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Agrobacterium Ti Plasmid Indoleacetic Acid Gene Is Required for Crown Gall Oncogenesis (Nopaline/Octopine/Oncogenesis/Tryptophan Aminotransferase) S

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Agrobacterium Ti Plasmid Indoleacetic Acid Gene Is Required for Crown Gall Oncogenesis (Nopaline/Octopine/Oncogenesis/Tryptophan Aminotransferase) S Proc. NatL Acad. Sci. USA Vol. 79, pp. 2812-2816, May 1982 Biochemistry Agrobacterium Ti plasmid indoleacetic acid gene is required for crown gall oncogenesis (nopaline/octopine/oncogenesis/tryptophan aminotransferase) S. -T. Liu, K. L. PERRY, C. L. SCHARDL, AND C. I. KADO* Davis Crown Gall Group, Department of Plant Pathology, University of California, Davis, California 95616 Communicated by I. C. GunsalusJJanuary 27, 1982 ABSTRACT A gene (iaaP) necessary for virulence and indole- MATERIALS AND METHODS acetic acid (IAA) production has been located on a nopaline Ti Media. A. tumefaciens plasmid ofAgrobacterium tumefaciens C58. iaaP function was es- Bacterial Strains, Plasmids, and tablished by using transformation. to insert nopaline or octopine 1D135, C58, and ACH-5 were described previously (12). Ti plasmids into an avirulent, Ti plasmid-free mutant 1D1293-3 Strains 1D1293 and CS11 are plasmid-free derivatives of strain that was defective in IAA synthesis (iaaC-). The resulting trans- C58 obtained by heat and ethidium bromide curing (13). Esch- formants produced increased levels of IAA and virulence was re- erichia coli 1830 pro- met- harboring Inc P1 plasmid pJB4JI stored. When these transformants were cured oftheir Ti plasmid, carrying Tn5 was obtained from A. W. B. Johnston (14). Phage virulence and high IAA production levels were concomitantly lost. Pl::Tn5 was kindly provided by A. D. Kaiser and D. Berg. All ATn5 mutagenized TiC58 plasmid, deficient in theabilityto direct A. tumefaciens strains were grown at 20°C or 29°C either in increased synthesis of IAA, wasiinserted by transformation into minimal medium 925 (15) supplemented with 0.05% or 0.005% mutant 1D1293-3. The resulting transformants 1D1293-3 filter-sterilized L-tryptophan or in complete medium 523 (15). (TiC58::Tn5) remained avirulent and iaaP-. Restriction analysis Octopine (Sigma) or nopaline [chemically synthesized as de- of the TiC58::Tn5 plasmid DNA identified the iaaP gene at'20.9 scribed by Jensen et aL (16)] medium consisted ofmedium 925 kilobases to the left of the T-DNA. A major aromatic-amino-acid salts with 2 mg of the opine per ml substituted for NH4C1. aminotransferase is coded by the iaaC gene, but not by the iaaP Construction of IAA Mutants. A. tumefaciens cells were gene. The possible reasons for the iaaP locus to be situated outside mutagenized with N-methyl-N'-nitro-N-nitrosoguanidine (K the T-DNA region are discussed. & K) as described (15). Individual colonies on minimal 925 agar plates were suspended in 5 ml ofmedium 925 containing 0.05% Crown gall tumor cells manifested through infection by Agro- filter-sterilized L-tryptophan and grown for 24-36 hr at 23°C. bacterium tumefaciens synthesize copious amounts of the Samples (50 1,u) ofeach culture were mixed in microtiter plates growth hormone indoleacetic acid (IAA) (1-6) and possess the with 100 ,1l of Salkowski reagent (17). IAA production by each ability to grow axenically in chemically defined culture media mutant was scored visually after 30 min of incubation at 23°C in the absence of IAA and cytokinin (7). On the other hand, by comparing color intensities. Quantitative analysis of IAA was normal plant cells are incapable of growth unless the medium determined spectrophotometrically at 590 nm and confirmed is supplemented with these phytohormones. The difference by gas/liquid chromatographic analysis as described (11). Mu- between normal and crown gall tumor cells may be explained tants were also constructed by inserting transposon Tn5 by con- by the unique ability ofA. tumefaciens to insert part of its ge- jugal transfer ofpJB4JI from E. coli 1830 to Agrobacterium re- netic material as a plasmid into plant cells. A specific portion cipients on agar medium 523 as described (18). of this plasmid (Ti plasmid), which is stably incorporated and Assay of Oncogenicity. Inoculations and virulence levels maintained in an integrated form in the nuclear DNA segment were assessed as described (15). known as the T-DNA (8, 9), carries either genetic information Enzyme and Product Assays. A. tumefaciens cells, grown in for the regulation of plant genes responsible for IAA synthesis 200 ml ofmedium 925 containing 0.01% L-tryptophan and 0.5% or structural genes for the direct synthesis of IAA. An alter- glucose but no NH4Cl, were harvested at 100 Klett units (green native hypothesis is that existing genetic components for IAA filter) by centrifugation and resuspended in 4 ml of buffer [20 synthesis in the plant cell are activated by the mere insertion mM Tris-HCl, pH 8.0/10 mM Na2EDTA/10% (vol/vol) glyc- ofthe T-DNA. Evidence in support ofthe first hypothesis comes erol/0. 1 mM dithiothreitoV20 mM pyridoxal phosphate]. They from the fact that A. tumefaciens cells themselves elaborate were broken with an Aminco pressure cell [15,000 pounds/ abnormally high amounts of IAA (10) and from the fact that and for 30 min at 27,000 x g. production of IAA is controlled by the Ti plasmid (11). A. tu- inch2 (103 MPa)] centrifuged mefaciens cells, when cured of the Ti plasmid, do themselves Samples (40 ,1d) ofthe supernatant were loaded on vertical poly- synthesize IAA. In all cases, transfer ofthe Ti plasmid into plas- acrylamide slab gels composed of 4.9% acrylamide and 0.13% mid-free A. tumefaciens directs IAA production to higher lev- methylene bisacrylamide in 375 mM Tris-HCl, pH 8.9, and els. These data have suggested that some aspect of IAA pro- polymerized with ammonium persulfate at 500 ,ug/ml and duction is directly controlled by Ti plasmid genes. In this paper N,N1,N',N'tetramethylethylenediamine at 0.75 ,ug/ml. Elec- we report evidence for the presence and lcdation ofan IAA gene trophoresis was carried out under constant current of28'mA for on the Ti plasmid and show that this gene is absolutely essential 3.5 hr at 4°C (19). Aromatic-aminoTacid aminotransferase activ- for tumor induction by A. tumefaciens. ity was measured as described by Lin et al. (20) and tryptophan aminotransferase (EC 2.6.1.27) andsecondary aromatic-amino- The publication costs ofthis article were defrayed in part by page charge 'payment. This article must therefore be hereby marked "advertise- Abbreviations: IAA, indoleacetic acid; kb, kilobase(s). ment" in accordance with 18 U. S. C. §1734 solely to indicate this fact. * To whom reprint requests should be addressed. 2812 Downloaded by guest on October 1, 2021 Biochemistry: Liu et al. Proc. Natl. Acad. Sci. USA 79 (1982) 2813 Table 1. Characteristics of IAA- mutants of A. tumefaciens IAA L-Tryptophan 3-Ketolactose Ti Utilization of synthesized,* uptake,t Strain formation plasmid Virulence nopaline 'tg/ml cpm hr-' per 106 cells 1D1293 (parental) + - - - 78 12,841 1D1293-1 (mutant) + - - - 42 12,986 1D1293-2 (mutant) + - - - 33 13,046 1D1293-3 (mutant) + - - - 15 12,859 * Average of three determinations taken after 60 hr of growth at 200C. L-Tryptophan (0.05%) was used in minimal medium 925. t For tryptophan uptake, exponentially grown cells were incubated in minimal medium containing 5 /ICi of L-[14C]tryptophan (60 mCi/mmol, New England Nuclear) for-60 min at 23TC and 50-uId samples were precipitated with ice-cold 10% trichloroacetic acid and collected on Whatman GF/A filter circles (2.5 cm). The precipitate was washed with 10% trichloroacetic acid followed by 95% (vol/vol) ethanol and dried, and the radioactivity remaining was measured in a Beckman LS-233 liquid scintillation spectrometer. The number of cells was concurrently measured by standard dilution plating on medium 925. acid aminotransferase activities were detected by staining the Starting with the Ti plasmid-free strain 1D1293, mutants de- gel, using 2-ketoglutarate as the amine acceptor (21). fective in IAA synthesis were constructed by three cycles ofN- IAA precursors from L-tryptophan were detected by induc- methyl-N'-nitro-N-nitrosoguanidinemutagenesis toobtain three ing cells in 20 ml of medium 925 containing 0.05% L-trypto- successive mutants. Examples of iaaC- mutants that were iso- phan. After 24 hr ofgrowth at 20'C, 1 mCi ofL-[3H]tryptophan lated with diminished production ofIAA are presented in Table (6.9 Ci/mmol, New England Nuclear; 1 Ci = 3.7 X 1010 1. The iaaC- mutant 1D1293-3 with the lowest IAA synthetic becquerels) was added and growth was continued for an addi- ability was selected for the experiments to follow. The data in tional 36 hr. The cells were then sonicated for 60 sec at 00C, Table 1 showed that this iaaC- mutant was not the result of using a Biosonik IV sonicator (Bronwill) at maximal setting. In- defective tryptophan uptake, because it accumulated trypto- dolepyruvate (1 mg/ml) in 1 ml of 10 mM Tris HCl, pH 8.0/ phan-as readily as the parental strain. Chromatographic analyses 1 mM Na2EDTA was added to each sample- immediately prior of cell extracts for enzyme products and activities of the IAA to derivatization with a saturated solution of 2,4-dinitrophen- pathway leading from tryptophan-i.e., tryptophan -* ylhydrazine in 2 M HC1 (22). The radioactive products (e.g., indolepyruvate -- indoleacetaldehyde --+IAA (10)-showed indolepyruvate) were resolved by thin-layer chromatography that L-[3H]tryptophan was indeed converted to [3H]indole- on silica gel 60 F254 plates (Merck). pyruvate (data not shown; see Materials and Methods). Fur- Plasmid Detection and Isolation. Ti plasmids harbored in A. thermore, analysis by electrophoresis in nondenaturing tumefaciens were analyzed by a mini-screen procedure (23). Ti polyacrylamide gels revealed that mutant 1D1293-3 carries a plasmid DNA was purified as described elsewhere (12). mutation in a principal 2-ketoglutarate-dependent aromatic- Mating and Transformation. Conjugative transfer of plas- amino-acid aminotransferase gene (Fig.
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