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. 1). Activities of other mids was performed as described (24) on agar medium 523 on which donor and recipient cells were mixed at a ratio of 10:1 and deposited on a 2.5-cm-diameter membrane filter. a b c d Transformation was performed according to Holsters et aL (25) with modifications. Cell pellets were frozen in a dry ice/ ethanol bath and then,immersed in a 37MC water bath for 3 min before plasmid DNA [100 ,j of dialyzed Ti plasmid DNA pre- pared by the mini-screen procedure (23)] was added. The mix- ture was refrozen and then incubated at 370C for 30 min. In- cubation was continued for 30 min at 30'C in 1 ml of523 broth. Serially diluted washed-cells were plated on selective media. Hybridization Analysis. Ti plasmid DNA fragments gener- ated by restriction endonucleases were fractionated by agarose gel electrophoresis (23) and then transferred to nitrocellulose membrane filter paper as described by Southern (26) as mod- ified by Wahl et at (27). Probe DNA was labeled by nick-trans- lation (28, 29) to a specific radioactivity of 107-10 cpm//1g of DNA by using deoxyadenosine5'-[32P]triphosphate and deoxy- cytosine 5'-[3 P]triphosphate (350 Ci/mmol each, Amersham), DNase I (Worthington), and DNA polymerase L(New England BioLabs). Hybridizations were performed in 50% (vol/vol) formamide, 420C, as described by Wahl et aL (27).

RESULTS A;.

..t:.:,!. -,;Z t ..: III *;:" 1.11-1'..,A. Characteristics ofiaa- Mutants. We have previously shown 0: .II.I O., that virulent A. tumefaciens C58 synthesize IAA in the presence -, .. oftryptophan or tyrosine and that levels ofIAA of50% or 10%, respectively, are still synthesized by its avirulent plasmid-free FIG. 1. Polyacrylamide gel electrophoretogram of tryptophan counterpart (11). This suggested that more than one gene must aminotransferase (arrow) and secondary aromatic-amino-acid ami- be involved in the synthesis of IAA and that these genes are notransferases fromA. tumefaciens. Lane a, 1D1293-3; lane b, 1D1293- located on the Ti plasmid and on the bacterial chromosome. 3(pTiC58); lane c, CS11; lane d; C58. Downloaded by guest on October 1, 2021 2814 Biochemistry: Liu et aL Proc. Natl. Acad. Sci. USA 79 (1982) Table 2. Ti plasmid-mediated IAA synthesis in IAA- mutant* IAA synthesized in presence of L-tryptophan Ti Opine /g/ml %above control Transformant plasmid utilization 0.05%t 0.005%t 0.05%t 0.005%t Virulence 1D1293-3 None None 10.2 2.2 0 0 rn1293-3-C58 TiC58 Nopaline 12.5 2.7 22.5 22.7 + 1D1293-3-ACH-5 TiACH-5 Octopine 15.8 2.8 54.9 27.2 + _1D1293-3-C58::Tn5 TiC58::Tn5 Nopaline 11.0 2.3 7.8 4.5 1D1293-2-135 Ti135 Nopaline 18.3§ - 81.1 - + 1D1293-2 cured None None 10.1§ 0 -- * IAA- mutant 1D1293-3 cells were grown in medium 925 containing either 0.05% or 0.005% L-tryptophan at 20TC for 60 hr or 48 hr, respectively. The cells were removed by centrifugation at 400, and the supernatant was analyzed for IAA content (11). Three.samples were measured for each supernatant analyzed. Standard deviation of the mean IAA concentration ranged between ±0.015 and ±0.073 Ag/ml of medium. t Data based on jtg of IAA per ml of culture medium. * Data based on ,ug of IAA per ml per .108 cells. § Data from a separate experiment was normalized accordingly. All data represent averages of three or more replicated samples *of three or more experiments.

aromatic-amino-acid aminotransferases that also convert, with iaaP and Its Function., Mutant 1D1293-3 was used as recip- low efficiency, tryptophan to indolepyruvate were apparent in ient for Ti plasmid DNAs isolated and purified from A. tume- this mutant, though some of these were also slightly altered faciens 1D135, C58, and ACH-5. The Ti plasmid DNA from (Fig. 1). This may explain why IAA levels were not reduced to each of these strains was introduced into mutant 1D1293-3 by zero and further suggests the need for several cycles of muta- transformation. Transformants were selected on the basis of genesis to lower IAA synthesis levels in strain 1D1293 (Table -nopaline- (strain 1D135 and C58) and octopine-.(strain ACH-5) 1). These results also showed that the observed tryptophan ami- utilizing ability. These transformants elaborated about 22-81% notransferase activity is coded by the Agrobacterium genome more IAA than-did the mutant 1D1293-3 (Table 2). These data rather than the- Ti plasmid because the enzyme was present in suggested that Ti plasmid gene(s) code for the synthesis of IAA Ti plasmid-free strain 1D1293 but not in the mutant 1D1293- or-for the regulation of IAA synthesis. An alternative hypothesis 3. Also, the enzyme activity was not restored in 1D1293-3 by may be that the Ti plasmid codes for an inactivator ofa-possible the insertion of the TiC58 plasmid (Fig. 1). inhibitor. or degrader of IAA. However, mutants, scored

a b C a b

C-51 -4. .4- 5

:j- 14 a

FIG. 3. Blot hybridization analyses of TiC58::Tn5 cleaved with restriction endonucleases Kpn I-(a) and HinduI (b). Left lanes of pairs a and b are restriction fragments of TiC58::Tn5. The right lanes of FIG. 2. Agarose gel electrophoretogram of TiC58::Tn5 and TiC58 each pair are restriction fragments ofTiC58. Phage P1:: Tn5DNA was plasmid DNAs cleaved with restriction endonucleases Kpn I (a), Sma used as 32P-labeled probe. Fragments carrying the Tn5 insertion have I (b), andHindfll (c). Numbers represent the fragment number missing been translocated as larger fragments as expected and revealed by in TiC58::Tn5 DNA (left lane of each pair). autoradiography. Downloaded by guest on October 1, 2021 Biochemistry: Liu et aL Proc. Natl. Acad. Sci. USA 79 (1982) 2815 Tn5 I T-iDNA i Kpn IF, 5 I I 13b |181 9 1 13a | 3 (,_ SinaI lb 12 13 t0 la HindMlf 9 18 14a 27 16 3 7 10 15 14b 19 (4 B ImHlf 2 1231 9 13411 13 1271 10 1151 3

\ '°e < I < l~~~~~~~~~~~Okb

,-~~~~150~ 30\ 4140 40~ pTi 130 50

120 60 110 7 100 9080

FIG. 4. Restriction cleavage map of that portion of fragments adjoining the left-hand terminus of the T-DNA. The critical point of Tn5 insertion is marked by the inserted triangle. through three cycles ofmutagenesis did not yield a hyper-IAA- shown). The presence ofTn5 sequences was confirmed by blot producing mutant. hybridization (Fig. 3). On the basis ofthe physical map ofTiC58 Relationship of iaaP and Virulence. Transformants harbor- of Depicker et al. (30), the insertion is located 41.8 kilobases ing a wild-type Ti plasmid and showing higher levels of IAA (kb) to the left of the arbitrary zero point (between Sma I frag- were checked for virulence. Unlike the iaaC- mutant 1D1293- ments 5 and 7) of TiC58 DNA. The insertion was thus located 3, which was totally avirulent, the transformants, now capable at about 20.9 kb to the left end of the T-DNA (Fig. 4). ofincreased IAA synthesis, induced crown gall tumor formation in sunflower, Kalanchoe, tomato, tobacco, and carrots. Each ofthe three transformants tested was as virulent as its parental DISCUSSION wild-type strain, asjudged by the crown gall size assay (15). The The molecular basis for crown gall oncogenesis may be ex- pTilD135 and pTiC58 transformants were then cured of their plained by the current hypothesis that integration ofthe T-DNA plasmids by serial growth passages at 370C (12). Transformants in the plant genome somehow contributes to crown gall for- freed ofthe Ti plasmid again showed complete loss ofvirulence mation. That a specific locus or loci in the T-DNA is all that is and opine utilization and showed reduced IAA levels (Table 2). required for oncogenesis remains to be clearly demonstrated. Location of iaaP. The location of the iaaP gene was deter- In fact, it remained unclear whether oncogenesis was the result mined by inserting the transposon Tn5 into various regions of ofthe mere insertion ofthe T-DNA or was the result ofcertain pTiC58. The resulting mutants were selected on the basis of products that are coded by T-DNA genes. We believe that our kanamycin resistance on minimal medium and for plasmid in- work supports the latter hypothesis, that gene products of the sertions by conjugatively transferring the mutagenized TiC58 Ti plasmid are essential for oncogenesis. plasmid to iaaC- mutant 1D1293-3. The presence ofTiC58::Tn5 Of interest is the location of the iaaP gene relative to the T- plasmids was confirmed by electrophoretic mini-screen analysis DNA; this gene was approximately 20.9 kb to the left ofthe T- (23). These Ti plasmid insertion mutants were further screened DNA. In the nopaline strain C58, the genetic determinants for for their inability to induce an increase in IAA. One mutant was virulence also map to the left ofthe T-DNA region but include both avirulent and characteristically defective in plasmid-coded the left portion of the T-DNA (24). This also seems to be the IAA, lending support to the above hypothesis, namely, that IAA case for octopine Ti plasmids (18, 31). Our results show that the synthesis is required for oncogenesis. Furthermore, the mutant iaaP gene maps in the virulence region far to the left of the T- Ti plasmid was inserted into a plasmid-free strain by transfor- DNA. This raises the question of how the iaaP gene on the Ti mation and the resulting transformants remained avirulent and plasmid functions to confer virulence. One possibility is that the defective in IAA. Analysis of the TiC58::Tn5 plasmid by cleav- iaaP gene, togetherwith the T-DNA, is integrated into the plant age with one of several restriction endonucleases followed by genome. It is then processed (possibly through DNA rearrange- fractionation ofthe plasmid DNA fragments by agarose gel elec- ment) into a site somewhere within the T-DNA and remains in trophoresis revealed the site of the Tn5 insertion. The Tn5 in- a transient state during crown gall tumorigenesis (see below). sertion was located in Kpn I fragment 5, Sma I fragment 13, A second possibility is that IAA synthesized by A. tumefaciens HindIII fragment 14a (Fig. 2), and BamHI fragment 9 (data not is essential for virulence because it is required for some type Downloaded by guest on October 1, 2021 2816 Biochemis": Liu et aL Proc. Natl. Acad. Sci. USA 79 (1982) of preconditioning of plant cells to make them competent for operating, whereby the portion with the iaaP gene becomes lost Ti DNA transfer. IAA is known to cause drastic plant cell wall as cell habituation occurs. changes, enhance permeability, cause swelling of the exposed cells, and stimulate the production of a second hormone, eth- We thank David Zaitlin, John Kao, Lisa McDaniel, Sandy Wood- ylene (32-34). ward, Jesse Dutra, and Jeff Hall for their capable technical assistance. Another possibility is that IAA plays a regulatory role on other This research was supported by Grant MV-102 from the American Can- loci needed for virulence and oncogenicity. Kline et aL (35) have cer Society, Grant CA-11526 from the National Cancer Institute, and shown that IAA circumvents the necessity for cAMP in eliciting a grant from the Science and Education Administration of the U.S. gene expression and very effectively regulates the L-arabinose Department of Agriculture. operon in E. coli. Thus in A. tumefaciens, IAA may also regulate the expression of those genes responsible for virulence and on- 1. Locke, S. B., Riker, A. J. & Duggar, R. M. (1938) J. Agric. Res. cogenicity. A. tumefaciens synthesizes little IAA in the absence 57, 21-39. of tryptophan but substantial amounts in the presence of this 2. Link, G. K. K. & Eggers, V. (1941) Bot. Gaz. 103, 87-106. substrate (10). Growth ofA. tumefaciens is necessary for infec- 3. DeRopp, R. S. (1947) Am. J. Bot. 34, 52-62. 4. Gautheret, R. J. (1947) C. R. Hebd. Seances Acad. Sci. 224, tion to take place, and growth in plant sap containing tryptophan 1728-1730. will generate IAA (36). IAA, in turn, may thus serve as the 5. Kulescha, Z. & Gautheret, R. J. (1948) C. R. Hebd. Seances Acad. "secondary messenger" to activate the genes for oncogenicity Sci. 227, 292-294. on the Ti plasmid. 6. Henderson, J. H. M. & Bonner, J. (1952) Am. J. Bot. 39, Early work on IAA biosynthesis in A. tumefaciens indicates 444-451. indolepyruvate and indoleacetaldehyde as intermediates ofthe 7. White, P. R. & Braun, A. C. (1942) Cancer Res. 2, 597-617. 8. Chilton, M.-D., Saiki, R. K., Yadav, N., Gordon, M. P. & Que- pathway from tryptophan (10). Our work using radioactive tryp- tier, F. (1980) Proc. Nati Acad. Sci. USA 77, 4060-4064. tophan confirmed the formation of indolepyruvate with tryp- 9. Willmitzer, L., DeBeuchkeleer, M., Lemmers, M., Van Mon- tophan. In addition to indolepyruvate and IAA, we observed tagu, M. & Schell, J. (1980) Nature (London) 287, 359-361. that, in the presence of tryptophan, A. tumefaciens synthesizes 10. Kaper, J. M. & Veldstra, H. (1958) Biochim. Biophys. Acta 30, a soluble brick-red pigment, which may be an indolepyruvate 401-420. conjugate, as observed in Klebsiella We assume 11. Liu, S.-T. & Kado, C. I. (1979) Biochem. Biophys. Res. Commun. aerogenes (19). 90, 171-178. that there are at most two or three genes required for the con- 12. Lin, B.-C. & Kado, C. I. (1977) Can.J. Microbiot 23, 1554-1561. version of tryptophan to IAA. We showed that the observed 13. Watson, B., Currier, T. C., Gordon, M. P., Chilton, M.-D. & principal tryptophan aminotransferase activity was not a Ti plas- Nester, E. W. (1975)J. Bacteriol 123, 255-264. mid-coded function (Fig. 1). Therefore the iaaP gene may code 14. Beringer, J. E., Beynon, J. L., Buchanan-Wellaston, A. V. & for enzymes that convert indolepyruvate to LAA. The residual Johnston, A. W. B. (1978) Nature (London) 276, 633-634. levels of IAA in Ti plasmid-free mutants may be explained by 15. Langley, R. A. & Kado, C. I. (1972) Mutat. Res. 14, 277-286. 16. Jensen, R. E., Zdybak, W. T., Yasuda, K. & Chilton, W. S. the low but detectable synthesis of indolepyruvate contributed (1977) Biochem. Biophys. Res. Commun. 75, 1066-1070. by secondary aromatic-amino-acid aminotransferases (Fig. 1) 17. Tang, Y. W. & Bonner, J. (1974) Arch. Biochem. 13, 11-25. (e.g., phenylalanine aminotransferase). This indolepyruvate 18. Garfinkle, D. J. & Nester, E. W. (1980) J. Bacteriol. 144, may then serve as substrate for iaaP-coded enzymes to convert 732-743. it to IAA. 19. Paris, C. G. & Magasanik, B. (1981)J. Bacteriot 145, 257-265. On the basis ofits T-DNA strain C58 20. Lin, E. C. C., Pitt, B. M., Civen, M. & Knox, W. E. (1958)J. size, the of has the cod- Biol Chem. 233, 668-673. ing potential of roughly 15 genes and may accommodate a few 21. Ryan, E., Bodley, F. & Fottrell, P. F. (1972) Phytochemistry 11, additional genes to its left during T-DNA processing. There is 957-963. no evidence, however, that the T-DNA region extends into the 22. Gibson, R. A., Schneider, E. A. & Wightman, F. (1972)J. Exp. iaaP gene, which is about 21 kb away. Polar mutational effects Bot. 23, 381-399. by the Tn5 insertion might help diminish this distance, but polar 23. Kado, C. I. & Liu, S.-T. (1981) J. Bacteriol 145, 1365-1373. effects are usually localized. An intriguing dilemma surfaces 24. Holsters, M., Silva, B., Van Vliet, F., Genetello, C., DeBlock, M., Dhaese, P., Depicker, A., Inge, D., Engler, G., Villarroel, from these findings. If the T-DNA is essential for oncogenesis, R., Van Montagu, M. & Schell, J. (1980) Plasmid 3, 212-230. then how does the iaaP locus located outside the T-DNA par- 25. Holsters, M., DeWaele, D., Depicker, A., Messens, E., Van ticipate in the process ofcrown gall tumor formation? One pos- Montagu, M. & Schell, J. (1980) Mol Gen. Genet. 163, 181-187. sibility is that the T-DNA is the vestigial segment of a much 26. Southern, E. M. (1975) J. Mol Biol 98, 503-517. larger piece of Ti plasmid that was incorporated in plant cells 27. Wahl, G. M., Stern, M. & Stark, G. R. (1979) Proc. Natl. Acad. and resulted from a complex ofinteractions during growth pro- Sci. USA 76, 3683-3687. 28. Rigby, P. W. J., Dieckmann, M., Rhodes, C. & Berg, P. (1977) cesses. The T-DNA may initially be a much larger segment of J. Mol Biol 113, 237-251. the Ti plasmid containing the iaaP gene. The resulting tumor 29. Maniatis, T., Jeffrey, A. & Kleid, D. (1975) Proc. Natl Acad. Sci. cells are initially auxin prototrophic by virtue of the insertion USA 72, 1184-1188. of the iaaP gene, but eventually they become habituated cell 30. Depicker, A., DeWilde, M., DeVos, R., Van Montagu, M. & lines harboring only vestigial segments ofthe Ti plasmid, known Schell, J. (1980) Plasmid 3, 193-211. currently as the T-DNA. Syono and Furuya (37) have shown that 31. Ooms, G., Klapwijk, P. M., Poulis, J. A. & Schilperoort, R. A. (1980)J. Bacteriol 144, 82-91. IAA-nonrequiring tobacco calli were induced with high fre- 32. Audus, L. J. (1959) Plant Growth Substances (Interscience, New quencies by short treatments with IAA. Thus, IAA prototrophy York). can be induced by brief exposures to the auxin. Although we 33. Lieberman, M. (1979) Annu. Rev. Plant. Physiol 30, 533-591. have no evidence to directly support this hypothesis, we have 34. Braun, A. C. (1954) Annu. Rev. Plant. Physiot 5, 133-162. shown that A. tumefaciens elaborates DNA-modifying enzymes 35. Kline, E. C., Brown, C. S., Banakaitis, V., Montefiori, D. C. that specifically recognize DNA molecules possessing super- & Craig, K. (1980) Proc. Nati Acad. Sci. USA 77, 1768-1772. 36. Dane, F. (1938) Zentrabl Bakteriol Parasitenkd. Infektionskr. helical conformation (38). These enzymes may be operating in 98, 385-429. processing the Ti plasmid during incorporation in plant cells. 37. Syono, K. & Furuya, T. (1974) Plant Cell Physiol. 15, 7-17. A coordinate interaction in the processing of the Ti plasmid, 38. LeBon, J. M., Kado, C. I., Rosenthal, C. J. & Chirikjian, J. G. particularly the iaaP gene, during infection may therefore be (1978) Proc. Natd Acad. Sci. USA 75, 4097-4101. Downloaded by guest on October 1, 2021