Mini–Ti: A New Vector Strategy for Plant Genetic Engineering

Abstract

Agrobacterium tumefaciens has the natural ability to insert a specific part of its Ti (tumor–inducing) plasmid, called T–DNA, into the chromosomal DNA of host plant cells, causing tumorous growth. Foreign DNA artificially introduced into T–DNA is inserted into the plant genome by this natural vector system. Genes on the Ti plasmid required for T–DNA transfer, called VIR (virulence) genes, are situated outside T–DNA. We have separated T–DNA from the VIR functions onto a MINI–Ti plasmid that replicates in E. coli and contains full–length T–DNA (including the 25 basepair border signals) from pTi T37, a nopaline–type Ti plasmid. We present evidence that MINI–Ti, when transferred to Agrobacterium by cointegration with a shuttle vector, in the presence of helper plasmid pAL4404 containing VIR genes, can incite nopaline positive tumors, evidence of T–DNA transfer. It is not clear whether MINI–Ti functions in trans to the VIR genes, or whether spontaneous cointegration of pAL4404 with MINI–Ti/pRK (the shuttle form of MINI–Ti) accounts for the tumor–inducing ability of the bacteria. The MINI–Ti approach simplifies procedures for exploiting T–DNA as a gene vector for plants.

Access options

Rent or Buy article

Get time limited or full article access on ReadCube.

from$8.99

All prices are NET prices.

References

  1. 1

    Smith, E.F. and Townsend, C.O. 1907. A plant tumor of bacterial origin. Science 25: 671–673.

  2. 2

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

  3. 3

    Zaenen, I., Van Larebeke, N., Teuchy, H., Van Montagu, M. and Schell, J. 1974. Supercoiled circular DNA in crown gall-inducing Agrobacterium strains. J. Mol. Biol. 86: 109–127.

  4. 4

    Watson, B., Currier, T.C., Gordon, M.P., Chilton, M.-D. and Nester, E.W. 1975. Plasmid required for virulence of Agrobacterium tumefaciens. J. Bacteriol. 123: 255–264.

  5. 5

    Chilton, M.-D., Drummond, M.H., Merlo, D.J., Sciaky, D., Montoya, A.L., Gordon, M.P. and Nester, E.W. 1977. Stable incorporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11: 263–271.

  6. 6

    Chilton, M.-D., Saiki, R.K., Yadav, N., Gordon, M.P. and Quétier, F. 1980. T-DNA from Agrobacterium Ti plasmid is in the nuclear fraction of crown gall tumor cells. Proc. Natl. Acad. Sci. USA 77: 4060–4064.

  7. 7

    Willmitzer, L., De Beuckeleer, M., Lemmers, M., Van Montagu, M. and Schell, J. 1980. DNA from Ti plasmid present in nucleus and absent from plastids of crown gall plant cells. Nature 287: 359–361.

  8. 8

    Drummond, M.H., Gordon, M.P., Nester, E.W. and Chilton, M.-D. 1977. Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumors. Nature 269: 535–536.

  9. 9

    Gelvin, S.B., Gordon, M.P., Nester, E.W. and Aronson, A.A. 1981. Transcription of the Agrobacterium Ti plasmid in the bacterium and in crown gall tumors. Plasmid 6: 17–29.

  10. 10

    Gurley, W.B., Kemp, J.D., Albert, M.J., Sutton, D.W. and Callis, J. 1979. Transcription of Ti plasmid-derived sequences in three octopine-type tumor lines. Proc. Natl. Acad. Sci. USA 76: 2828–2832.

  11. 11

    Petit, A., Delhaye, S., Tempé, J. and Morel, G. 1970. Recherches sur les guanidines des tissues de crown gall. Mise en évidence d'une relation biochimique spécifique entre les souches d'Agrobacterium tumefaciens et les tumeurs qu'elles induisent. Physiol. Vég. 8: 205–213.

  12. 12

    Bomhoff, G., Klapwijk, P.M., Kester, H.C.M., Schilperoort, R.A., Hernalsteens, J.P. and Schell, J. 1976. Octopine and nopaline synthesis and breakdown genetically controlled by a plasmid of Agrobacterium tumefaciens. Mol. Gen. Genet. 145: 177–181.

  13. 13

    Schröder, J., Schröder, G., Huisman, H., Schilperoort, R.A. and Schell, J. 1981. The mRNA for lysopine dehydrogenase in plant tumor cells is complementary to a Ti plasmid fragment. FEBS. Lett. 129: 166–168.

  14. 14

    Murai, N. and Kemp, J.D. 1982. Octopine synthase mRNA isolated from sunflower crown gall callus is homologous to the Ti plasmid of Agrobacterium tumefaciens. Proc. Natl. Acad. Sci. USA 79: 86–90.

  15. 15

    Petit, A. and Tempé, J. 1978. Isolation of Agrobacterium Ti plasmid regulatory mutants. Mol. Gen. Genet. 167: 147–155.

  16. 16

    Holsters, M., Silva, B., Van Vliet, F., Genetello, C., De Block, M., Dhaese, P., Depicker, A., Inze, D., Engler, G., Villarroel, R., Van Montagu, M. and Schell, J. 1980. The functional organization of the nopaline A. tumefaciens plasmid pTi C58. Plasmid 3: 212–230.

  17. 17

    De Greve, H., Decraemer, H., Seurinck, J., Van Montagu, M. and Schell, J. 1981. The functional organization of the octopine Agrobacterium tumefaciens plasmid pTi B6S3. Plasmid 6: 235–248.

  18. 18

    Hernalsteens, J.P., Van Vliet, F., De Beuckeleer, M., Depicker, A., Engler, G., Lemmers, M., Holsters, M., Van Montagu, M. and Schell, J. 1980. The Agrobacterium tumefaciens Ti plasmid as a host vector for introducing foreign DNA in plant cells. Nature 287: 654–656.

  19. 19

    Leemans, J., Shaw, C., Deblaere, R., De Greve, H., Hernalsteens, J.P., Maes, M., Van Montagu, M. and Schell, J. 1981. Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells. J. Mol. Appl. Genet. 1: 149–164.

  20. 20

    Matzke, A.J.M. and Chilton, M.-D. 1981. Site-specific insertion of genes into T-DNA of the Agrobacterium Ti plasmid: an approach to genetic engineering of higher plant cells. J. Mol. Appl. Genet. 1: 39–49.

  21. 21

    Barton, K.A., Binns, A.N., Matzke, A.J.M. and Chilton, M.D. 1983. Regeneration of intact tobacco plants containing full-length copies of genetically engineered T-DNA and transmission of T-DNA to R1 progeny. Cell, in press.

  22. 22

    Barton, K.A. and Chilton, M.-D. 1982. Agrobacterium Ti plasmids as vectors for plant genetic engineering. Methods in Enzymology, in press.

  23. 23

    Ruvkun, G.B. and Ausubel, F.M. 1981. A general method for site-directed mutagenesis in prokaryotes. Nature 289: 85–88.

  24. 24

    Bevan, M.W. and Chilton, M.-D. 1982. T-DNA of the Agrobacterium Ti and Ri plasmids. Ann. Rev. Genet. 16: 357–384.

  25. 25

    Yadav, N.S., Vanderleyden, J., Bennet, D., Barnes, W.M. and Chilton, M.-D. 1982. Short direct repeats flank the T-DNA on a nopaline Ti plasmid. Proc. Natl. Acad. Sci. USA 79: 6322–6326.

  26. 26

    Koekman, B.P., Hooykaas, P.J.J. and Schilperoort, R.A. 1982. A functional map of the replicator region of the octopine Ti plasmid. Plasmid 7: 119–132.

  27. 27

    Kingsbury, D.T. and Helinski, D.R. 1973. Temperature-sensitive mutants for the replication of plasmids in Escherichia coli:Requirement for deoxyribonucleic acid polymerase I in the replication of the plasmid Col El. J. Bacteriol. 114: 1116–1124.

  28. 28

    Engler, G., Depicker, A., Maenhaut, R., Villarroel-Mandiola, R., Van Montagu, M. and Schell, J. 1981. Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens. J. Mol. Biol. 152: 183–208.

  29. 29

    Simpson, R., O'Hara, P., Lichtenstein, C., Montoya, A.L., Kwok, W., Gordon, M.P. and Nester, E.W. 1982. The DNA from A6S/2 tumor contains scrambled Ti plasmid sequence near its junction with plant DNA. Cell 29: 1005–1014.

  30. 30

    Garfinkel, D.J., Simpson, R.B., Ream, L.W., White, F.F., Gordon, M.P. and Nester, E.W. 1981. Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27: 143–153.

  31. 31

    Leemans, J., Deblaere, R., Willmitzer, L., De Greve, H., Hernalsteens, J.P., Van Montagu, M. and Schell, J. 1982. Genetic identification of functions of TL-DNA transcripts in octopine crown galls. The EMBO Journal 1: 147–152.

  32. 32

    Murray, N.E., Brammer, W.J. and Murray, K. 1977. Lambdoid phages that simplify the recovery of in vitro recombinants. Mol. Gen. Genet. 150: 53–61.

  33. 33

    Tacon, W. and Sherratt, D. 1976. Col E plasmid replication in DNA polymerase I deficient strains of Escherichia coli. Mol. Gen. Genet. 147: 331–335.

  34. 34

    Ooms, G., Hooykaas, P.J.J., Van Veen, R.J.M., Van Beelen, P., Regensburg-Tuink, T.J.G. and Schilperoort, R. 1982. Octopine Ti plasmid deletion mutants of Agrobacterium tumefaciens with emphasis on the right side of the T-region. Plasmid 7: 15–29.

  35. 35

    Sciaky, D., Montoya, A.L. and Chilton, M.-D. 1978. Fingerprints of Agrobacterium Ti plasmids. Plasmid 1: 238–253.

  36. 36

    Yadav, N.S., Postle, K., Saiki, R.K., Thomashow, M.F. and Chilton, M.-D. 1980. T-DNA of a crown gall teratoma is covalently joined to host plant DNA. Nature 287: 458–461.

  37. 37

    Ditta, G., Stanfield, S., Corbin, D. and Helinski, D. 1980. Broad host range DNA cloning system for gram-negative bacteria: construction of a gene bank of Rhizobium meliloti. Proc. Natl. Acad. Sci. USA 77: 7347–7351.

  38. 38

    Jacoby, G.A., Jacob, A.E. and Hedges, R.W. 1976. Recombination between plasmids of incompatibility groups P-1 and P-2. J. Bacteriol. 127: 1278–1285.

  39. 39

    Cohen, S.N., Chang, A.C.Y. and Hsu, L. 1972. Non-chromosomal antibiotic resistance in bacteria: genetic transformation of Escherichia coli by R-factor DNA. Proc. Natl. Acad. Sci. USA 69: 2110–2114.

  40. 40

    Holsters, M., de Waele, D., Depicker, A., Messens, E., Van Montagu, M., and Schell, J. 1978. Transfection and transformation of Agrobacterium tumefaciens. Mol. Gen. Genet. 163: 181–187.

  41. 41

    Blair, D.G., Sherratt, D.J., Clewell, D.B. and Helinski, D.R. 1972. Isolation of supercoiled colicinogenic factor E1 DNA sensitive to ribonuclease and alkali. Proc. Natl. Acad. Sci. USA 69: 2518–2522.

  42. 42

    Gill, R. Ph.D. University of Washington, Seattle, Washington, 1980.

  43. 43

    Currier, T.C. and Nester, E.W. 1976. Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biochem. 76: 431–441.

  44. 44

    Casse, F., Boucher, C., Julliot, J.S., Michel, M. and Dénarié, J. 1979. Identification and characterization of large plasmids in Rhizobium meliloti using agarose gel electrophoresis. J. Gen. Microbiol. 113: 229–242.

  45. 45

    Southern, E.M. 1975. Detection of specific sequences among DNA fragments separated by gel electrophoresis. J. Mol. Biol. 98: 503–517.

  46. 46

    Wahl, G.M., Stern, M. and Stark, G.R. 1979. Efficient transfer of large DNA fragments from agarose gels to diazobenzyloxymethyl paper and rapid hybridization by using dextran sulfate. Proc. Natl. Acad. Sci. USA 76: 3683–3687.

  47. 47

    Thomashow, M.F., Nutter, R., Montoya, A.L., Gordon, M.P. and Nester, E.W. 1980. Integration and organization of Ti plasmid sequences in crown gall tumors. Cell 19: 729–739.

  48. 48

    Murashige, T. and Skoog, F. 1962. A revised method for rapid growth and bioassays with tobacco tissue cultures. Physiol. Plant. 15: 473–497.

  49. 49

    Yamada, S. and Itano, H.A. 1966. Phenanthrenequinone as an analytical reagent for arginine and other monosubstituted guanidines. Biochem. Biophys. Acta 130: 538–540.

  50. 50

    Koekman, B.P., Ooms, G., Klapwijk, P.M. and Schilperoort, R.A. 1979. Genetic map of an octopine Ti plasmid. Plasmid 2: 347–357.

Download references

Author information

Rights and permissions

Reprints and Permissions

About this article

Cite this article

de Framond, A., Barton, K. & Chilton, M. Mini–Ti: A New Vector Strategy for Plant Genetic Engineering. Nat Biotechnol 1, 262–269 (1983). https://doi.org/10.1038/nbt0583-262

Download citation