Summary
The T-DNA genes of Ti plasmids of Agrobacterium tumefaciens can induce tumorous growth on a wide range of dicotyledonous plants. We subcloned the individual onc genes of the pTiC58 T-DNA and reintroduced them in the T-region of the Ti plasmid gene vector pGV3850 (from which the onc genes had been removed (Zambryski et al. 1983)). These experiments were designed to analyze the contribution of each onc gene to the development of a tumor and have fulfilled two purposes. First, it was found that only the strains carrying gene 4 produced tumors without the aid of other T-DNA genes; in cell culture these tumors sprout shoots. Second, the shoot-forming phenotype of tumors induced by agrobacteria carrying Ti plasmids defective in either gene 1 or gene 2 can be restored to wildtype phenotype by simple coinfection with Agrobacterium strains whose Ti plasmids contain respectively only gene 2, or only gene 1 in their T-region. A parallel experiment demonstrated that the combined action of genes 1 and 2 is sufficient to induce tumor formation on tobacco plantlets.
The external addition of α-naphthalene acetic acid (NAA) restores to wild-type the phenotype of tumors induced by mutants in gene 1 or in gene 2. However, α-naphthalene acetamide can only restore to wild-type the phenotype of mutants in gene 1. These data indicate that the product of the T-DNA gene 2 participates in the conversion of α-naphthalene acetamide to a biologically active auxin, presumably NAA, and suggest that gene 1 codes for an enzyme involved in the synthesis of an indole-3-acetyl derivative.
Similar content being viewed by others
References
Akiyoshi DE, Morris RO, Hinz R, Mischke PS, Kosuge T, Garfinkel DJ, Gordon MP, Nester EW (1983) Cytokinin/auxin balance in crown gall tumors is regulated by specific loci in the T-DNA. Proc Natl Acad Sci USA 80:407–411
Amasino RM, Miller CO (1982) Hormonal control of tobacco crown gall tumor morphology. Plant Physiol 69:389–392
Anand VK, Heberlein GT (1977) Crown gall tumorigenesis in potato tuber tissue. Am J Bot 64:153–158
Bevan MW, Chilton M-D (1982) Multiple transcripts of T-DNA detected in nopaline crown gall tumors. J Mol Appl Genet 1:539–546
Binns AN, Sciaky D, Wood HN (1982) Variation in hormone autonomy and regenerative potential of cells transformed by strain A66 of Agrobacterium tumefaciens. Cell 31:605–612
Birnboim HC, Doly J (1979) A rapid alkaline extraction procedure for screening recombinant plasmid DNA. Nucl Acids Res 7:1512–1523
Bolivar F, Betlach M, Heynecker HL, Shine J, Rodriguez R, Boyer HW (1977) Construction and characterization of new cloning vehicles. I. Ampicillin-resistant derivatives of the plasmid pMB9. Gene 2:75–93
Boyer HW, Roulland-Dussoix D (1969) A complementation analysis of the restriction and modification of DNA in E. coli. J Mol Biol 41:459–472
Chilton M-D, Drummond MH, Merlo DJ, Sciaky D, Montoya AL, Gordon MP, Nester EW (1977) Stable incroporation of plasmid DNA into higher plant cells: the molecular basis of crown gall tumorigenesis. Cell 11:263–271
Chilton M-D, Drummond MH, Merlo DJ, Sciaky D (1978) Highly conserved DNA of Ti-plasmids overlaps T-DNA, maintained in plant tumors. Nature (London) 275:147–149
Chilton M-D, Saiki RK, Yadav N, Gordon MP, Quetier F (1980) T-DNA from Agrobacterium Ti plasmid is in the nuclear DNA fraction of crown gall tumor cells. Proc Natl Acad Sci USA 77:4060–4064
Colson C, Glover SW, Symonds N, Stacey KA (1965) The location of genes for host controlled modification and restriction in Eschrichia coli K12. Genetics 52:1043–1050
De Beuckeller M, Lemmers M, De Vos G, Willmitzer L, Van Montagu M, Schell J (1981) Further insight on the transferred-DNA of octopine crown gall. Mol Gen Genet 183:283–288
De Cleene M, De Ley J (1976) The host range of crown-gall. Bot Rev 42:389–466
Depicker A, Van Montagu M, Schell J (1978) Homologous DNA sequences in different Ti-plasmids are essential for oncogenicity. Nature (London) 275:150–153
Depicker A, De Wilde M, De Vos G, De Vos R, Van Montagu M, Schell J (1980) Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Plasmid 3:193–211
De Vos G, De Beuckeller M, Van Montagu M, Schell J (1981) Restriction endonuclease mapping of the octopine tumor inducting pTiAch5 of Agrobacterium tumefaciens. Plasmid 6:249–253
Dhaese P, De Greve H, Decraemer H, Schell J, Van Montagu M (1979) Rapid mapping of transposon insertion and deletion mutations in the large Ti-plasmids of Agrobacterium tumefaciens. Nucl Acids Res 7:1837–1949
Drummond MH, Gordon MP, Nester EW, Chilton M-D (1977) Foreign DNA of bacterial plasmid origin is transcribed in crown gall tumours. Nature (London) 269:535–536
Engler G, Depicker A, Maenhaut R, Villarroel-Mandiola R, Van Montagu M, Schell J (1981) Physical mapping of DNA base sequence homologies between an octopine and a nopaline Tiplasmid of Agrobacterium tumefaciens. J Mol Biol 152:183–208
Garfinkel DJ, Simpson RB, Ream LW, White FF, Gordon MP, Nester EW (1981) Genetic analysis of crown gall: fine structure map of the T-DNA by site-directed mutagenesis. Cell 27:143–153
Gelvin SB, Thomashow MF, McPherson JC, Gordon MP, Nester EW (1982) Sizes and map positions of several plasmid-DNA-encoded transcripts in octopine-type crown gall tumors. Proc Natl Acad Sci USA 79:76–80
Gurley WB, Kemp JD, Albert MJ, Sutton DW, Callis J (1979) Transcription of Ti plasmid-derived sequences in three octopine-type crown gall tumor lines. Proc Natl Acad Sci USA 76:2828–2832
Guyon P, Chilton M-D, Petit A, Tempé J (1980) Agropine in “null type” crown gall tumors: evidence for the generality of the opine concept. Proc Natl Acad Sci USA 77:2693–2697
Hamilton RH, Fall MZ (1971) The loss of tumor initiating ability in Agrobacterium tumefaciens by incubation at high temperature. Experientia 27:229–230
Holsters M, Silva B, Van Vliet F, Genetello C, De Block M, Dhaese P, Depicker A, Inzé D, Engler G, Villarroel R, Van Montagu M, Schell J (1980) The functional organization of the nopaline A. tumefaciens plasmid pTiC58. Plasmid 3:212–230
Joos H, Inzé D, Caplan A, Sormann M, Van Montagu M, Schell J (1983) Genetic analysis of T-DNA transcripts in nopaline crown galls. Cell 32:1057–1067
Klapwijk PM, Van Beelen P, SchiIperoort RA (1979) Isolation of a recombinant deficient Agrobacterium tumefaciens mutant. Mol Gen Genet 173:171–175
Kosuge T, Heskett MG, Wilson EE (1968) Microbial synthesis and degradation of indole-3-acetic acid. I. The conversion of L-tryptophan. J Biol Chem 241:3738–3744
Leemans J, Shaw C, Deblaere R, De Greve H, Hernalsteens JP, Maes M, Van Montagu M, Schell J (1981) Site-specific mutagenesis of Agrobacterium Ti plasmids and transfer of genes to plant cells. J Mol Appl Genet 1:149–164
Leemans J, Deblaere R, Willmitzer L, De Greve H, Hernalsteens JP, Van Montagu M, Schell J (1982) Genetic identification of functions of TL-DNA transcripts in octopine crown galls. EMBO J 1:147–152
Lemmers M, De Beuckeleer M, Holsters M, Zambryski P, Depicker A, Hernalsteens JP, Van Montagu M, Schell J (1980) Internal organization, boundaries and integration of Ti-plasmid DNA in nopaline crown gall tumours. J Mol Biol 144:353–376
Letham OS, Goodwin PB, Higgins TJV (1978) Phytohormones and the development of higher plants, vol I and II. Elsevier/ North-Holland Biomedical Press, Amsterdam
Linsmaier EM, Skoog F (1965) Organic growth factor requirements of tobacco tissue cultures. Physiol Plant 18:100–127
Liu ST, Kado CI (1979) Indoel acetic acid production: a plasmid function of Agrobacterium tumefaciens C58 Biochem Biophys Res Comm 20:171–178
Maniatis T, Fritsch EF, Sambrook J (1982) Mocular cloning, a laboratory manual. Cold Spring Harbor Laboratory, New York, pp 545
Miller JH (1972) Experiments in molecular genetics. Cold Spring Harbor Laboratory, New York, pp 466
Morris RO, Akiyoshi DE, MacDonald EMS, Morris JW, Regier DA, Zaerr JB (1982) Cytokinin metabolism in relation to tumor induction by Agrobacterium tumefaciens. In: Wareing PF (ed) Plant growth substances 1982. Academic Press, London, pp 175–183
Ooms G, Hooykaas PJ, Moleman G, Schilperoort RA (1981) Crown gall plant tumors of abnormal morphology, induced by Agrobacterium tumefaciens carrying mutated octopine Ti plasmids; analysis of T-DNA functions. Gene 14:33–50
Ooms G, Molendijk L, Schilperoort RA (1982) Double infection of tobacco plants by two complementing octopine T-region mutants of Agrobacterium tumefaciens. Plant Mol Biol 1:217–226
Otten LA, Schilperoort RA (1978) A rapid microscale method for the detection of lysopine-and nopaline dehydrogenase activities. Biochim Biophys Acta 527:497–500
Petit A, Delhaye S, Tempé J, Morel G (1970) Recherches sur les guanidines des tissus de crown gall. Mise en évidence d'une relation biochemique spécifique entre les souches d'Agrobacterium et les tumeurs qu'elles induisent. Physiol Vég 8:205–213
Rao RN, Rogers SG (1979) Plasmid pKC7: a vector containing ten restriction endonuclease sites suitable for cloning DNA fragments. Gene 7:79–82
Schneider EA, Wightman F (1978) Auxines. In: Letham DS, Goodwin PB, Higgins TJV (eds) Phytohormones and related compounds. A comprehensive treatise, vol I. Elsevier/North-Holland, Amsterdam, pp 29–92
Schröder G, Schröder J (1982) Hybridization selection and translation of T-DNA encoded mRNAs from octopine tumors. Mol Gen Genet 185:51–55
Schröder G, Klipp W, Hillebrand A, Ehring R, Koncz C, Schröder J (1983) The conserved part of the T-region in Ti-plasmids expresses four proteins in bacteria. EMBO J 2:403–409
Schröder G, Waffenschmidt S, Weiler EW, Schröder J (1984) The T-region of Ti plasmids codes for an enzyme synthesizing indole-3-acetic acid. Eur J Biochem, in press
Smith EF, Townsend CO (1907) A plant tumor óf bacterial origin. Science 25:671–673
Stonier T (1962) Normal, abnormal, and pathological regeneration in Nicotiana. In: Rudnick D (ed) Regeneration (XXth Annual Symp Soc for the Study of Growth and Development). Ronald Press, New York, pp 85–115
Thomashow MF, Nutter R, Montoya AL, Gordon MP, Nester EW (1980) Integration and organisation of Ti-plasmid se-quences in crown gall tumors. Cell 19:729–739
Van Haute E, Joos H, Maes M, Warren G, Van Montagu M, Schell J (1983) Intergeneric transfer and exchange recombination of restriction fragments cloned in pBR322: a novel strategy for the reversed genetics of the Ti plasmids of Agrobacterium tumefaciens. EMBO J 2:411–417
Van Larebeke N, Engler G, Holsters M, Van den Elsacker S, Zaenen I, Schilperoort RA, Schell J (1974) Large plasmid in Agrobacterium tumefaciens essential for crown gall-inducing ability. Nature (London) 252:169–170
Van Larebeke N, Genetello C, Hernalsteens JP, Depicker A, Zaenen I, Messens E, Van Montagu M, Schell J (1977) Transfer of Ti plasmids between Agrobacterium strains by mobilization with the conjugative plasmid RP4. Mol Gen Genet 152:119–124
Vervliet G, Holsters M, Teuchy H, Van Montagu M, Schell J (1975) Characterization of different plaque-forming and defective temperate phages in Agrobacterium strains. J Gen Virol 26:33–48
Willmitzer L, De Beuckeleer M, Lemmers M, Van Montagu M, Schell J (1980) DNA from Ti-plasmid is present in the nucleus and absent from plastids of plant crown-gall cells. Nature (London) 287:359–361
Willmitzer L, Otten L, Simons G, Schmalenbach W, Schröder J, Schröder G, Van Montagu M, De Vos G, Schell J (1981) Nuclear and polysomal transcripts of T-DNA in octopine crown gall suspension and callus cultures. Mol Gen Genet 182:255–262
Willmitzer L, Simons G, Schell J (1982) The TL-DNA in octopine crown gall tumours coles for seven well-defined polyadenylated transcripts. EMBO J 1:139–146
Willmitzer L, Dhaese P, Schreier PH, Schmalenbach W, Van Montagu M, Schell J (1983) Size, location, and polarity of T-DNA-encoded transcripts in nopaline crown gall tumors; evidence for common transcripts present in both octopine and nopaline tumors. Cell 32:1045–1056
Zaenen I, Van Larebeke N, Teuchy H, Van Montagu M, Schell J (1974) Supercoiled circular DNA in crown gall inducing Agrobacterium strains. J Mol Biol 86:109–127
Zambryski P, Joos H, Genetello C, Leemans J, Van Montagu M, 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
Author information
Authors and Affiliations
Additional information
Communicated by G. Melchers
Rights and permissions
About this article
Cite this article
Inzé, D., Follin, A., Van Lijsebettens, M. et al. Genetic analysis of the individual T-DNA genes of Agrobacterium tumefaciens; further evidence that two genes are involved in indole-3-acetic acid synthesis. Molec. Gen. Genet. 194, 265–274 (1984). https://doi.org/10.1007/BF00383526
Received:
Issue Date:
DOI: https://doi.org/10.1007/BF00383526