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Transposon-mediated generation of marker free rice plants containing a Bt endotoxin gene for insect resistance

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Transposon-mediated generation of marker free rice plants containing a Bt endotoxin gene for insect resistance
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  Transposon-mediated generation of T-DNA- and marker-free rice plantsexpressing a  Bt  endotoxin gene Olivier Cotsaftis 1 , Christophe Sallaud 1 , Jean Christophe Breitler 1 , Donaldo Meynard 1 ,Rafaella Greco 2 , Andy Pereira 2 and Emmanuel Guiderdoni 1, * 1  Biotrop Programme, Cirad-Amis, TA40/03, F-34398, Montpellier, Cedex 5, France;  2 Plant Research International, PO Box 16, 6700 AA, Wageningen, The Netherlands; *Author for correspondence (e-mail:guiderdoni@cirad.fr; fax: 33 (0)4 67 61 56 05) Received 11 December 2001; accepted in revised form 15 April 2002 Key words: Ac/Ds ,  Bacillus thuringiensis , Insect resistance, Repositioning, Selectable marker free, Transgenicrice Abstract Transposon-mediated repositioning of transgenes is an attractive strategy to generate plants that are free of se-lectable markers and T-DNA inserts. By using a minimal number of transformation events a large number of transgene insertions in the genome can be obtained so as to benefit from position effects in the genome that cancontribute to higher levels of expression. We constructed a  Bacillus thuringiensis  synthetic  cry1B  gene expressedunder control of the maize ubiquitin promoter between minimal terminal inverted repeats of the maize  Ac-Ds transposon system, which was cloned in the 5’ untranslated sequence of a  gfp  gene used as an excision marker.The T-DNA also harboured the  Ac  transposase gene driven by the CaMV 35S promoter and the  hph  gene con-ferring resistance to the antibiotic hygromycin. Sixty-eight independent rice ( Oryza sativa  L.) transformants wereregenerated and molecularly analysed revealing excision and reinsertion of the  Ds-cry1B  element in 37% and25% respectively of the transformation events. Five independent transformants harbouring 2–4 reinserted  Ds-Cry1B  copies were analysed in the T1 progeny, revealing 0.2 to 1.4 new transpositions per plant. Out segregationof the  cry1B  gene from the T-DNA insertion site was observed in 17 T1 plants, representing 10 independentrepositioning events without selection. Western analysis of leaf protein extracts of these plants revealed detect-able Cry1B in all the plants indicating efficient expression of the transgene reinsertions. Stability of position andexpression of the  cry1B  transgene was further confirmed in T2 progeny of T-DNA-free T1 plants. New T-DNA-free repositioning events were also identified in T2 progenies of T1 plants heterozygous for the T-DNA. Further-more, preliminary whole plant bioassay of T-DNA-free lines challenged with striped stem borer larvae suggestedthat they are protected against SSB attacks. These results indicate that transposon mediated relocation of thegene of interest is a powerful method for generating T-DNA integration site-free transgenic plants and exploitingfavourable position effects in the plant genome. Introduction Development of an engineered crop relies on theidentification of a transformation event which expressthe integrated transgene at both a high and stable levelover generations and environments. This ideal eventis usually selected among a large number of primarytransformants (T0) exhibiting a range of expressionlevels corresponding to unique sites of integration of the transgene in the host genome. Qualitative andquantitative variation in transgene expression is de-termined by the intrinsic features of the promoter se-quence and modulated by the transcriptional activityof the region where the transgene is inserted – the so-called gene position effect – and further transcrip-tional and post-transcriptional regulation of expres-sion of foreign DNA by cell mechanisms. The lattercontrol – referred as gene silencing phenomenon – 165  Molecular Breeding 1 0: 1 65 –1 80 , 2002.© 2002  Kluwer Academic Publishers. Printed in the Netherlands.  may occur early during T0 plant development but alsolater, in subsequent generations of selection of thetransgenic event. These unpredictable variations ne-cessitate production of multiple independent transfor-mants in an initial transformation effort which is gen-erally labour- and time-consuming and particularlydifficult in recalcitrant cultivars or species.Development of a transgenic plant also relies onan early and stringent selection of the transformedcell line which is obtained through transfer and ex-pression of a selectable marker gene usually linked tothe transgene of interest. This results in the persis-tence of both genes at a single genetic locus in thetransgenic crop, both in microprojectile- and  Agro-bacterium -mediated transformation systems. Eventhough alternative marker genes with no possiblyharmful biological activities of their product, havebeen recently developed (Hansen and Wright (1999)for review) presence of the selectable marker gene inreleased transgenics appears undesirable for biosafetyauthorities and consumer stand points. Furthermore,it prevents further transformation of the transgeniccultivar using the same selection procedure. Technol-ogies for producing selectable marker-gene free trans-genic plants have so far been based on either co-integration at unlinked sites of the gene of interest andthe selectable marker gene in the primary transfor-mant that can be genetically out segregated in theprogenies (Mc Knight et al. 1987; De Block and De-brouwer 1991; Komari et al. 1996; Daley et al. 1998),excision of the selectable marker gene by site-speci fi crecombinases (Dale and Ow 1991; Sugita et al. 1999;Gleave et al. 1999; Zuo et al. 2001) or intrachromo-somal recombination (Zubko et al. 2000) (Hohn et al.(2001), for recent review). However, in these meth-ods the srcinal transgene integration site which mayinclude T-DNA borders, recombinase site and vectorssequences, as well as rearrangements of host DNAinduced by T-DNAintegration is retained in the trans-formation event (Smith et al. 2001).Transposon-mediated repositioning of a transgeneof interest has been proposed as an alternative forgenerating a wide range of expression levels in se-lectable-marker gene-free transgenic plants (Yoderand Goldsbrough 1994). Reinsertion of the transpo-son-borne transgene of interest indeed permits to ex-plore a large range of position effects thereby gener-ating a wide qualitative and quantitative variation inexpression levels from a single transpositionnaly ac-tive transformant line. This considerably limits theinitial effort required for production of primary trans-formants in species or cultivars poorly amenable totransformation. Provided that reinsertion of the trans-gene of interest occurs at unlinked sites, relocationallows elimination through recombination in theprogeny of all sequences co-integrated at the srcinalintegration site. Furthermore, a relocated transposon-borne transgene may be less prone to gene silencingphenomenon than at the srcinal integration site. Thetwo former advantages have been exempli fi ed oncethrough  Ds -mediated repositioning of a  gusA  trans-gene in tomato (Goldsbrough et al. 1993). The latteradvantage has been illustrated in a very recent reportof monitoring of expression of a  Ds -mediated relo-cated  bar   transgene compared to that at its srcinalintegration site in the F3-F4 progenies of biolisticbarley transformants following trans-activation ob-tained by crossing with a  Ac  transposase-expressingplant (Koprek et al. 2001).So far transposon – mediated repositioning of atransgene of interest has not been attempted in rice( Oryza sativa  L.), although functionality of the maize  Ac/Ds  system as a gene tagging tool has been beingsuccessfully demonstrated since the early 90s (Izawaet al. (1991, 1997); Chin et al. 1999; Enoki et al.1999). To exemplify its feasibility thereafter, wechoose a  Bacillus thuringiensis  synthetic  cry1B  genewhose product is not selectable. Insect pest resistancemediated by expression of   cry  endotoxin genes hasbeen early and extensively investigated in  japonica and  indica  rices (Tyagi and Mohanty (2000), for re-view), including a recent successful  fi eld trial of anelite hybrid cultivar (Tu et al. 2001). As Bt-mediatedresistance will likely be among the  fi rst trait engi-neered in a commercial rice crop, there is a strikinginterest in adopting methods avoiding persistence of so called super fl uous DNA in the released events.In our study, the  cry1B  gene cassette was clonedbetween the inverted terminal repeats of the maize  Ac transposon, which was inserted in the 5 ’  untranslatedsequence of a  gfp  gene that could be used as an ex-cision marker. The T-DNA also harboured the geneencoding Ac transposase driven by the CaMV 35Spromoter and the  hph  gene conferring resistance tothe antibiotic hygromycin. We further report here thesuccessful transfer of the T-DNA and subsequent re-location of the  Ds-cry1B  element in the rice genome.T-DNA integration site free-repositionings were ob-served in T1 and T2 generation in several transposi-tionaly active transgenic rice lines. Stability of ex-pression of the relocated  cry1B  transgenes wascon fi rmed in T1 and T2 generations through immuno- 166  blot analyses. Furthermore, a preliminary bioassaysuggested that the T-DNA free relocation events ex-hibit a level of resistance to a major rice insect pest, Chilo suppressalis . Materials and methods Construction of the pC  DS-cry1B  vector  The pC  DS-cry1B  vector was assembled by multi-point ligations in which individual fragments with ap-propriate compatible cohesive ends were ligated to-gether and inserted in between the  EcoR I and  Sal Isites of the T-DNA of the pC5300 binary vector (apCAMBIA1300 (R. Jefferson, CAMBIA, Canberra,Australia) derivative where the T-DNA  hph  codingsequence has been replaced by a  hph  castor bean cat-alase ( cat1 ) gene intron-containing  hph  coding se-quence (Wang et al. 1997) (P. Ouwerkerk, LeidenUniversity, The Netherlands)).The fragments used for simultaneous insertion inthe pC5300 multiple cloning site consisted in (from5 ’  to 3 ’ ): The CaMV35S promoter and double en-hancer (CaMV35Sde) including an AMV leader se-quence after removal of the ATG initiation site as a 1kb  EcoR I/   Apa I fragment derived from pMOG18 (Sij-mons et al. 1990): A 0.3 kb  Apa I/  Spe I fragment cor-responding to the left junction of the maize transpo-son  Ac7   (AcLj) (Greco et al. 2001b); a 4.3 kb  Spe I/   Hind  III fragment containing the coding sequence of the codon-optimized  cry1B  gene (Bohorova et al.2001) controlled by the promoter region,  fi rst exonand  fi rst intron of the maize ubiquitin gene (Chris-tensen and Quail 1996) and terminated by the termi-nator of the  nos  gene: A 1.3 kb  Hind  III/  Sac I fragmentconsisting in a fusion between the  Ac7   transposonright junction (AcRj), the coding sequence the hu-manized red-shifted GFP  SgfpS65T   (Chiu et al. 1996)and a 4.2 kb  Sac I/  Sal I fragment containing a fusionbetween the CaMV35S promoter and the transposasegene of the maize Ac transposon (Greco et al. 2001a).All these 5 fragments were cloned in pBluescript ® SK+ vectors before restriction and multi-point liga-tion in the 9.2 kb pCAMBIA5300 vector, resulting inthe 20.3 kb pC  DS-cry1B  bearing a 14 kb-large T-DNA (Figure 1). The pC  DS-cry1B  binary vector wastransferred into  Agrobacterium  strain EHA105, anEHA101 derivative (Hood et al. 1993) by electropo-ration (Sambrook et al. 1989).  Agrobacterium  cellswere plated on solid AB medium containing 50 mg/lkanamycin sulfate and incubated at 28  ° C for 3 days.The bacteria were then collected with a  fl at spatulaand resuspended in liquid cocultivation medium bygentle vortexing prior transforming rice tissues. Sta-bility of the binary vector was controlled by restric-tion analyses using  BamH  I,  EcoR I,  EcoR V,  Spe I,  Nco I and  Pvu I enzymes after extraction from the ac-tual  Agrobacterium  clone which was used for co-cul-ture.  Agrobacterium-mediated transformation of rice Dehulled seeds of the mediterranean elite  japonica rice ( Oryza sativa  L.) cultivar Ariete were sterilized,inoculated on NB medium and incubated for 18 – 21days in the dark as described by Chen et al. (1998).0.5 – 1 mm embryogenic nodular units released fromthe primary, embryo scutellum-derived callus at theexplant/medium interface were transferred on freshNB medium and incubated for 15 additionnal days.300 embryogenic calli were co-cultured in presenceof EHA105 cells bearing the pC  DS-cry1B  binaryplasmid at a density of 3 – 5 10 9 cells/ml (OD600 of 1). The procedure for evolving transgenic rice plantsusing a selection based on resistance to 50 mg/l hy-gromycin will be reported elsewhere. 151 (ca. 50%)of the co-cultured calli yielded hygromycin-resistantcell lines, 5 weeks after co-culture. Independent trans-formation events were regenerated from 68 co-cul-tured calli (transformation efficiency of 22.7%).  Molecular characterization of T-DNA integrationand   Ds-cry1B  transposition Total genomic DNAwas extracted from 500 mg bladetissue of a batch sample of the ante penultimate leavesof each tiller of transgenic rice plants at the tilleringstage using a modi fi ed CTAB method (Hoisington1992). Five   g of DNA were digested with  BamH  Ior  EcoR I restriction endonucleases and DNA frag-ments were separated on 0.8% agarose gels, andtransferred to nylon membranes (Hybond-N+ (Amer-sham ® )).  T-DNA organisation ; Integration of non T-DNA sequences of the pC  DS-cry1B  binary plasmid(Figure 1) in the rice genome was monitored throughsubsequent hybridization of membrane of   BamH  I di-gests of T0 plants using two PCR probes correspond-ing to regions extending 237 bp from the left (senseprimer: 5 ’ -GATCACA GGCAGCAACGCTC-3 ’ ; an-tisense primer: 5 ’ -ACCAGCCAGCCA ACAGCTC-CCCG AC-3 ’ ) and right (sense primer: 5 ’ -ATTA- 167  GAATAACGGATATTTAAAAGGGC-3 ’ ; antisenseprimer: 5 ’ -GACAAGAAAACGCCAGGAAA-3 ’ )borders of the T-DNA. The number of T-DNA copiesintegrated was determined through hybridization of a556 bp PCR probe corresponding to the  hph  codingsequence(sense probe: 5 ’ -TACTTCTACACAGC-CATCGG-3 ’ ; antisense probe: 5 ’ -AAGGAATCGGT-CAATACACT-3 ’ ) and of a 3623 pb probe corre-sponding to the  BamH  I fragment of the  AcTpase gene, on  BamH  I and  EcoR I membranes rerspectively.Integrity of the  AcTpase  and  gfp  genes was assessedthrough hybridization of   BamH  I membranes with the  AcTpase  probe and a 1005 pb  BamH  I fragment con-taining the  gfp  coding sequence as probe respectively. Transposition of the Ds-cry1B element : Effectivetransposition of the  Ds-cry1B  element was  fi rst moni-tored by PCR analyses. Presence of an empty donorsite (EDS) resulted in ampli fi cation of a 734 pb re-gion situated between the 35S promoter and the  gfp coding sequence (sense primer: 5 ’ -ATCCCACTATC-CTTCGCAAGACCC-3 ’ ; antisense primer: 5 ’ -GCT-TGTCGGCCATGATATAGACG-3 ’ ) whereas noproduct is formed from an intact full donor site (FDS)(Figure 1). EDS can also be discriminated from anFDS through hybridisation of   EcoR I membranes withthe gfp probe. Reinsertion of the  Ds-cry1B  elementwas monitored through hybridization of PCR probecorresponding to a 475 pb region of the  cry1B  gene(sense primer: 5 ’ -GAGGACTCCTTGTGCATCGC-3 ’ ; antisense primer:5 ’ -AGAGGTGCAAGTTG-GCAGCC-3 ’ ) on  BamH  I membranes. [  - 32 P] la-belled probes were synthesized through randompriming from the mentionned templates using theAmersham ® kit RPN 1607. Following hybridization,the membrane was washed twice (SSC2x, SDS 1%and SSC 0.1x, SDS 0.1% both at 65  ° C for 10 min)and analyzed by autoradiography.  Analyses of transgene expression Green  fl uorescent protein activity was observed ongrowing, hygromycin-resistant cell lines then root andleaf tissues of the 68 primary transformants using aLEICA MZ FLIII stereomicroscope through a LEICAGFP3  fi lter (   excitation: 470 nm +/− 20;    excita-tion stop: 525 nm +/− 25. 10.6% of the hygromycin- Figure 1.  Schematic representation of the  Ds-cry1B  T-DNA. The  Ubi-cry1B-nos3’  cassette was inserted by multi-point ligation between theterminal inverted repeats (Rj and Lj) of the maize  Ac7   element and cloned in the 5’ untranslated region of the  gfp  gene between the left (LB)and right (RB) borders of the T-DNA as described in  Materials and Methods  The restriction map of the  Ds-cry1B  T-DNA indicates the sitesand probes used in Southern blot analyses of the organization of the T-DNA in transgenic rice plants. Hybridisation of blots of   BamH  I and  EcoR I digests of total DNA of plants harbouring the  Ds-cry1B  T-DNA with a  hph  and  AcTpase  probes respectively served at determining thenumber of insertion sites and minimum number of copies of the T-DNA.  BamH  I blots hybridised to the  AcTpase  probe served at assessingthe integrity of the  AcTpase  coding region (upper panel). The excision of the  Ds-cry1B  element is assayed by detection of a PCR amplifi-cation product of 734 bp using primers specific to the 35S promoter and the  gfp  coding sequence (middle panel) and by occurrence of newbands following hybridisation of blots of   BamH  I digests with a transposon-specific  cry1B  probe (lower panel). 168  resistant cell lines were sectorially or fully  fl uores-cent, suggesting that early excision of the transposonoccurred in the transformed cells (Figures 2A and2B). Out of 68 plant-forming hygromycin-resistantcell lines, thirteen exhibited sectors of GFP activity.Examination of the 68 test-tube grown rooted plantsallowed detection of GFP activity in root and leaf tis-sues of 11 events but none of them was found to de-rive from the above-mentioned GFP positive callus.Segregation of expressing copy (ies) of the  hph  genewas assayed  in planta  in following a procedure mod-i fi ed from Speulman et al. (1999), in inoculating thelast expanded leaf with a 2   l droplet of a 25 mg/mlhygromycin 0,001% Triton X-100 and 0,5% gelatinsolution and monitoring necrotic lesions on the leaf 4 – 6 days following the treatment. The procedureproved highly reliable in rice since in some lines, itwas even possible to score a 1:2:1 segregation basedon lesion development using this assay, indicating a hph  transgene copy dosage effect on phenotypic ex-pression of the resistance to hygromycin.Expression of the  cry1B  was monitored in leaf tis-sues by immunoblot analyses: Proteins were extractedby direct grounding of frozen leaf tissues in extrac-tion buffer consisting in 50 mM Tris-HCl, pH 8.0, 1mM EDTA, 5% v/v glycerol, 1 mM DTT and 0.1%Triton X100. The extract was centrifuged at 15 000rpm at 4  ° C for 15 mn. The supernatant was retained,centrifuged at 15 000 rpm at 4  ° C for 5 mn and pro-tein concentration was determined (Bradford 1976).From several plant extracts, 50   g protein were sub- jected to SDS-PAGE on 8% acrylamide gels in dena-turating conditions (Laemmli 1970). After electro-phoresis, the protein was electro-blotted onto anitrocellulose  fi lter ((Biorad ® ). The blots were devel-oped using immuno affinity-puri fi ed rabbit antibodiesspeci fi c to the Cry1B protein. Goat anti-rabbit IgGlinked to alkaline phosphatase (Sigma ® ) was used tobind to the primary antibody and the complex wasdetected with a NBT-BCIP solution.  Insect feeding bioassays Control Ariete plants and western-positive, T2 prog-eny plants of the T-DNA free T1 plants 136.2/2,136.2/17, 149.1/26, 150.2/26 which harboured eachan srcinal repositioning event of the  DS-cry1B  ele-ment, were assayed for resistance to the Striped StemBorer (SSB) ( Chilo suppressalis  Walker). We used aspositive control, plants of a transgenic T3 Ariete 3.4homozygous line harbouring a T-DNA containing thesame fusion between the  cry1B  gene and the maizeubiquitin promoter than the  DS-cry1B  element. The3.4 line was previously found to stably express Cry1Bat more than 0.4% tsp in leaf tissues over generationsand to be fully protected against SSB attacks both ingreenhouse bioassays and arti fi cial infestation in the fi eld (unpublished results to be reported elsewhere).Bioassays were carried out on 8 plants at the tilleringstage per line which were infested each with 6 third(L3) instar SSB larvae. Plants were grown under con-tainment greenhouse conditions in pots placed in anylon mesh cage. After 9 days, the infested plantswere  fi rst screened for insect leaf and pith damage,then dissected starting with the infested tiller, to de-termine the number of surviving larvae and their de-velopmental stage. Insect antibiosis was assessed bythe presence/absence of head capsules and sloughs,the recovery of living larvae, and the developmentalstage and body weight of living larvae. Results T-DNA organisation in transgenic rice plants Organisation of pC  Ds-cry1B  T-DNA was investigatedby DNA-blot hybridisation in the 68 primary trans-formants. Additional plants (150.1, 150.2 and 150.3in one hand and 38.2a and 38.2b on the other hand)regenerated respectively from 3 callus pieces derivedfrom fragmentation of the same hygromycin resistantcell line #150 and from two sectors of a single, re-generating hygromycin-resistant callus #38.2 werealso included in the analysis.To determine  fi rst the frequency of integration of non-T-DNA sequences in the regenerated plants, ge-nomic DNA was restricted by  BamH  I and the result-ing DNA blots were hybridised successively withprobes extending 237 bp from the left (LB-out) andthe right (RB-out) border of the pC  DS-cry1B  T-DNA.23.2% and 30.5% of the transformants showed hy-bridisation signals with the RB-out and LB-outprobes respectively, while 22% of the transformantsexhibited bands hybridising to both probes (data notshown).The number and integrity of T-DNA copies in-serted was further assessed in transgenic rice DNAdigested with  Bam HI and  Eco RI restriction enzymes(Figure 3). Thirty- fi ve plants were found to harbour asingle band hybridising to the  hph  probe suggestingthat they contain a single copy of the T-DNA. For a 169
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