mg/l carbenicillin, 200 mg/l Claforan, 1 mg/l BAand 100 mg/l kanamycin sulfate. Shoot initials weregrown on similar medium, without BA. Shoots wererooted on MS 20 medium, containing 8 g/l agarose,200 mg/l carbenicillin, 200 mg/l Claforan, 250 mg/lcasamino acids and 100 mg/l kanamycin sulfate andtransferred to soil in the greenhouse for further analy-sis by PCR (described below). PCR-positive plantswere designated primary transformants.

Transformation of A. thaliana

Binary vector plasmids were transferred to A. tumefa-ciens strain LBA4404 (Hoekema et al., 1983) usinga cold shock (Chen et al., 1994). A. thaliana line‘No O’ root explants were transformed according toValvekens et al. (1988). Primary transformants weretransferred to soil in the greenhouse. The numberof (independent) T-DNA integrations was estimatedby germinating T1 seeds of primary transformantson kanamycin-containing medium. Selfing popula-tions with a single segregating transgene locus wereanalysed by PCR.

Plant DNA isolation

Leaf tissue (4 g) was ground in liquid nitrogen and,after addition of 25 ml cold extraction buffer (0.35 Msorbitol, 20 mM Na2S2O3, 0.1 M Tris-HCl, 5 mMEDTA, pH 7.5), the mixture was centrifuged (10 000×g) for 20 min at 4 ◦C. The pellet was re-suspended in1.25 ml extraction buffer, before addition of 1.75 mlnuclei lysis buffer (2% w/v hexadecyltrimethyl ammo-nium bromide (CTAB), 2 M NaCl, 0.2 M Tris-HCl,50 mM EDTA, pH 7.5) and 0.6 ml 5% w/v sarko-syl. After brief mixing, the solution was incubatedat 65 ◦C for 1 h. After extraction with 10 ml chloro-form: isomylalcohol (24:1), the DNA was precipitatedfrom the aqueous phase with an equal volume ofisopropanol and dissolved in 500 µl TE (10 mM Tris-HCl, 1 mM EDTA, pH 8.0) with 2 µg RNase A at60 ◦C. For PCR analysis, plant DNA was isolatedusing a downscaled version of the method describedabove.

PCR assays

Resistant putative transformants were checked bymeans of PCR with primers npt F (5′-CCGGTTCTTT-TTGTCAAGAC-3′) and npt R (5′-AGAAGAACTC-GTCAAGAAGG-3′) after kanamycin selection, or

primers hpt F (5′-GAACTCACCGCGACGTCTGT-3′) and hpt R (5′-GTCGGCATCTACTCTATTCCT-3′)after hygromycin selection. All putative Cre trans-formants were checked with primers cre top (5′-ATGTCCAATTTACTGACCGTA-3′) and cre bot (5′-TCAATCGCCATCTTCCAGCAGGC-3′). To moni-tor deletion efficiency in D3 plants, primers mas3′ out (5′-GCGATATTATTGCCTTTCGCC-3′) andbar out (5′-CTGCCGGTACCGCCCCGTCC-3′), aliasprimer 2 and primer 3, respectively, were used.To check for deletion in T933 plants, primersmas 3′ out (primer 2, see above) and CAMVenh-2 (5′-CTCCACTGACGTAAGGGATGACG-3′), aliasprimer 11, were used. The annealing position in con-structs D3 and T933 is shown in Figure 1. Primerswere manufactured by Isogen Bioscience (Nether-lands). PCR reactions were performed in 50 µl vol-umes with ca. 70 ng genomic DNA, 0.1 U Taqpolymerase (MRC Netherlands) with the suppliedbuffer, 0.2 mM dNTPs and 1.0 µM primers in 500 µltubes. All primer combinations were used in the sameregime: first, the mixtures were kept at 94 ◦C for4 min, followed by 35 cycles of 60 s at 94 ◦C, 45 sat 59 ◦C, 60 s at 72 ◦C, concluded by 10 min at 72 ◦C,in the time-regulated thermo block of a DNA ThermalCycler (Perkin Elmer, USA).

Antibiotic assays

Progeny of pMH2626 tomato transformants weresprayed about 4 weeks after sowing in flat trays con-taining soil. Leaves of seedlings were sprayed with a400 mg/l kanamycin sulfate (Duchefa, Netherlands)solution in double-distilled water, according to Weideet al. (1989). Progeny of pMM222 tomato transfor-mants were sown in vitro on MS 15 medium, con-taining 7.5 g/l agarose and 100 mg/l hygromycin-B(Duchefa). Seeds were dipped in 70% v/v ethanolprior to sterilization in 1 g/l sodium hypochlorite so-lution for 10 min. Seeds were sown after rinsing insterile demineralised water for 30 min.

Southern blot preparation

Total DNA (8 µg) was digested with restriction en-donucleases (Boehringer, Germany) and the resultingfragments were size-fractionated by electrophoresisthrough 0.8% w/v agarose gels. After depurination in0.25 M HCl solution and denaturation in 0.4 M NaOHsolution, the DNA was transferred onto Hybond-N+membranes (Amersham, UK) by vacuum blotting

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Figure 1. Schematic structures of the T-DNAs of constructs used. Schematic representation of the essential elements in the T-DNAs of theconstructs used (not to scale). Positions and directions of relevant primers are indicated by arrowheads 2, 3 and 11 (see Materials and methods).Constructs pMH2626, pMM222 and pJB30 harbour each a cre gene, cloned in between the cauliflower mosaic virus (CaMV) 35S promoterand the nopalin synthase (nos) terminator. pJB40 contains a cre gene driven by the phaseolin (Ph5′) promoter fragment of the French bean(Phaseolus vulgaris) fused to the phaseolin terminator (Ph3′) sequence (Odell et al., 1994). pJB110 contains a cre gene driven by a 284 bpplastocyanin (petE) promoter fragment of pea (Pisum sativum) (Pwee and Gray, 1993), fused to a nopalin synthase terminator (nos) sequence.Abbreviations: npt, the neomycin phosphotransferase (nptII) gene; hpt, the hygromycin phosphotransferase (hpt) gene; bar, the phosphinotricinacetyl transferase gene; gus, the β-glucoronidase gene; gus-int, the gus gene interrupted by a plant intron; ORI, a p15A bacterial origin ofreplication; CAM, chloramphenicol resistance gene for selection in Escherichia coli; Ds 5′ and Ds 3′: borders of the Ds transposable elementfrom maize, forming a functional transposable element that embodies the interjacent DNA.

(LKB) for 1 h with 0.4 M NaOH. The blot was neu-tralized in 5× SSC (0.75 M NaCl, 0.075 M sodiumcitrate) and the DNA was fixed to the membrane bybaking at 60 ◦C for 1 h, prior to hybridization.

PFG electrophoresis

Megabase-size DNA was isolated from plant nucleiin agarose plugs according to Zhang et al. (1995).

Plugs of 50 µl were digested with restriction en-donucleases (Boehringer, Germany) and the resultingfragments were size-fractionated by electrophoresisthrough 0.8% w/v SeaKem LE agarose in 0.5× TAEgels (Sambrook et al., 1989), using a CHEF DR IIIdevice (BioRad, USA) at 0.2–7 s ramped switch timefor 7 h at 14 ◦C. Gels were stained for 3 h with 250 µgethidium bromide in 250 ml 0.5× TAE and nicked at312 nm UV light for 5 min during photography prior to

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depurination in 0.25 M HCl solution and denaturationin 0.4 M NaOH solution. The DNA was transferredto Hybond-N+ membranes (Amersham, UK) by cap-illary blotting for at least 48 h using 0.4 M NaOH.The blot was neutralized in 5± SSC (0.75 M NaCl,0.075 M sodium citrate) and the DNA was fixated tothe membrane by baking at 60 ◦C for 1 h, prior tohybridization.

RNA analysis

Total RNA was isolated from 0.5 g of leaf tissue,ground in liquid nitrogen. After transfer to 2 ml tubes,containing 750 µl phenol and 750 µl extraction buffer(0.1 M NaCl, 1% w/v sarkosyl, 0.1 M Tris-HCl,20 mM EDTA, pH 8.5) tubes were mixed. After a phe-nol/chloroform (1:1) extraction, and an extraction withonly chloroform, nucleic acids in the aqueous phasewere precipitated with an equal volume of isopropanoland washed with 70% w/v ethanol. Air-dried pelletswere re suspended in 0.5 ml double-distilled water andRNA was precipitated in 2 M LiCl for 16 h at 0 ◦C.After precipitation and centrifugation, pellets werere-suspended in 400 µl double-distilled water, precipi-tated and washed in 70% v/v ethanol and air dried pel-lets were re suspended in 100 µl double-distilled wa-ter. Total RNA (20 µg) was size-fractionated by elec-trophoresis through 1.5% w/v agarose gels containing16% v/v formaldehyde in 20 mM 3-[N-morpholino]-propane-sulfonic acid (MOPS), 5 mM sodium acetate,0.5 mM EDTA, pH 8.0 (Sambrook et al., 1989). TheRNA was transferred onto Hybond-N+ membranes(Amersham, UK) by capillary blotting for at least 24 hwith 20 ◦SSC. The RNA was fixated to the mem-brane by rinsing in 0.04 M NaOH for 1 min and UVirradiation (302 nm) for 1 min prior to hybridization.

Hybridizations

Hybridizations of both RNA and DNA blots were car-ried out in 1 mM EDTA, 0.5 M sodium phosphatepH 7.2, 7% w/v SDS and 1% w/v BSA (Churchand Gilbert, 1984) at 65 ◦C for 16 h in a HY-BAID hybridization oven, with [32P]-dCTP (Amer-sham, UK) radiolabelled probes, prepared by randomprimed DNA (Feinberg and Vogelstein, 1983). Af-ter hybridization, the filters were washed with 0.1×SSC, 0.1% w/v SDS at 60 ◦C, exposed to PhosphorImager screens (Molecular Dynamics, USA) for 16–48 h and scanned on a Phosphor Imager with ImageQuant software. Probes were stripped off by boilingthe membranes in 0.5% w/v SDS solution for 1 min.

Histochemical in situ GUS staining

Young primary leaves of in vitro grown seedlingswere submerged in staining solution (10 mM 5-bromo-4-chloro-3-indolyl β-D-glucuronic acid (X-gluc, Duchefa), 0.1% v/v Triton X-100, 50 mMsodium phosphate, 10 mM EDTA, pH 7.0) and vac-uum infiltrated three times for 1 h and incubated for16 h at 37 ◦C in the dark and bleached with 70% v/vethanol.

Fluorometric MUG assay

Leaf material was harvested by punching a leaf discwith a cap of a 1.5 ml Eppendorf centrifuge tube.Leaf discs were thoroughly ground in 100 µl MUGextraction buffer (0.1% w/v sarkosyl, 0.1% v/v Tri-ton X-100, 50 mM sodium phosphate, 1 mM EDTA,pH 7.0) Samples were centrifuged at 4 ◦C for 1 minand 10 µl of the clear supernatant was added to 40 µlGUS reaction buffer (0.1% w/v sarkosyl, 1 mM 4-methylumbelliferyl-β-D-glucuronide (MUG, Sigma,USA), 50 mM sodium phosphate, 1 mM EDTA,pH 7.0). After incubation at 37 ◦C for 3 h, β-glucuronidase activity was quantified by measuringthe fluorescence of the product, with a FluoroFast 96Fluorometer (3M, USA).

Results

Introduction of the cre gene in plants can induceaberrant phenotypes

To conduct site-specific recombination experiments inpetunia, N. tabacum, A. thaliana and tomato, the cregene was introduced in these species by Agrobac-terium tumefaciens-mediated transformation using thedifferent T-DNA containing constructs shown in Fig-ure 1.

To express the cre gene in petunia, line V26was transformed with pMH2626 (Figure 1). In total,12 different kanamycin-resistant T0 plants (primarytransformants) were obtained, only one of which hada wild-type appearance. The primary transformantsshowed a variety of phenotypes ranging from yellowchlorosis and deformations of the leaves (Figure 2A)to short internodes, stunted growth and delayed flow-ering. Since one of these plants was male-sterile all T0lines were out-crossed by fertilization with pollen ofline V26 to obtain the T1 generation. In the T1 gener-

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Table 1. Overview of the phenotypic observations made in two generations after transformation of the plant species tomato,petunia, N. tabacum and A. thaliana with different cre-containing constructs. In tomato the ‘Cre phenotype’ is rarelyobserved in T0 plants, while its presence is clear in the next generation (T1) of pMH2626 and pMM222 transformants.In petunia and N. tabacum, aberrant phenotypes are already visible in T0 plants and are mostly transmitted to the nextgeneration. A mild ‘Cre phenotype’ is observed in A. thaliana in 50% of the kanamycin resistant progeny of an out-crossof a T1 plant (∗∗∗). Single T-DNA insertion: primary transformants were analysed on Southern blot using both the cre geneand the antibiotic selection gene as a probe, except for ∗∗ A. thaliana, were the number of lines with a single segregatingantibiotic resistance locus was determined in T1 progeny. ND = not determined. ∗ In this single-copy line (C7) the cregene is not expressed.

Number of T0 plants Single Number of T1 lines

plant T-DNA from Total with Cre Totally Only male T-DNA Total with Cre Totally Only male

species construct number phenotype sterile sterile insertion number phenotype sterile sterile

Petunia pMH2626 12 11 0 1 0 10 10 0 ND

Tobacco pJB30 25 5 0 0 ND 25 5 0 0

pJB110 30 6 0 0 ND 30 6 0 0

Arabidopsis pMH2626 10 0 0 0 2∗∗ 2 1∗∗∗ 0 0

Tomato pMH2626 16 2 1 3 1∗ 12 11 2 0

pMM222 4 0 0 1 0 3 3 0 0

pJB40 12 0 0 0 6 6 0 0 0

ation, ten lines showed aberrant phenotypes (overviewin Table 1).

To express the cre gene in N. tabacum, cv. SamsunNN was transformed with pJB110 and pJB30 (Fig-ure 1). In the T-DNA of pJB110 (Figure 1), a strongplastocyanin promoter (Pwee and Gray, 1993) drivesthe cre gene. Out of 30 primary transformants, sixshowed clear chlorosis and (in some plants) severelystunted growth, compared to the wild-type control(Figure 2B). Chlorosis was observed throughout allleaves of the six primary transformants and their off-spring after self-fertilization. In pJB30 transformants,the cre gene is expressed from a 35S promoter (Fig-ure 1). Out of 25 primary transformants, five showedchlorosis and stunted growth (not shown), albeit lesssevere than in plants transformed with pJB110. In allprimary transformants, the construct was passed onto the next generation after self-fertilization. Aberrantphenotypes were only visible in the offspring of thefive affected primary transformants.

To express the cre gene in A. thaliana, line No Owas transformed with pMH2626 (Figure 1). The pri-mary transformants all had a normal phenotype. Twolines were selected on kanamycin-containing mediumcarrying a single segregating transgene locus. In out-crossed progeny of a T1 plant of one particular Creline, a mildly aberrant phenotype (Figure 2C) was ob-served in half of the plants of the kanamycin-resistantT1 population. Before flowering, the leaves showedchlorosis, the leaf edges were deformed and remained

smaller than the wild-type leaves. Although growthand development were slower, the Cre plants flowerednormally and were fertile.

To express the cre gene in tomato, cv. Moneymakerwas transformed with the constructs pJB40, pMH2626and pMM222 (Figure 1). In pJB40 transformants (Fig-ure 1), the cre gene is expressed from a phaseolinpromoter (Odell et al., 1994). All 12 primary transfor-mants were phenotypically normal and six single-copyplants were selected to generate T1 lines. All plantsin the T1 lines were phenotypically normal. Of 16primary pMH2626 transformants, 14 had a normalphenotype. Two T0 plants showed yellow chlorosison the slightly smaller leaves, less vigorous growthcompared to untransformed plants (not shown), andone of these plants was completely sterile. Since ger-minal transmission of the cre construct via the pollenwas essential to most applications, all 15 male-fertileprimary transformants were both self-pollinated andcrossed out at least five times per primary transformantto untransformed Moneymaker plants. Transmissionof the pMH2626 T-DNA occurred in twelve of theseout-crossed progeny, while in the progeny of self-fertilized plants, the construct was transmitted in all 15cases. This indicates that in three cases (including theremaining T0 plant with a chlorotic phenotype) therehas been a selection against the construct either duringmale gametogenesis or in the process of fertilization.

To obtain Cre lines with a different selectablemarker, pMM222 was used (Figure 1), containing

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Figure 2. ‘Cre phenotypes’ in petunia, N. tabacum and A. thaliana.A. Petunia hybrida leaf of an untransformed ‘V26’ plant (left) andtwo leaves of lines transformed with construct pMH2626. In themiddle a leaf of line 7b showing overall chlorosis and deformationof the mature leaves and on the right a leaf of line 3, showing mildmosaic-like chlorosis, growth reduction and deformation of the ma-ture leaves. B. N. tabacum leaf of an untransformed ‘Samsun NN’plant (left). In the middle a leaf of line JB110. 1, showing clear,mosaic-like chlorosis and growth reduction of the mature leaves.On the right a leaf of line JB110. 8, showing overall chlorosisand growth reduction of the mature leaves. C. A. thaliana leaf ofan untransformed ‘No O’ plant (left), and two mature leaves of apMH2626-transformed T1 plant after out-crossing, showing mildmosaic-like chlorosis and growth reduction.

35S-cre with the hpt gene that confers resistance to theantibiotic hygromycin B. The four independent pri-mary pMM222 transformants that were obtained hada normal phenotype and were out-crossed to Mon-eymaker as well as self-fertilized. In one line, thepMM222 construct was not transmitted through themale germline.

Although the majority of the primary transfor-mants of both pMH2626 and pMM222 was phe-notypically normal, their offspring, both after self-fertilization and after crossing to Moneymaker, ger-minated poorly and showed distinct phenotypes (Fig-ure 3A). Chlorotic spots were observed sometimes inthe cotyledons and young leaves, but were usuallymore apparent in the older leaves. Short internodesand smaller, wrinkled leaves were typical character-istics of the plants showing a more severe phenotype.In spite of their appearance, some of the Cre plantswith a severely altered phenotype bore normal flowersand fruits. Based on the timing of the occurrence andthe severity of the phenotypic aberrations, six classesof Cre lines, ranging from normal in class I to a severephenotype in class VI, were demarcated as is indicatedin Figure 3A.

In different plant species similar phenotypic abnor-malities are observed after the introduction of T-DNAscarrying a cre gene. Since different constructs wereused to confer the cre gene, aberrant phenotypes mightbe associated with cre expression, and not with uniquecharacteristics of one of the constructs. To be able tostudy this presumptive ‘Cre phenotype’ in more detail,the following analyses were focused on tomato.

cre gene steady-state mRNA levels and severity of thephenotype

To address whether the phenotypes that were observedin tomato were caused by cre gene expression, the Creplants were analysed molecularly. Figure 3B shows anorthern blot with total RNA of T1 tomato plants, rep-resenting the six phenotypic classes described above.In line C7, which lacks the ‘Cre phenotype’, cremRNA was not detected. In all plants with a phe-notype, cre mRNA was detected, although a strictcorrelation between the level of steady-state mRNA(in mature leaves) and the severity of the phenotypewas not observed. To study the function of the Creprotein in planta, the timing of cre gene expressionduring shoot development and the exact location ofexpression within specific plant organs, two ‘Tester’lines (T933 and G3-3-20) were used to visualize theactivity of the Cre protein in vivo.

The first Tester line, T933, contains constructpMV933 (Figure 1), harbouring a functional GUS-expressing cassette, bracketed by two lox sites indirect repeat. The timing of cre gene expression wascompared between lines by histochemical staining ofyoung primary leaves of in vitro grown F1 seedlings

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Figure 3. The correlation between ‘Cre phenotype’ and cre gene expression in T1 lines of tomato. A. Six classes of ‘Cre phenotypes’ observedin T1 tomato lines. All plants shown were 6 weeks old, except for plant C62.1 which was 10 weeks old. The primary leaves and the cotyledonsare shown in detail in the lower panel. ‘MM’ represents the untransformed control, cv. Moneymaker. Class I: wild type or no ‘Cre phenotype’.This class consisted of one line, C7, which contained a single-copy insertion of the pMH2626 T-DNA (Figure 5C), but did not express the cregene (Figure 3B). Class II: consists of line C43, which showed no ‘Cre phenotype’ in young plants, but clear symptoms in older leaves (notshown) starting at the time of flowering. Class III: represented by line C13, which showed hardly any ‘Cre phenotype’ in the cotyledons, butclear chlorotic spots in all leaves, starting from the primary leaf. The phenotype remained clearly visible throughout the life of the plant. Fourout of 12 pMH2626 lines and the three pMM222 lines (including C22) fell into this class. Class IV: is represented by line C5, showed a clearphenotype in cotyledons and the first five leaves, but a gradual disappearance of the phenotype and recovery of normal growth after formationof leaf 6. Line C59 also fell into this class. Class V: is represented by line C62. This class had narrow and chlorotic cotyledons and small lightgreen leaves with yellow chlorotic spots and a severely stunted and retarded growth. Although the plant remained small, flowers and fruits wereonly little smaller than normal size. Class V also contained Line C60. Class VI: is represented by lines C6 and C18 (panel B), showing poorgermination. Self-fertilized seedlings of the primary transformant had swollen hypocotyls and small cotyledons. Growth was usually arrestedat this stage and the seedlings remained light green or yellow until senescence, mostly within 10 weeks after sowing. Plants that survived thisstage formed small leaves and had short internodes. The older plants look like class V, but growth was even slower. All T1 plants of C18 and C6were sterile. B. Northern blot of total RNA isolated from mature leaves of flowering T1 plants of six Cre lines (and Moneymaker, in lane MM),representing the six classes of Cre transformants. Total RNA was isolated from mature leaves after flowering; the cre transcripts are detectedby a cre probe in the upper panel. After stripping, the same blot was hybridized with the labelled glutaraldehyde phosphodehydrogenase probe(gapdh), as a control for equal loading of the lanes.

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Figure 4. The timing of cre gene expression in tomato. A. Histochemically GUS-stained primary leaves of in vitro cultured F1 seedlings of theCre lines indicated at the top, after crossing T2 plants to line T933. The ‘T933’ leaf represents the positive control, containing a functional,undeleted GUS cassette. The ‘MM’ leaf represents the untransformed control, cv. Moneymaker. Larger white areas indicate earlier deletion ofthe GUS gene during leaf development. B. Upper panel: schematic representation of plant G3-3-20. Linked transposition of the Ds elementfrom the pD3 T-DNA, which was integrated on chromosome 7 of plant D3-3, yielding plant G3-3-20. The positions of the Ds element andthe T-DNA, the distance between the lox sites in G3-3-20 and sizes of the exons of the putative prenyl transferase gene are indicated to scale.M represents the translation start of the putative prenyl transferase gene, ∗ is the stop codon. Lower panel: representative mature leaves of F1plants of line G3-3-20 (see upper panel) crossed to homozygous T2 plants of C5, C13, C22, C43, and C7, respectively. Larger white areasindicate earlier deletion of the putative prenyl transferase gene during leaf development. C. Northern blot of total RNA of mature leaves takenat three or four different time points of Cre transformants, C5, C13 and C43 (see text). The first (1), second (2), fourth (4) and sixth (6) leaves,or the first leaf after flowering (N) were taken for northern blotting and hybridization with the cre probe (upper panel). After stripping, the sameblot was hybridized with gapdh, as a control for equal loading of the lanes (lower panel).

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of T2 plants of the twelve male-fertile pMH2626 Crelines and pMM222 line C22 crossed to Tester lineT933. The gus gene was thus deleted upon expres-sion of the cre gene, which was verified using theprimers indicated as 2 and 11 in Figure 1 (results notshown). In Figure 4A a set of primary leaves are de-picted which are representative of the lines indicated,showing clear differences in GUS staining patternsamong the lines. When F1 seedlings of the Cre plantscrossed to T933 were grown in soil, measurementsof β-glucoronidase activity with methylumbelliferylglucoronidase (MUG) as a substrate confirmed the his-tochemical GUS staining data obtained from in vitrogrown seedlings (not shown).

A smaller selection of lines was used for further ex-periments. Lines C7, C43, C13, C5 transformed withpMH2626 represent phenotypic classes I, II, III andIV respectively (see also Figure 3). Line C22 (trans-formed with pMM222) belongs to class III. T2 plantsof these five lines were crossed to a second Tester line,line G3-3-20.

Tester line G3-3-20 is the result of linked transpo-sition of the Ds element from the pD3 T-DNA (Fig-ure 1). In G3-3-20 a putative prenyl transferase geneinvolved in chlorophyll biosynthesis is in this wayflanked by the lox sites present in the pD3 T-DNA andthe Ds element as schematically shown in Figure 4B.Cre protein activity induces the deletion of this puta-tive prenyl transferase gene, giving rise to chlorophylldeficiency that results in a dominant ‘white’ phenotypein the heterozygous F1 plant (details to be publishedelsewhere). In this way the effectiveness of cre geneexpression was assessed, by comparing the severity ofthe induced ‘white’ phenotypes (Figure 4B). As ex-pected, the results obtained with the ‘white’ mutantsof the G3-3-20 × Cre crosses were comparable to theprogeny of the T933 × Cre crosses that were sownboth in vitro and in soil. Both tester lines show thatin plants producing cre mRNA, functional Cre proteinwas made and that clear differences exist between Crelines in the onset of cre gene expression.

The timing of cre gene expression and the severity ofthe phenotype

Tomato line C43 has no ‘Cre phenotype’ before flow-ering (Figure 3A) and there is little Cre activity inyoung primary leaves as shown by crossing to T933(Figure 4A). In mature leaves of flowering F1 plantsof a C43 × G3-3-20 cross (Figure 4B) many small

yellow sectors suggest a relatively late induction ofCre activity during leaf development.

Figure 4C visualizes the cre steady-state mRNAlevels in mature leaves of lines C5, C13 and C43 dur-ing plant development. No cre mRNA was observedin C7 (shown in Figure 3B) and stable steady-statemRNA levels were shown in lines C5 and C13. Al-though in line C43 there was a clear increase in cregene transcription after the formation of leaf number4, the transcription levels in leaves 1 and 4 were com-parable to the levels in lines C5 and C13. Probablythe timing of cre transcription initiation during leafdevelopment affects the severity of the ‘Cre pheno-type’ more than the final steady-state mRNA levels inmature leaves as shown in Figure 4C.

In this same figure, the difference in quality ofmRNA, both between the cre mRNA and the gapdhcontrol and between line C43 and the lines C5 andC13 is apparent. When poly(A)+ mRNA was isolated,the smears below the cre mRNA bands were lost (datanot shown), suggesting either a mRNA degradationprocess, starting at the poly(A) tail, or premature stopsin the transcription process.

Physical characterization of multiple-copy cre T-DNAlines in tomato

None of the 16 independent primary pMH2626 trans-formants obtained contained a single, functional insertwhile 25% of the primary transformants had 5 ormore insertions (results not shown). Also in petunia,Southern analyses showed that none of the 11 primarytransformants with an aberrant phenotype appearedto have a single-copy T-DNA integration (results notshown).

To study the character of the cre T-DNA inserts intomato in more detail, the T-DNA integration patternsin these lines were examined both genetically and bySouthern analysis. Genetically, the T-DNA insertionsin C5, C7, C13 and C22 were found to be a singlelocus, based on the segregation of the antibiotic re-sistance in progenies of selfings and outcrosses. The‘Cre phenotype’ in lines C5, C13 and C22 co segre-gated with resistance to the antibiotic applied, whileC7 has no ‘Cre phenotype’. Southern analysis showedco segregation of all T-DNA copies in 10 individualsof out-crossed progeny of each of the four Cre linesexamined (data not shown).

To resolve the physical linkage of the multiple T-DNA inserts in tomato lines C5, C7, C13 and C22,high-molecular-weight DNA was digested with the re-

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striction endonuclease SpeI, which does not cut withinthe T-DNA constructs. Pulsed-field gel electrophoresis(PFGE), Southern blotting and hybridization with thelabelled cre gene as a probe showed that the T-DNAintegrations in these lines were all located on singleDNA fragments ranging from 12 to 38 kb (Figure 5A).

To determine the order and relative orientationof the T-DNAs in Cre lines C5, C7, C13 and C22,high-molecular-weight DNA was digested with SpeI,combined with enzymes that cut only once in the T-DNA (Figure 5). Panels B and C show Direct Repeats(DR) of T-DNA insertions in C5 and C13 and invertedrepeats (IR, with the cre gene in the centre of the re-peat) in lines C5 and C22. Incomplete T-DNAs werefound in C5 and C13 as indicated by vertical, stag-gered lines in Figure 5C. In C5, 400 bp were missing,which presumably did not affect the function of thegenes in the T-DNAs. In C13, 1.3 kb could not beaccounted for, which may either include most of thenptII gene or a part of the nos terminator of the cregene, or parts of both. The cre and nptII genes arestill expressed in C13 plants, since there are two other,probably complete copies of both genes present. InC22 one AatII site is lost but at least one copy of boththe cre genes and the hpt genes are still functional.C7 is the only tomato line with a single copy of thepMH2626 T-DNA, but this cre gene is not expressedas shown above.

The ‘Cre phenotype’ can be segregated away

For most research purposes, the ‘Cre phenotype’ isnot a problem, as long as the germinal transmissionof recombination events is not jeopardized and the cregene can be segregated away. Homozygous T3 plantsof Cre lines C5, C22, C13 and C43 were all crossedto at least 12 lines containing lox sites and in all casesan independent segregation of the cre locus and thelocus containing the lox sites was observed in the F2.Roughly 25% of the F2 plants of these crosses lackedthe ‘Cre phenotype’ which correlated with absenceof the cre gene, as determined by PCR with primerscre-top and cre-bot (data not shown). In these F2plants, lacking the cre gene, the intended site-specificrearrangements were transmitted germinally.

Germinal transmission efficiency of cre-mediatedgenome deletions

To study the efficiency of germinal transmission ofsite-specific deletions, homozygous plants of tomato

Cre line C5 were crossed to lines D3-3 and D3-5 con-taining the D3 construct (Figure 1). Lines D3-3 andD3-5 have a single-copy insertion of the D3 T-DNAon chromosome 7 and 6 respectively. In three inde-pendent experiments, a total of 325 F2 plants of bothD3-3 and D3-5 lines crossed to line C5 were analysed.Of these 325 plants, 25% did not inherit the cre gene,which was determined by PCR with primers cre topand cre bot. The fraction of kanamycin-resistant plantswithout cre gene that inherited the 2.7 kb deletion wasestimated by means of PCR with primers mas 3′ outand bar out (primer 2 and 3 respectively in Figure 1).The germinal transmission of the D3 T-DNA with2.7 kb deletion was estimated at 91.0% (SD 3.75%).

When homozygous plants of the Cre lines C5,C13, C22, C43 and C7 were crossed to line G3-3-20(Figure 4B), germinal transmission of the 8 kb dele-tion is visible in F2 populations as completely whiteseedlings. Reciprocal test crosses to wild-type plantsshowed that the dominant, white phenotype is onlytransmitted via the female germline to the F2 plantsand not via pollen. For C5, C13, C22, C43 and C7 theobserved number of completely white seedlings pertotal number of F2 seedlings were 12/29, 22/98, 20/99,0/98 and 0/100 respectively. The observed numbers ofgerminally transmitted deletion events correspond tothe severity of the somatic phenotype of the F1 plants(Figure 4B), except for C5. Since most F1 progenyof the G3-3-20 × C5 cross died within weeks due tolack of chlorophyll, seeds could only be obtained ofF2 progeny of F1 plants with relatively low somaticCre activity. Transmission via the germline for C5 istherefore underestimated.

These data show that Cre can be efficient both ingenerating site-specific deletions and in the transmis-sion of these deletions via the germline in spite of theaberrant phenotypes associated with its presence.

Discussion

The phenotype described for tomato, petunia andN. tabacum is observed among transformants with cregene containing T-DNAs. In tomato this presump-tive ‘Cre phenotype’ always co segregates with theantibiotic resistance marker in hundreds of progenyseedlings of different primary transformants. Al-though no conclusive proof could be established, thedata for tomato strongly suggest a correlation betweenthe phenotype and cre steady-state mRNA levels.

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Figure 5. Physical maps of cre loci in tomato. To obtain physical maps indicating the number and relative orientation of T-DNA inserts in fourCre lines, C5, C7, C13 and C22, high-molecular-weight DNA was digested with restriction endonucleases, size-fractionated on pulsed-field gels(PFG) and Southern-blotted. A. PFG Southern analysis of four Cre lines, cut with SpeI. The membrane was hybridised, using the radiolabelledcre gene as a probe. B. PFG Southern analysis of C22, cut with the indicated enzymes. Results of the hybridizations of the membrane with theradiolabelled cre probe and the hpt probe, respectively, are shown on the right. The physical map deduced from these autoradiograms is givenat the left bottom of this panel. In this map, horizontal, black bars indicate the position of the probes. C. PFG Southern analysis of C5, C7 andC13, cut with SpeI (S) and HindIII (H). The membranes were hybridized with the radiolabelled nos 5′ probe (from the nos 5′-npt II fusion) andthe cre probe respectively, as shown on the right, resulting in the physical maps shown at the left hand side of the panel. In this map, horizontal,black bars indicate the position of the probes and a bold staggered line in the map indicates incomplete integrations of the cre genes in C5 andC13. Abbreviations: B, BamHI; S, SpeI; A, AatII; H, HindIII; M, size marker, ∗, incomplete digestion; ?, lost AatII site; IRcre , inverted repeatwith the cre gene in the middle; DR, directly repeated T-DNAs.

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In petunia chlorosis and growth retardation inleaves, reduced internode elongation and increasedincidence of (male) sterility were observed. Thesedata confirm the observation of Que, et al. (1998),who report a ‘surprisingly high proportion’ of pri-mary transformants with abnormal leaf phenotypesand stunted growth after transforming petunia with aCaMV 35S-cre construct.

In N. tabacum similar aberrant phenotypes are ob-served, but (male) sterility was not found. Althoughseveral research groups expressed the cre gene in N.tabacum, using the CaMV 35S promoter, there are noreports of a high incidence of aberrant phenotypes inprimary N. tabacum transformants (reviewed by Odelland Russell, 1994; Ow and Medberry, 1995). Alsoin A. thaliana no aberrant phenotypes were reportedfor plants that express the cre gene driven by theCaMV 35S promoter (Russell et al., 1992; Osborneet al., 1995). The reason is obviously that the majorityof the transformants in N. tabacum and A. thalianaappear normal. The contrary is observed in petunia(Que et al., 1998) and in tomato transformants, wherethe majority of the obtained plants displayed growthaberrations.

In A. thaliana Cre-related phenotypes are probablymore exceptional and, if visible at all, less severe. KirkSchnorr (personal communication) independently ob-served an aberrant leaf phenotype after expression ofthe cre gene mediated by a CaMV 35S promoter, inwhich the Cre protein is targeted to the nucleus with anuclear localization signal. In this case, several inde-pendent transformants showed small but clear necroticlesions in the leaves.

Interestingly, in the T0 plants of the pMH2626transformation of tomato, only 2 out of 16 primarytransformants had a mild ‘Cre phenotype’. In gener-ation T1, however, 11 out of 12 tomato lines displayedsometimes very severe ‘Cre phenotypes’. T0 and T1tomato plants are hard to compare since the T0 plantswere regenerated in vitro while T1 plants germinatedfrom seed. In N. tabacum and petunia the aberrantphenotypes were already clearly visible in the pri-mary transformants and there are no clear differenceswith the T1 plants. Apparently, manifestation of the‘Cre phenotype’ is regulated somehow, so future ex-periments should be aimed at revealing the possiblerole of transcriptional gene silencing (TGS), post-transcriptional gene silencing (PTGS) or methylationin the presumed suppression of the ‘Cre phenotype’ intomato.

The timing of cre expression affects germinaltransmission of deletions

In tomato, the level of cre mRNA steady-state levelsin older leaves appears to be poorly correlated withthe ‘Cre phenotype’. However, the earlier the cre geneis expressed during development, the more severe the‘Cre phenotype’ is. Probably, the developmental stageof a plant organ at which the cre gene is switched ondetermines the severity of the observed ‘Cre pheno-type’. The differences in timing of the activation of thecre gene also seem to correlate with the transmissionof recombination events via the germline. In tomatolines where the ‘Cre phenotype’ is observed in veryyoung seedlings (like C5), the germinal transmissionfrequency of a deletion in tester constructs T933 andD3 is much higher, compared to lines that show the‘Cre phenotype’ later (like C43). Presumably, induc-tion of deletions in germline cells is more likely whenthe cre gene is switched on earlier in organogenesis.This suggests that if Cre lines were found without aphenotype, they are expected to express the cre generelatively late or at a low level.

This deduction is supported by observations ofQue et al. (1998) in petunia. They selected an almostnormal transformant with a single copy T-DNA thatmissed part of the 35S promoter driving the cre gene.Using this plant, they observed frequent somatic re-arrangements, but virtually no germinally transmittedrearrangement events. Similar to our observations fortomato, selection against the ‘Cre phenotype’ mostlikely implied selection against early expression ofthe cre gene and indirectly against efficient germinaltransmission of recombination events. To obtain thedesired inversion, Que et al. (1998) had to regener-ate petunia plants from somatic tissue that containedthe inversion. This emphasises the need for testingthe germinal transmission of recombination events asthe most important selection criterion, before look-ing at phenotypes, T-DNA copy number or somaticrecombination frequencies.

Does recombination between pseudo lox sites explainthe ‘Cre phenotype’?

Hypothetically, the ‘Cre phenotype’ could be causedby Cre-mediated recombination events between cryp-tic or pseudo-lox sites. Assuming that two or morepseudo-lox sites are present in the genome, recombi-nation between these sites should leave an alterationbehind. In other words, the ‘Cre phenotype’ would

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become a genetic trait that would segregate indepen-dently from the Cre-producing construct.

However, among hundreds of progeny of both self-fertilized tomato plants and out-crosses of 15 indepen-dent Cre lines, the ‘Cre phenotype’ co-segregates withthe antibiotic resistance marker linked to the cre gene.To account for the co-segregation, recombination be-tween pseudo-lox sites may take place only in somaticcells or causes 100% sterility in the gametes or eventhe complete germline. Although (male) sterility wasobserved in 25% of the tomato plants transformed withpMH2626, Cre-induced deletions in tester constructs(D3 and T933) are transmitted through meiosis withhigh frequencies. This indicates that germline cellsthat have been exposed to Cre protein activity werestill fertile.

Is Cre toxic above a certain threshold level?

Possibly, the Cre protein itself has deleterious effectson plant development when expressed at certain crit-ical stages of development, thereby causing similaraberrant phenotypes in different plant species. Loon-stra et al. (2001) report that the toxicity of Cre inmouse embryonic fibroblast cell cultures depends onthe strand exchange activity and the dose of the activeprotein. They show that there is a certain thresholdconcentration of Cre molecules, above which delete-rious effects appear. Below this threshold level, site-specific recombination over a short distance betweentwo perfect lox sites still occurs without growth in-hibitory effects. In tomato and petunia it can thereforenot be excluded that the cases of (male) sterility arecaused by Cre toxicity during specific stages of thesporogenesis, similar to the male sterility observed intransgenic mice by Schmidt et al. (2000).

The sensitivity to Cre toxicity is not equal in alltissues or stages of development

In tomato, there is a positive correlation between earlyexpression of the cre gene and the severity of the‘Cre phenotype’, which suggests a more toxic ef-fect of the Cre protein when active earlier during theprocess of organogenesis. Similarly, in Drosophilamelanogaster, Cre recombinase toxicity is primar-ily apparent after prolonged expression in mitoticallyproliferating cells (Heidmann and Lehner, 2001). Intomato, when the tissue specific phaseolin promoteris used to drive the cre gene, recombination occursefficiently but the ‘Cre phenotype’ is not observed.Van der Geest et al. (1995) may provide a reason

for the absence of a phenotype in vegetative tomatoplant parts of these pJB40 transformants. They showedthat expression of the phaseolin promoter from Frenchbean (Phaseolus vulgaris) is confined to embryoge-nesis and microsporogenesis in N. tabacum. Besidesthis, the level of expression in the embryos may be be-low the threshold level for visible deleterious effects inseedlings. Apparently, the problems caused by the Creprotein can be circumvented by using promoters lessactive than the CaMV 35S promoter, by using (weak)tissue-specific promoters or inducible promoters likethe heat-shock-inducible promoter used by Sieburthet al. (1998). Earlier experiments using other re-combinases also showed aberrations in higher plants.Expression in N. tabacum and A. thaliana cells ofGin, an invertase-group member from bacteriophageMu, appeared to have adverse effects (Maeser andKahmann, 1991). Although we have not been able totransform tomato with a 35S-FLP construct (data notshown), two other reports (Lloyd and Davies, 1994;Kilby et al., 1995) show that it is possible to ex-press the integrase FLP in A. thaliana from a CaMV35S promoter, but they observed low recombinationfrequencies. As shown by Schmidt et al. (2000), Loon-stra et al. (2001), Silver and Livingston (2001) andHeidmann and Lehner (2001), the difficulties associ-ated with the expression of the cre gene as describedhere are not restricted to plants. When developmentaldeformations are caused by the activity of the protein,aberrations are also to be expected in other heterolo-gous systems. It seems therefore advisable to limit creexpression to specific tissues and to a short period oftime to avoid undesired side-effects of this powerfultool.

Acknowledgements

We wish to thank Nunhems Zaden (Netherlands) forthe N. tabacum data, David Ow (PGEC, Albany, CA)for providing plasmid pED23, Van der Have Researchfor providing us with binary vectors pVdH275 andpVdH212, and Kirk Schnorr for discussing unpub-lished results. Jan Peter Nap is acknowledged for crit-ically reading the manuscript and many discussions,Maartje Kuijpers for nursing the plants, Marian Noor-dam for technical assistance and Jeroen Stuurman forstimulating discussions. This work was supported bya grant from the Netherlands Technology Foundation(STW), project 349-3190.

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