Skip to main content
SpringerLink
Log in

Transgene structures in T-DNA-inserted rice plants

  • Published:
Plant Molecular Biology Aims and scope Submit manuscript

Abstract

T-DNA is commonly used for delivery of foreign genes and as an insertional mutagen. Although ample information exists regarding T-DNA organization in dicotyledonous plants, little is known about the monocot rice. Here, we investigated the structure of T-DNA in a large number of transgenic rice plants. Analysis of the T-DNA borders revealed that more than half of the right ends were at the cleavage site, whereas the left ends were not conserved and were deleted up to 180 bp from the left border (LB) cleavage site. Three types of junctions were found between T-DNA and genomic DNA. In the first, up to seven nucleotide overlaps were present. The frequency of this type was much higher in the LB region than at the right border (RB). In the second type, which was more frequent in RB, the link was direct, without any overlaps or filler DNA. Finally, the third type showed filler DNA between T-DNA and the plant sequences. Out of 171 samples examined, 77 carried the vector backbone sequence, with the majority caused by the failure of T-strand termination at LB. However, a significant portion also resulted from co-integration of T-DNA and the vector backbone to a single locus. Most linkages between T-DNA and the vector backbone were formed between two 3′ ends or two 5′ ends of the transferred DNAs. The 3′ ends were mostly linked through 3–6 bp of the complementing sequence, whereas the 5′ ends were linked through either precise junctions or imprecise junctions with filler DNA.

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

Similar content being viewed by others

References

  • An, G., Watson, B.D., Stachel, S., Gordon, M.P. and Nester, E.W. 1985. New cloning vehicles for transformation of higher plants. EMBO J. 4: 277-284.

    Google Scholar 

  • Bevan, M. 1984. Binary Agrobacterium vectors for plant transformation. Nucl. Acids Res. 12: 8711-8721.

    Google Scholar 

  • Binns, A.N. 2002. The T-DNA of Agrobacterium tumefaciens: 25 years and counting. Trends Plant Sci. 7: 231-233.

    Google Scholar 

  • Birch, R.G. 1997. Plant transformation: problems and strategies for practical application. Annu. Rev. Plant Physiol. Plant Mol. Biol. 48: 297-326.

    Google Scholar 

  • Brunaud, V., Balzergue, S., Dubreucq, B., Aubourg, S., Samson, F., Chauvin, S., Bechtold, N., Cruaud, C., de Rose, R., Pelletier, G., Lepiniec, L., Caboche, M. and Lecharny, A. 2002. T-DNA integration into the Arabidopsis genome depends on sequences of pre-insertion sites. EMBO Rep. 3: 1152-1157.

    Google Scholar 

  • Bytebier, B., Deboeck, F., De Greve, H., Van Montagu, M. and Hernalsteens, J.P. 1987. T-DNA organization in tumor cultures and transgenic plants of monocotyledon Asparagus officianalis. Proc. Natl. Acad. Sci. USA 84: 5345-5349.

    Google Scholar 

  • Chen, D.H. and Ronald, P.C. 1999. A rapid DNA minipreparation method suitable for AFLP and other PCR applications. Plant Mol. Biol. Rep. 17: 53-57.

    Google Scholar 

  • Cluster, P.D., O'Dell, M., Metzlaff, M. and Flavell, R.B. 1996. Details of T-DNA structural organization from a transgenic Petunia population exhibiting co-suppression. Plant Mol. Biol. 32: 1197-1203.

    Google Scholar 

  • De Buck, S., Jacobs, A., Van Montagu, M. and Depicker, A. 1999. The DNA sequences of T-DNA junctions suggest that complex T-DNA loci are formed by a recombination process resembling T-DNA integration. Plant J. 20: 295-304.

    Google Scholar 

  • De Buck, S., De Wilde, C., Van Montagu, M. and Depicker, A. 2000. T-DNA vector backbone sequences are frequently integrated into the genome of transgenic plants obtained by Agrobacterium-mediated transformation. Mol. Breed. 6: 459-468.

    Google Scholar 

  • De Cleene, M. 1985. The susceptibility of monocotyledons to Agrobacterium tumefaciens. Phytopath. Z. 113: 81-89.

    Google Scholar 

  • De Cleene, M. and De Ley, J. 1976. The host range of crown gall. Bot. Rev. 42: 389-466.

    Google Scholar 

  • De Neve, M., De Buck, S., Jacobs, A., Van Montagu, M. and Depicker, A. 1997. T-DNA integration patterns in cotransformed plant cells suggest that T-DNA repeats originate from co-integration of separate T-DNAs. Plant J. 11: 15-29.

    Google Scholar 

  • Durrenberger, F., Crameri, A., Hohn, B. and Koukolikova-Nicola, Z. 1989. Covalently bound VirD2 protein of Agrobacterium tumefaciens protects the T-DNA from exonucleolytic degradation. Proc. Natl. Acad. Sci. USA 86: 9154-9158.

    Google Scholar 

  • Galbiati, M., Moreno, M.A., Nadzan, G., Zourelidou, M. and Dellaporta, S.L. 2000. Large-scale T-DNAmutagenesis in Arabidopsis for functional genomic analysis. Funct. Integr. Genom. 1: 25-34.

    Google Scholar 

  • Gelvin, S.B. 2000. Agrobcaterium and plant genes involved in TDNA transfer and integration. Annu. Rev. Plant Physiol. Plant Mol. Biol. 51: 223-256.

    Google Scholar 

  • Gheysen, G., Villarroel, R. and Van Montagu, M. 1991. Illegitimate recombination in plants: a model for T-DNA integration. Genes Dev. 5: 287-297.

    Google Scholar 

  • Hiei, Y., Ohta, S., Komari, T. and Kumashiro, T. 1994. Efficient transformation of rice (Oryza sativa L.) mediated by Agrobacterium and sequence analysis of the boundaries of the T-DNA. Plant J. 6: 271-282.

    Google Scholar 

  • Hoekema, A., Hirsch, P.R., Hooykaas, P.J.J. and Schilperoort, R.A. 1983. A binary plant vector strategy based on separation of vir-and T-regions of the Agrobacterium tumefaciens Ti-plasmid. Nature 303: 179-180.

    Google Scholar 

  • Ishida, Y., Saito, H., Ohta, S., Hiei, Y., Komari, T. and Kumashiro, T. 1996. High efficiency transformation of maize (Zea mays L.) mediated by Agrobacterium tumefaciens. Nature Biotechnol. 14: 745-750.

    Google Scholar 

  • Jeon, J.S., Lee, S., Jung, K.H., Jun, S.H., Jeong, D.H., Lee, J., Kim, C., Jang, S., Yang, K., Nam, J., An, K., Han, M.J., Sung, R.J., Choi, H.S., Yu, J.H., Choi, J.H., Cho, S.Y., Cha, S.S., Kim, S.I. and An, G. 2000. T-DNA insertional mutagenesis for functional genomics in rice. Plant J. 22: 561-570.

    Google Scholar 

  • Jorgensen, R., Snyder, C. and Jones, J.G.D. 1987. T-DNA is organized predominantly in inverted repeat structure in plants transformed with Agrobacterium tumefaciens C58 derivatives. Mol. Gen. Genet. 207: 478-485.

    Google Scholar 

  • Kononov, M.E., Bassuner, B. and Gelvin, S.B. 1997. Integration of T-DNA binary vector ‘backbone’ sequences into the tobacco genome: evidence for multiple complex patterns of integration. Plant J. 11: 945-957.

    Google Scholar 

  • Krizkova, L. and Hrouda, M. 1998. Direct repeats of T-DNA integrated in tobacco chromosome: characterization of junction regions. Plant J. 16: 673-680.

    Google Scholar 

  • Kumar, S. and Fladung, M. 2000. Transgene repeats in aspen: molecular characterization suggests simultaneous integration of independent T-DNAs into receptive hotspots in the host genome. Mol. Gen. Genet. 264: 20-28.

    Google Scholar 

  • Kumar, S. and Fladung, M. 2002. Transgene integration in aspen: Structures of integration sites and mechanism of T-DNA integration. Plant J. 31: 543-551.

    Google Scholar 

  • Lee, S., Jeon, J.S., Jung, K.H. and An, G. 1999. Binary vectors for efficient transformation of rice. J. Plant Biol. 42: 310-316.

    Google Scholar 

  • Mayerhofer, R., Koncz-Kalman, Z., Nawrath, C., Bakkeren, G., Crameri, A., Angelis, K., Redei, G.P., Schell, J., Hohn, B. and Koncz, C. 1991. T-DNA integration: a model of illegitimate recombination in plants. EMBO J. 10: 697-704.

    Google Scholar 

  • Miranda, A., Janssen, G., Hodges, L., Peralta, E.G. and Ream, W. 1992. Agrobacterium tumefaciens transfers extremely long TDNAs by a unidirectional mechanism. J. Bact. 174: 2288-2297.

    Google Scholar 

  • Nester, E.W. and Kosuge, T. 1981. Plasmids specifying plant hyperplasias. Annu. Rev. Microbiol. 35: 531-561.

    Google Scholar 

  • Ochman, H., Gerber, A.S. and Hartl, D.L. 1988. Genetic applications of an inverse polymerase chain reaction. Genetics 120: 621-623.

    Google Scholar 

  • Ooms, G., Bakker, A., Molendijk, L., Wullems, G.J., Gordon, M.P., Nester, E.W. and Schilperoort, R.A. 1982. T-DNA organization in homogeneous and heterogeneous octopine-type crown gall tissues of Nicotiana tabacum. Cell. 30: 589-597.

    Google Scholar 

  • Otten, L., Canaday, J., Gerard, J.C., Fournier, P., Crouzet, P. and Paulus, F. 1992. Evolution of agrobacteria and their Ti plasmids: a review. Mol. Plant-Microbe Interact. 5: 279-287.

    Google Scholar 

  • Puchta, H. 1998. Repair of genomic double-strand breaks in somatic plant cells by one-sided invasion of homologous sequences. Plant J. 13: 331-339.

    Google Scholar 

  • Ramanathan, V. and Veluthambi, K. 1995. Transfer of non-TDNA portions of the Agrobacterium tumefaciens Ti plasmid pTiA6 from the left terminus of TL-DNA. Plant Mol. Biol. 28: 1149-1154.

    Google Scholar 

  • Salomon, S. and Puchta, H. 1998. Capture of genomic and T-DNA sequences during double-strand break repair in somatic plant cells. EMBO J. 17: 6086-6095.

    Google Scholar 

  • Sheng, J. and Citovsky, V. 1996. Agrobacterium-plant cell DNA transport: have virulence proteins, will travel. Plant Cell 8: 1699-1710.

    Google Scholar 

  • Stachel, S.E., Messens, E., Van Montagu, M. and Zambryski, P. 1985. Identification of the signal molecules produced by wounded plant cells that activate T-DNA transfer in Agrobacterium tumefaciens. Nature 318: 624-629.

    Google Scholar 

  • Stachel, S.E., Timmerman, B. and Zambryski, P. 1987. Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5? virD gene products. EMBO J. 6: 857-863.

    Google Scholar 

  • Stahl, R., Horvath, H., van Fleet, J., Voetz, M., von Wettstein, D. and Wolf, N. 2002. T-DNA integration into the barley genome from single and double cassette vectors. Proc. Natl. Acad. Sci. USA 99: 2146-2151.

    Google Scholar 

  • Tingay, S., McElroy, D., Kalla, R., Fieg, S., Wang, M., Thornton, S. and Bretell, R. 1997. Agrobacterium tumefaciens-mediated barley transformation. Plant J. 11: 1369-1376.

    Google Scholar 

  • Tinland, B. 1996. The integration of T-DNA into plant genomes. Trends Plant Sci. 1: 178-184.

    Google Scholar 

  • Triglia, T., Peterson, M.G. and Kemp, D.J. 1988. A procedure for in vitro amplification of DNA segments that lie outside the boundaries of known sequences. Nucl. Acids Res. 16: 8186.

    Google Scholar 

  • Ursic, D., Slightom, J.L. and Kemp, J.D. 1983. Agrobacterium tumefaciens T-DNA integrates into multiple sites of the sun-flower crown gall genome. Mol. Gen. Genet. 190: 494-503.

    Google Scholar 

  • van der Graaff, E., den Dulk-Ras, A. and Hooykaas, P.J.J. 1996. Deviating T-DNA transfer from Agrobacterium tumefaciens to plants. Plant Mol. Biol. 31: 677-681.

    Google Scholar 

  • Wang, K., Herrera-Estrella, L., Van Montagu, M. and Zambryski, P. 1984. Right 25 bp terminus sequence of the nopaline T-DNA is essential for and determines direction of DNA transfer from Agrobacterium to the plant genome. Cell. 38: 455-462.

    Google Scholar 

  • Wenck, A., Czako, M., Kanevski, I. and Marton, L. 1997. Frequent collinear long transfer of DNA inclusive of the whole binary vector during Agrobacterium-mediated transformation. Plant Mol. Biol. 34: 913-922.

    Google Scholar 

  • Wolters, A.M.A., Trindade, L.M., Jacobsen, E. and Visser, R.G.F. 1998. Fluorescence in situ hybridization on extended DNA fibres as a tool to analyse complex T-DNA loci in potato. Plant J. 13: 837-847.

    Google Scholar 

  • Yanofsky, M.F., Porter, S.G., Young, C., Albright, L.M., Gordon, M.P. and Nester, E.W. 1986. The virD operon of Agrobacterium tumefaciens encodes a site-specific endonuclease. Cell. 47: 471-477.

    Google Scholar 

  • Yu, J., Hu, S.N., Wang, J., Wong, G.K.S., Li, S.G., Liu, B., Deng, Y.J., Dai, L., Zhou, Y., Zhang, X.Q., Cao, M.L., Liu, J., Sun, J.D., Tang, J.B., Chen, Y.J., Huang, X.B., Lin, W., Ye, C., Tong, W., Cong, L.J., Geng, J.N., Han, Y.J., Li, L., Li, W., Hu, G.Q., Huang, X.G., Li, W.J., Li, J., Liu, Z.W., Li, L., Liu, J.P., Qi, Q.H., Liu, J.S., Li, L., Li, T., Wang, X.G., Lu, H., Wu, T.T., Zhu, M., Ni, P.X., Han, H., Dong, W., Ren, X.Y., Feng, X.L., Cui, P., Li, X.R., Wang, H., Xu, X., Zhai, W.X., Xu, Z., Zhang, J.S., He, S.J., Zhang, J.G., Xu, J.C., Zhang, K.L., Zheng, X.W., Dong, J.H., Zeng, W.Y., Tao, L., Ye, J., Tan, J., Ren, X.D., Chen, X.W., He, J., Liu, D.F., Tian, W., Tian, C.G., Xia, H.G., Bao, Q.Y., Li, G., Gao, H., Cao, T., Wang, J., Zhao, W.M., Li, P., Chen, W., Wang, X.D., Zhang, Y., Hu, J.F., Wang, J., Liu, S., Yang, J., Zhang, G.Y., Xiong, Y.Q., Li, Z.J., Mao, L., Zhou, C.S., Zhu, Z., Chen, R.S., Hao, B.L., Zheng, W.M., Chen, S.Y., Guo, W., Li, G.J., Liu, S.Q., Tao, M., Wang, J., Zhu, L.H., Yuan, L.P., Yang, H.M. 2002. A draft sequence of the rice genome (Oryza sativa L. ssp. indica). Science 296: 79-92.

    Google Scholar 

  • Zambryski, P., Joos, H., Genetello, C., Leemans, J., Van Montagu, M. and Schell, J. 1983. Ti plasmid vector for the introduction of DNA into plant cells without alteration of their normal regeneration capacity. EMBO J. 2: 2143-2150.

    Google Scholar 

  • Ziemienowicz, A., Tinland, B., Bryant, J. Gloeckler, V. and Hohn, B. 2000. Plant enzymes but not Agrobacterium VirD2 mediate T-DNA ligation in vitro. Mol. Cell. Biol. 20: 6317-6322.

    Google Scholar 

  • Zupan, J., Muth, T.R., Draper, J. and Zambryski, P. 2000. The transfer of DNA from Agrobacterium tumefaciens into plants: a feast of fundamental insights. Plant J. 23: 11-23.

    Google Scholar 

Download references

Author information

Authors and Affiliations

  1. National Research Laboratory of Plant Functional Genomics, Division of Molecular and Life Sciences, Pohang University of Science and Technology (POSTECH), Pohang, 790-784, Republic of Korea

    Sung-Ryul Kim, Jinwon Lee, Sung-Hoon Jun, Sunhee Park, Hong-Gyu Kang & Gynheung An

  2. Department of Horticulture and Breeding, Andong National University, Andong, 760-749, Republic of Korea

    Sung-Ryul Kim & Soontae Kwon

Authors
  1. Sung-Ryul Kim
    View author publications

    You can also search for this author in PubMed Google Scholar

  2. Jinwon Lee
    View author publications

    You can also search for this author in PubMed Google Scholar

  3. Sung-Hoon Jun
    View author publications

    You can also search for this author in PubMed Google Scholar

  4. Sunhee Park
    View author publications

    You can also search for this author in PubMed Google Scholar

  5. Hong-Gyu Kang
    View author publications

    You can also search for this author in PubMed Google Scholar

  6. Soontae Kwon
    View author publications

    You can also search for this author in PubMed Google Scholar

  7. Gynheung An
    View author publications

    You can also search for this author in PubMed Google Scholar

Corresponding author

Correspondence to Gynheung An.

Rights and permissions

Reprints and permissions

About this article

Cite this article

Kim, SR., Lee, J., Jun, SH. et al. Transgene structures in T-DNA-inserted rice plants. Plant Mol Biol 52, 761–773 (2003). https://doi.org/10.1023/A:1025093101021

Download citation

  • Issue Date:

  • DOI: https://doi.org/10.1023/A:1025093101021

Search

Navigation