Skip to main content
SpringerLink
Log in

Expression of active Streptomyces phage phiC31 integrase in transgenic wheat plants

  • Genetic Transformation and Hybridization
  • Published:
Plant Cell Reports Aims and scope Submit manuscript

Abstract

Site-specific recombination systems are becoming an important tool for the genetic modification of crop plants. Here we report the functional expression of the Streptomyces phage-derived phiC31 recombinase (integrase) in wheat. T-DNA constructs containing a phiC31 integrase transgene were stably transformed into wheat plants via particle gun bombardment. A plant-virus-based assay system was used to monitor the site-specific recombination activity of the recombinant integrase protein in vivo. We established several independent doubled haploid (DH) inbred lines that constitutively express an active integrase enzyme without any apparent detrimental effects on plant growth and development. The potential of phiC31 integrase expression in crop plants related to transgene control technologies or hybrid breeding systems is discussed.

This is a preview of subscription content, log in via an institution to check access.

Access this article

Log in via an institution

Price excludes VAT (USA)
Tax calculation will be finalised during checkout.

Instant access to the full article PDF.

Institutional subscriptions

Fig. 1
Fig. 2
Fig. 3
Fig. 4

Similar content being viewed by others

References

  • Albert H, Dale EC, Lee E, Ow DW (1995) Site-specific integration of DNA into wild-type and mutant lox sites placed in the plant genome. Plant J 7:649–659

    Article  PubMed  CAS  Google Scholar 

  • Andreas S, Schwenk F, Kuter-Luks B, Faust N, Kuhn R (2002) Enhanced efficiency through nuclear localization signal fusion on phage PhiC31-integrase: activity comparison with Cre and FLPe recombinase in mammalian cells. Nucleic Acids Res 30:2299–2306

    Article  PubMed  CAS  Google Scholar 

  • Araki K, Araki M, Yamamura K (1997) Targeted integration of DNA using mutant lox sites in embryonic stem cells. Nucleic Acids Res 25:868–872

    Article  PubMed  CAS  Google Scholar 

  • Chawla R, Ariza-Nieto M, Wilson AJ, Moore SK, Srivastava V (2006) Transgene expression produced by biolistic-mediated, site-specific gene integration is consistently inherited by the subsequent generations. Plant Biotechnol J 4:209–218

    Article  PubMed  CAS  Google Scholar 

  • Christensen AH, Quail PH (1996) Ubiquitin promoter-based vectors for high-level expression of selectable and/or screenable marker genes in monocotyledonous plants. Transgenic Res 5:213–218

    Article  PubMed  CAS  Google Scholar 

  • Clausen M, Krauter R, Schachermayr G, Potrykus I, Sautter C (2000) Antifungal activity of a virally encoded gene in transgenic wheat. Nat Biotechnol 18:446–449

    Article  PubMed  CAS  Google Scholar 

  • Coppoolse ER, de Vroomen MJ, Roelofs D, Smit J, van Gennip F, Hersmus BJ, Nijkamp HJ, van Haaren MJ (2003) Cre recombinase expression can result in phenotypic aberrations in plants. Plant Mol Biol 51:263–279

    Article  PubMed  CAS  Google Scholar 

  • Corneille S, Lutz K, Svab Z, Maliga P (2001) Efficient elimination of selectable marker genes from the plastid genome by the CRE-lox site-specific recombination system. Plant J 27:171–178

    Article  PubMed  CAS  Google Scholar 

  • Cornejo MJ, Luth D, Blankenship KM, Anderson OD, Blechl AE (1993) Activity of a maize ubiquitin promoter in transgenic rice. Plant Mol Biol 23:567–581

    Article  PubMed  CAS  Google Scholar 

  • Dale EC, Ow DW (1991) Gene transfer with subsequent removal of the selection gene from the host genome. Proc Natl Acad Sci USA 88:10558–10562

    Article  PubMed  CAS  Google Scholar 

  • De Buck S, Peck I, De Wilde C, Marjanac G, Nolf J, De Paepe A, Depicker A (2007) Generation of single-copy T-DNA transformants in Arabidopsis by the CRE/loxP recombination-mediated resolution system. Plant Physiol 145:1171–1182

    Article  PubMed  Google Scholar 

  • Dekker EL, Woolston CJ, Xue YB, Cox B, Mullineaux PM (1991) Transcript mapping reveals different expression strategies for the bicistronic RNAs of the geminivirus wheat dwarf virus. Nucleic Acids Res 19:4075–4081

    Article  PubMed  CAS  Google Scholar 

  • Dellaporta SL, Wood J, Hicks JB (1983) A plant DNA minipreparation: version II. Plant Mol Biol 1:19–29

    Article  CAS  Google Scholar 

  • Djukanovic V, Orczyk W, Gao H, Sun X, Garrett N, Zhen S, Gordon-Kamm W, Barton J, Lyznik LA (2006) Gene conversion in transgenic maize plants expressing FLP/FRT and Cre/loxP site-specific recombination systems. Plant Biotechnol J 4:345–357

    Article  PubMed  CAS  Google Scholar 

  • Eliby S, Kandzia R, Ismagul A, Karabaev M, Sasakuma T, Klimyuk V (2000) Biolistic transformation of wheat utilizing a new DNA/gold coating procedure. Abstracts of the 6th International Wheat Conference, Budapest, p 313

  • Gidoni D, Bar M, Gilboa N (2001) FLP/FRT-mediated restoration of normal phenotypes and clonal sectors formation in rolC transgenic tobacco. Transgenic Res 10:317–328

    Article  PubMed  CAS  Google Scholar 

  • Gils M, Marillonnet S, Werner S, Grutzner R, Giritch A, Engler C, Schachschneider R, Klimyuk V, Gleba Y (2008) A novel hybrid seed system for plants. Plant Biotechnol J 6:226–235

    Article  PubMed  CAS  Google Scholar 

  • Gleba Y, Marillonnet S, Klimyuk V (2004a) Design of safe and biologically contained transgenic plants: tools and technologies for controlled transgene flow and expression. Biotechnol Genet Eng Rev 21:325–367

    PubMed  Google Scholar 

  • Gleba Y, Marillonnet S, Klimyuk V (2004b) Engineering viral expression vectors for plants: the ‘full virus’ and the ‘deconstructed virus’ strategies. Curr Opin Plant Biol 7:182–188

    Article  PubMed  CAS  Google Scholar 

  • Gutierrez C (2000) DNA replication and cell cycle in plants: learning from geminiviruses. EMBO J 19:792–799

    Article  PubMed  CAS  Google Scholar 

  • Gutierrez C (2002) Strategies for geminivirus DNA replication and cell cycle interference. Physiol Mol Plant Pathol 60:219–230

    Article  CAS  Google Scholar 

  • Hoa TT, Bong BB, Huq E, Hodges TK (2002) Cre/lox site-specific recombination controls the excision of a transgene from the rice genome. Theor Appl Genet 104:518–525

    Article  PubMed  CAS  Google Scholar 

  • Hu Q, Kononowicz-Hodges H, Nelson-Vasilchik K, Viola D, Zeng P, Liu H, Kausch AP, Chandlee JM, Hodges TK, Luo H (2008) FLP recombinase-mediated site-specific recombination in rice. Plant Biotechnol J 6:176–188

    Article  PubMed  CAS  Google Scholar 

  • Jia H, Pang Y, Chen X, Fang R (2006) Removal of the selectable marker gene from transgenic tobacco plants by expression of Cre recombinase from a tobacco mosaic virus vector through agroinfection. Transgenic Res 15:375–384

    Article  PubMed  CAS  Google Scholar 

  • Kittiwongwattana C, Lutz K, Clark M, Maliga P (2007) Plastid marker gene excision by the phiC31 phage site-specific recombinase. Plant Mol Biol 64:137–143

    Article  PubMed  CAS  Google Scholar 

  • Lazarowitz (1992) Geminiviruses: genome structure and gene function. Crit Rev Plant Sci 11:327–349

  • Luo H, Lyznik LA, Gidoni D, Hodges TK (2000) FLP-mediated recombination for use in hybrid plant production. Plant J 23:423–430

    Article  PubMed  CAS  Google Scholar 

  • Luo K, Duan H, Zhao D, Zheng X, Deng W, Chen Y, Stewart CN Jr, McAvoy R, Jiang X, Wu Y, He A, Pei Y, Li Y (2007) ‘GM-gene-deletor’: fused loxP-FRT recognition sequences dramatically improve the efficiency of FLP or CRE recombinase on transgene excision from pollen and seed of tobacco plants. Plant Biotechnol J 5:263–274

    Article  PubMed  CAS  Google Scholar 

  • Lutz KA, Corneille S, Azhagiri AK, Svab Z, Maliga P (2004) A novel approach to plastid transformation utilizes the phiC31 phage integrase. Plant J 37:906–913

    Article  PubMed  CAS  Google Scholar 

  • Lutz KA, Svab Z, Maliga P (2006) Construction of marker-free transplastomic tobacco using the Cre-loxP site-specific recombination system. Nat Protoc 1:900–910

    Article  PubMed  CAS  Google Scholar 

  • Lyznik LA, Gordon-Kamm WJ, Tao Y (2003) Site-specific recombination for genetic engineering in plants. Plant Cell Rep 21:925–932

    Article  PubMed  CAS  Google Scholar 

  • Lyznik LA, Mitchell JC, Hirayama L, Hodges TK (1993) Activity of yeast FLP recombinase in maize and rice protoplasts. Nucleic Acids Res 21:969–975

    Article  PubMed  CAS  Google Scholar 

  • Lyznik LA, Rao KV, Hodges TK (1996) FLP-mediated recombination of FRT sites in the maize genome. Nucleic Acids Res 24:3784–3789

    Article  PubMed  CAS  Google Scholar 

  • Malpartida F, Zalacain M, Jimenez A, Davies J (1983) Molecular cloning and expression in streptomyces lividans of a hygromycin B phosphotransferase gene from Streptomyces hygroscopicus. Biochem Biophys Res Commun 117:6–12

    Article  PubMed  CAS  Google Scholar 

  • Marillonnet S, Giritch A, Gils M, Kandzia R, Klimyuk V, Gleba Y (2004) In planta engineering of viral RNA replicons: efficient assembly by recombination of DNA modules delivered by Agrobacterium. Proc Natl Acad Sci USA 101:6852–6857

    Article  PubMed  CAS  Google Scholar 

  • Matsumura M, Katakura Y, Imanaka T, Aiba S (1984) Enzymatic and nucleotide sequence studies of a kanamycin-inactivating enzyme encoded by a plasmid from thermophilic bacilli in comparison with that encoded by plasmid pUB110. J Bacteriol 160:413–420

    PubMed  CAS  Google Scholar 

  • Matzeit V, Schaefer S, Kammann M, Schalk HJ, Schell J, Gronenborn B (1991) Wheat dwarf virus vectors replicate and express foreign genes in cells of monocotyledonous plants. Plant Cell 3:247–258

    Article  PubMed  CAS  Google Scholar 

  • Matzk F, Mahn A (1994) Improved techniques for haploid production in wheat using chromosome elimination. Plant Breed 113:125–129

    Article  Google Scholar 

  • Mengiste T, Revenkova E, Bechtold N, Paszkowski J (1999) An SMC-like protein is required for efficient homologous recombination in Arabidopsis. EMBO J 18:4505–4512

    Article  PubMed  CAS  Google Scholar 

  • Mlynarova L, Conner AJ, Nap JP (2006) Directed microspore-specific recombination of transgenic alleles to prevent pollen-mediated transmission of transgenes. Plant Biotechnol J 4:445–452

    Article  PubMed  CAS  Google Scholar 

  • Mlynarova L, Nap JP (2003) A self-excising Cre recombinase allows efficient recombination of multiple ectopic heterospecific lox sites in transgenic tobacco. Transgenic Res 12:45–57

    Article  PubMed  CAS  Google Scholar 

  • Murashige T, Skoog F (1962) A revised medium for rapid growth and bio assays with tobacco tissue cultures. Physiol Plant 15:473–497

    Article  CAS  Google Scholar 

  • Nanto K, Ebinuma H (2007) Marker-free site-specific integration plants. Transgenic Res 17:337–344

    Article  PubMed  Google Scholar 

  • Odell JT, Nagy F, Chua NH (1985) Identification of DNA sequences required for activity of the cauliflower mosaic virus 35S promoter. Nature 313:810–812

    Article  PubMed  CAS  Google Scholar 

  • Ow DW (2002) Recombinase-directed plant transformation for the post-genomic era. Plant Mol Biol 48:183–200

    Article  PubMed  CAS  Google Scholar 

  • Ow DW (2007) GM maize from site-specific recombination technology, what next? Curr Opin Biotechnol 18:115–120

    Article  PubMed  CAS  Google Scholar 

  • Puchta H (2003) Towards the ideal GMP: homologous recombination and marker gene excision. J Plant Physiol 160:743–754

    Article  PubMed  CAS  Google Scholar 

  • Que Q, Wang HR, Jorgensen A (1998) Distinct patterns of pigment suppression are produced by allelic sense and antisense chalcone synthase transgenes in petunia flowers. Plant J 13:401–409

    Article  CAS  Google Scholar 

  • Radhakrishnan P, Srivastava V (2005) Utility of the FLP-FRT recombination system for genetic manipulation of rice. Plant Cell Rep 23:721–726

    Article  PubMed  CAS  Google Scholar 

  • Ream TS, Strobel J, Roller B, Auger DL, Kato A, Halbrook C, Peters EM, Theuri J, Bauer MJ, Addae P, Dioh W, Staub JM, Gilbertson LA, Birchler JA (2005) A test for ectopic exchange catalyzed by Cre recombinase in maize. Theor Appl Genet 111:378–385

    Article  PubMed  Google Scholar 

  • Rooke L, Byrne D, Salgueiro S (2000) Marker gene expression driven by the maize ubiquitin promoter in transgenic wheat. Annals of Applied Biology 136:167–172

    Article  CAS  Google Scholar 

  • Sambrook J, Fritsch EF, Maniatis T (1989) Molecular cloning. A laboratory manual, 2nd edn. Cold Spring Harbor Laboratory Press, New York

    Google Scholar 

  • Sanford JC, Smith FD, Russell JA (1993) Optimizing the biolistic process for different biological applications. Methods Enzymol 217:483–509

    Article  PubMed  CAS  Google Scholar 

  • Schalk HJ, Matzeit V, Schiller B, Schell J, Gronenborn B (1989) Wheat dwarf virus, a geminivirus of graminaceous plants needs splicing for replication. EMBO J 8:359–364

    PubMed  CAS  Google Scholar 

  • Smith MC, Till R, Brady K, Soultanas P, Thorpe H (2004) Synapsis and DNA cleavage in phiC31 integrase-mediated site-specific recombination. Nucleic Acids Res 32:2607–2617

    Article  PubMed  CAS  Google Scholar 

  • Southern EM (1992) Detection of specific sequences among DNA fragments separated by gel electrophoresis 1975. Biotechnology 24:122–139

    PubMed  CAS  Google Scholar 

  • Sreekala C, Wu L, Gu K, Wang D, Tian D, Yin Z (2005) Excision of a selectable marker in transgenic rice (Oryza sativa L.) using a chemically regulated Cre/loxP system. Plant Cell Rep 24:86–94

    Article  PubMed  CAS  Google Scholar 

  • Srivastava V, Anderson OD, Ow DW (1999) Single-copy transgenic wheat generated through the resolution of complex integration patterns. Proc Natl Acad Sci USA 96:11117–11121

    Article  PubMed  CAS  Google Scholar 

  • Srivastava V, Ow DW (2001) Single-copy primary transformants of maize obtained through the co-introduction of a recombinase-expressing construct. Plant Mol Biol 46:561–566

    Article  PubMed  CAS  Google Scholar 

  • Srivastava V, Ariza-Nieto M, Wilson AJ (2004) Cre-mediated site-specific gene integration for consistent transgene expression in rice. Plant Biotechnol J 2:169–179

    Article  PubMed  CAS  Google Scholar 

  • Thorpe HM, Smith MC (1998) In vitro site-specific integration of bacteriophage DNA catalyzed by a recombinase of the resolvase/invertase family. Proc Natl Acad Sci USA 95:5505–5510

    Article  PubMed  CAS  Google Scholar 

  • Tremblay A, Beauchemin C, Seguin A, Laliberte JF (2007) Reactivation of an integrated disabled viral vector using a Cre-loxP recombination system in Arabidopsis thaliana. Transgenic Res 16:213–222

    Article  PubMed  CAS  Google Scholar 

  • Tungsuchat T, Kuroda H, Narangajavana J, Maliga P (2006) Gene activation in plastids by the CRE site-specific recombinase. Plant Mol Biol 61:711–718

    Article  PubMed  CAS  Google Scholar 

  • Vergunst AC, Jansen LE, Hooykaas PJ (1998) Site-specific integration of Agrobacterium T-DNA in Arabidopsis thaliana mediated by Cre recombinase. Nucleic Acids Res 26:2729–2734

    Article  PubMed  CAS  Google Scholar 

  • Zhang W, Subbarao S, Addae P, Shen A, Armstrong C, Peschke V, Gilbertson L (2003) Cre/lox-mediated marker gene excision in transgenic maize (Zea mays L.) plants. Theor Appl Genet 107:1157–1168

    Article  PubMed  CAS  Google Scholar 

Download references

Acknowledgments

The authors thank Icon Genetics GmbH (Halle/Saale, Germany) for kindly providing the plasmid-vectors used in this study. We particularly thank Dr. Serik Eliby for his contribution in vector construction and his distinguished advices for wheat transformation. Also, we thank Dr. Romy Kandzia for vector cloning and Dr. Sylvestre Marillonnet for many helpful conversations. We are mostly grateful to Corinna Moritz for excellent technical assistance and greenhouse management. The support provided by Dr. Heike Schmuths (Saaten-Union Resistenzlabor GmbH) is gratefully acknowledged. Additionally, we like to thank Wolf v. Rhade and Dr. Ralf Schachschneider (Nordsaat GmbH) for constant encouragement. Especially, the authors wish to thank Dr. Renate Schmidt for comments on the manuscript and for many inspiring discussions. The research was funded by the Bundesministerium für Bildung und Forschung (BMBF, GABI-FUTURE grant 0315043A) at the Leibniz Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben.

Author information

Authors and Affiliations

  1. Leibniz-Institut für Pflanzengenetik und Kulturpflanzenforschung (IPK) Gatersleben, Corrensstr. 3, 06466, Gatersleben, Germany

    Myroslava Rubtsova, Katja Kempe & Mario Gils

  2. Nordsaat Saatzucht GmbH, Saatzucht Langenstein, Hauptstr. 1, 38895, Böhnshausen, Germany

    Angelika Gils

  3. Australian Centre for Plant Functional Genomics (ACPFG), University of Adelaide, Waite Campus, Waite Main Building, Waite Rd, Urrbrae, Adelaide, SA, 5064, Australia

    Ainur Ismagul

  4. Saaten-Union Resistenzlabor GmbH, Hovedisser Str. 92, 33818, Leopoldshöhe, Germany

    Jens Weyen

Authors
  1. Myroslava Rubtsova
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Katja Kempe
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Angelika Gils
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Ainur Ismagul
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Jens Weyen
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Mario Gils
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Mario Gils.

Additional information

Communicated by H. Jones.

M. Rubtsova and K. Kempe contributed equally to the paper.

Electronic supplementary material

Below is the link to the electronic supplementary material.

MOESM1 [INSERT CAPTION HERE] (DOC 41 kb)

Rights and permissions

Reprints and permissions

About this article

Cite this article

Rubtsova, M., Kempe, K., Gils, A. et al. Expression of active Streptomyces phage phiC31 integrase in transgenic wheat plants. Plant Cell Rep 27, 1821–1831 (2008). https://doi.org/10.1007/s00299-008-0604-z

Download citation

  • Received:

  • Revised:

  • Accepted:

  • Published:

  • Issue Date:

  • DOI: https://doi.org/10.1007/s00299-008-0604-z

Keywords

Search

Navigation