Proc. Natl. Acad. Sci. USA Vol. 81, pp. 5071-5075, August 1984 Biochemistry Crown gall oncogenesis: Evidence that a T-DNA from the Ti pTiA6 encodes an enzyme that catalyzes synthesis of indoleacetic acid (/indoleacetamide/oncogenes) LINDA S. THOMASHOW*, SUZANNE REEVES+, AND MICHAEL F. THOMASHOW* *Bacteriology and Public Health and tProgram in Genetics and Biology, Washington State University, Pullman, WA 99164-4340 Communicated by Arthur Kelman, April 30, 1984

ABSTRACT Stable incorporation of tumor-inducing (Ti) the pTiA6 transcript 2 gene in auxin independence. The nu- plasmid sequences, the T-DNA, into the of dicotyle- cleotide sequence of the gene has been determined (22, 23). donous results in the formation of crown gall tumors. The results indicate that there is a 1404-base-pair open read- Previous genetic studies have suggested that the products of ing frame that could code for a polypeptide of Mr 49,800, a the encoding transcripts 1 and 2, which are encoded by value in agreement with the results of Schroder et al. (21). the TL-DNA region of pTiA6, are responsible for inducing the We now have constructed a plasmid that produces the tran- auxin-independent phenotype of crown gall tissues. Here we script 2 gene product at detectable levels in E. coli. Bacterial report the construction of a plasmid, pMTlacT2, which directs extracts containing this protein can convert indoleacetamide the synthesis of the Mr 49,800 polypeptide encoded by the tran- to indoleacetic acid, the natural auxin of plants. script 2 gene. Cell-free extracts prepared from Escherichia coli harboring this plasmid converted indoleacetamide to indole- MATERIALS AND METHODS acetic acid, the natural auxin of plants; extracts prepared from Bacterial Strains. E. coli strains MH3000 [araD139 A(ara, plasmidless strains of E. coli or strains harboring the cloning leu)7697 A(lac)X74 galU galK rpsL (Strr) ompR101] and vehicle pBR322 did not carry out this reaction. We conclude TK1046 [araD.139 A(argF-lac)U169 rpsL150 (Strr) relAl that the transcript 2 gene of pTiA6 codes for an enzyme that flbB5301 deoCi ptsF25 malPQ: :TnS ompBcsl] were ob- participates in auxin biosynthesis, probably an indoleaceta- tained from G. Weinstock and used as described (24) to con- mide hydrolase. struct LacZ+ clones and produce high levels of,galacto- sidase fusion proteins. E. coli MC1061 [araD139 A(ara, leu)7697 A(lac)X74 galU galK hsdR(r-) rpsL (Strr)] was ob- Agrobacterium tumefaciens can infect wound sites on a wide tained from M. Casadaban (University of Chicago) and used range of dicotyledonous plants and cause the formation of to construct pMTlacT2. E. coli SG20251 [araD139 A(lacIPO- crown gall tumors (for reviews, see refs. 1-3). The disease ZYA)U169 A(lon)100 rpsL (Strr) thi cps3::TnlO] was ob- process is characterized by a virulence mechanism that is tained from S. Gottesman (National Institutes of Health) and unique among described procaryotic-eucaryotic cell-cell 2 interactions: it involves the transfer of genetic information used for expression of the cloned transcript gene. from bacterium to . All virulent A. tumefaciens strains . The open reading frame vehicle pORF2 (24) was harbor one of a diverse group of tumor-inducing (Ti) plas- provided by G. Weinstock. This vector (see Fig. 1) contains mids. During the course of infection, a portion of the Ti plas- the promoter, ribosome binding site, and the first 33 codons mid, the T-DNA, is stably transferred to the plant cells of the E. coli ompF gene fused out of frame to the ninth where it becomes integrated into the nuclear (4-10). codon of the lacZ gene of E. coli. There are restriction sites Expression of specific genes encoded by the T-DNA (11-17) at the point of the ompF-lacZ fusion into which DNA seg- causes an alteration in the normal metabolism of and ments can be inserted. If the DNA segment has an open , two classes of plant hormones that have key reading frame and if the insert puts the ompF and lacZ se- roles in controlling plant cell growth and development. quences into frame, a multihybrid protein having 03-galacto- Whereas normal plant tissues generally require both of these sidase activity is synthesized. pGL101 (25) was from G. phytohormones for in vitro propagation, crown gall tissues Lauer. This plasmid contains a "portable" promoter-i.e., can be propagated indefinitely without them (18-20). Three the ribosome binding sequences and promoter-operator re- genes encoded by the TL-DNA of -type Ti plasmids gion of the E. coli lac . pMT40 contains the HindIII such as pTiA6 are believed to be responsible for this phyto- fragment X of pTiA6 (7) inserted into the HindIII site of hormone-independent phenotype: genes encoding tran- pBR325. To construct pMTaT2 (Fig. 1), this T-DNA frag- Willmitzer et al. are ment was ligated into the HindIII site of pAC1 (26). This scripts 1 and 2, as numbered by (14), latter plasmid is a pBR322 derivative that contains tandem involved in auxin independence; and the transcript 4 gene lac UV5 promoters (including the first eight codons of lacZ) has a role in independence (11-15). Protein prod- in the EcoRI site and gives increased expression of the tetra- ucts from these genes have not yet been detected in tumor cycline-resistance gene. However, transcript 2 gene expres- tissues. However, Schroder et al. (21) have shown that they sion is not significantly enhanced in this vehicle (see text). are expressed at low levels in Escherichia coli minicells or in Media and Reagents. LB broth was as described (27). For coupled in vitro - systems prepared plasmid maintenance, ampicillin was included at 150 ,ug/ml from either E. coli or A. tumefaciens. Their data indicate in plates and liquid media. LacZ+ clones were detected on that transcripts 1, 2, and 4 encode Mr 74,000, Mr 49,000, and plates containing 40 ,ug of 5-bromo-4-chloro-3-indolyl-p3-D- Mr 29,000 polypeptides, respectively. galactoside per ml. We have focused our efforts on understanding the role of DNA Manipulations. Restriction enzymes, BAL-31 exonu- clease and T4 DNA ligase were from New England Biolabs The publication costs of this article were defrayed in part by page charge payment. This article must therefore be hereby marked "advertisement" Abbreviations: Ti plasmid, tumor-inducing plasmid; T-DNA, Ti in accordance with 18 U.S.C. §1734 solely to indicate this fact. plasmid DNA sequences stably transferred to the plant. 5071 Downloaded by guest on September 26, 2021 5072 Biochemistry: Thomashow et al. Proc. NatL Acad Sci. USA 81 (1984)

FIG. 1. Construction of plasmids that code for an ompF-transcript 2-lacZ gene fusion (pLTt2-6) and synthesis of the transcript 2 M, 49,800 protein (pMTlacT2). In step 1, recombinant molecules that coded for the production of OmpF-transcript 2-LacZ proteins were constructed by digesting pMT40 with Bgl II, treating the DNA fragments with BAL-31 and inserting them into the Sma I site of pORF2. These molecules were then transformed into E. coli MH3000, and colonies were screened for f-galactosidase production on plates containing 5-bromo-4-chloro-3- indolyl-f3-D-galactoside. Plasmid pLTt2-6 contained a transcript 2 gene fragment of about 750 base pairs with the same 5'-to-3' orientation as the ompF and lacZ genes. Step 2 shows the construction of pMTaT2Z, a plasmid containing tandem lac promoters upstream ofthe transcript 2-lacZ gene fusion. pLTt2-6 and pMTaT2 were digested with Pst I and Sma I, mixed, ligated, and used to transform E. coli MC1061. E. coli MC1061 harboring pMTaT2Z produced 50 units of /3-galactosidase activity, whereas E. coli with a fully induced lac operon produced about 1000 to 2000 units of the activity. Step 3 shows placement of a lac "portable" promoter upstream to the transcript 2-lacZ gene fusion. pGL101 was digested with Pst I and Pvu II, and the DNA fragment containing the lac promoter and ribosome binding site was gel-purified. pMTaT2Z was linearized by HindIII digestion, treated with BAL-31 for varying amounts of time, digested with Pst I, and the large DNA fragments containing the transcript 2 open reading frame fused to lacZ were gel purified. This DNA was then mixed with the lac promoter fragment, ligated, and used to transform E. coli MC1061. Transformants were identified using indicator plates as before. The bluest colonies were picked and assayed for 3- galactosidase activity. E. coli MC1061 harboring pMTlacT2Z-12 produced 5000 to 6000 units of ,B-galactosidase activity and synthesized a novel protein ofabout M, 153,000, the approximate size expected for the transcript 2-LacZ fusion protein (data not shown). Step 4 shows construction of pMTlacT2, a plasmid producing the Mr 49,800 transcript 2 gene product. pMTaT2 was digested with Pst I and Sma I, and the DNA fragments containing the 3' end of the transcript 2 open reading frame were gel-purified. Plasmid pMTlacT2Z-12 was digested with Pst I and Sma I, mixed with the purified pMTaT2 fragments, ligated, and used to transform E. coli MC1061. Plasmid DNA was isolated from a number of white transformants on the indicator plates, and the desired plasmid, pMTlacT2, was identified by restriction enzyme analysis. It contained the transcript 2 gene preceded by the lac promoter and ribosome binding site. bla, 3-lactamase; tet, tetracycline resistance; P, Pst I; E, EcoRI; B, BamHI; S, Sma I; H, HindIII; PV, Pvu II; lacPSD, the lac promoter and ribosome binding site; lacP, the tandem lac promoters from pAC1. Numbers below the names of the plasmids denote size in kilobase pairs.

and were used as specified. DNA transformation (28) and transferase activity was determined by the method of Gel- plasmid isolation (29) were as described. fand and Steinberg (34). Tryptophan monooxygenase and Preparation of Antiserum. Cultures of TK1046 that con- indoleacetamide hydrolase activities were assayed as de- tained LacZ+ plasmids were grown and lysates were loaded scribed by Comai and Kosuge (35), except that extraction on NaDodSO4/polyacrylamide gels as described by Wein- was into diethyl ether instead of ethyl acetate. Recovery of stock et al. (24) except that the polyacrylamide concentra- indoleacetic acid was monitored by addition of radioactively tion was reduced to 4.5%. /3-Galactosidase-fusion protein labeled indoleacetic acid to incubation mixtures prior to ex- bands were visualized in ice-cold 0.2 M KCl (30). The bands traction of the indoles. Samples were applied to silica gel G were excised, minced, and emulsified with an equal volume TLC plates, and the plates were developed in either n-pro- of Freund's incomplete adjuvant. Rabbits were injected sub- panol/methyl acetate/7 M NH40H, 45:35:20 (vol/vol), or n- cutaneously at multiple sites with approximately 100 ,ug of butanol/acetic acid/water, 65:20:22 (vol/vol). Indole com- the fusion proteins at 2-wk intervals and were bled 1 wk after pounds were detected with a modified Ehrlich's reagent (36). the third injection. Cells were grown and assayed for 83-galactosidase as de- Gel Electrophoresis and Protein Blotting. Bacterial cell ly- scribed by Miller (27). sates were prepared in lysis buffer (125 mM Tris HCl, pH 6.8/2% NaDodSO4/10% glycerol/0.7 M 2-mercapto- RESULTS ethanol/0.003% bromphenol blue). NaDodSO4/polyacryla- mide gel electrophoresis was as described (31). Proteins The auxin-independent phenotype of crown gall tissues incit- were transferred from the gels to nitrocellulose sheets by ed by A. tumefaciens harboring pTiA6 could be explained electrophoresis (32) at 500 mA for 2 hr and visualized essen- relatively simply if the transcripts 1 and 2 TL-DNA genes tially as described (33). encode enzymes for the biosynthesis of indoleacetic acid. To Enzyme Assays. Cultures were grown to an Ao00 of 0.6, test this hypothesis, we chose to raise antisera that could centrifuged, suspended to an A600 of 15, and passed twice recognize the Mr 49,800 transcript 2 gene product, to use the through a French pressure cell at 15,000 pounds per square antisera to identify an E. coli strain that could produce the inch (1 psi = 6.89 kPa). Unlysed cells and debris were re- Mr 49,800 protein, and then to assay cell-free extracts of the moved by centrifugation at 37,000 x g for 30 min. Amino- for the various possible activities known to be in- Downloaded by guest on September 26, 2021 Biochemistry: Thomashow et aL Proc. NatL. Acad. Sci. USA 81 (1984) 5073 volved in indoleacetic acid synthesis. antigen preparations were relatively pure because the two Production of Antisera. Since we did not have a biochemi- hybrid proteins were well-resolved from most other E. coli cal marker for the transcript 2 gene product, we could not polypeptides in the gels. The antiserum prepared against the isolate it from tumor tissues. Therefore, we constructed gene Mr 145,000 protein could recognize both the Mr 145,000 and fusions between transcript 2 sequences and the E. coli en- the Mr 120,000 hybrid proteins as judged by protein blotting zyme 0-galactosidase, isolated the fusion proteins, and pre- (Fig. 2B). The antisera directed against the Mr 120,000 hy- pared the desired antisera. brid protein gave similar results (not shown). When, howev- We used the open reading frame vehicle pORF2 (24) to er, the antiserum directed against the Mr 145,000 protein was construct a plasmid, pLTt2-6, that contained a portion of the first adsorbed with cell lysates containing the Mr 120,000 hy- transcript 2 gene fused in frame between the E. coli ompF brid protein, it still reacted well with the Mr 145,000 multihy- and lacZ genes (see Fig. 1). E. coli TK1046 cells harboring brid protein but only very weakly with the Mr 120,000 hybrid this plasmid produced a novel polypeptide of about Mr protein (Fig. 2C). These data indicate that the antiserum 145,000, the mass expected for the desired multihybrid pro- against the Mr 145,000 protein contains antibodies directed tein (Fig. 2A). Overproduction of the protein was tempera- against the transcript 2 gene product determinants. ture-dependent and lethal to TK1046. This lethality was con- Expression of the Transcript 2 Gene Product in E. coli. Nu- sistent with translation being initiated within the ompF se- cleotide sequence data (22, 23) indicate that HindI11 frag- quences, since these sequences provide a signal for protein ment X of pTiA6 (7) contains the entire coding sequence for export. Overproduction of p-galactosidase fused to such se- the transcript 2 gene. The data of Schroder et al. (21) show quences is lethal to the cell (24). As a control, we also creat- that this fragment has sequences that can promote transcrip- ed a derivative of pORF2 that produced an ompF-lacZ hy- tion of the transcript 2 gene in E. coli. However, when brid gene product containing no transcript 2 gene sequences. pMT40 (a plasmid containing HindlIl fragment X) was trans- This was accomplished by digesting pORF2 with Bgl II and formed into E. coli MC1061, we could not detect the produc- BamHI (see Fig. 1), religating the molecules, and screening tion of the Mr 49,800 transcript 2 polypeptide by protein blot- for /-galactosidase production. Plasmids that had lost the 17 ting with our antisera. This was also true for E. coli trans- base pairs between the Bgl II and BamHI sites had the ompF formed with pMTaT2, a plasmid containing the transcript 2 sequences in frame with the lacZ sequences. E. coli TK1046 gene located downstream from tandem lac promoters (see cells harboring this plasmid, pLTAORF2, produced a novel Materials and Methods). Therefore, we used the strategy de- Mr 120,000 protein, the size expected for the hybrid polypep- vised by Guarente et al. (25) to construct a plasmid, tide (Fig. 2A). pMTlacT2, that produced the Mr 49,800 protein at detectable Antisera that could recognize the hybrid Mr 145,000 and levels (see Fig. 1). Mr 120,000 proteins were prepared from antigen isolated First we created a molecule, pMTaT2Z, that had the 5' from preparative NaDodSO4/polyacrylamide gels. Such end of the transcript 2 gene, including 53 flanking bases and =85% of the coding sequence, fused in-frame with the lacZ

CN 04 rl4 gene (Fig. 1). Next, we positioned a DNA fragment contain- ing the lacZ promoter and Shine-Dalgarno (37) ribosome CN 0 binding site at various distances from the 5' end of the tran- O' - No a0 __ L-l script 2 gene and screened for optimal promoter placement A CL CL B a- a- a a- Cf CL by assaying E. coli transformants for -galactosidase activi- ty. This created pMTlacT2Z-12. Finally, we replaced the

205- e- transcript 2-lacZ fusion sequences with the normal 3' end of the transcript 2 gene. This resulted in pMTlacT2, a plasmid having the complete transcript 2 gene preceded by the lac promoter and ribosome binding site. To demonstrate that pMTlacT2 could direct the synthesis 97-00- of the Mr 49,800 transcript 2 protein, we subjected lysates of 116-* @ E. coli SG20251 harboring this plasmid to NaDodSO4/poly- acrylamide gel electrophoresis and protein blot analysis. The -8 _ "t. data indicate that this E. coli strain did indeed produce a protein of about Mr 50,000 that reacted with antisera against the OmpF-transcript 2-LacZ protein (Fig. 3) but not with preimmune serum or antisera to the ompF-lacZ protein (data not shown). This Mr 50,000 protein was not synthe- sized in plasmidless E. coli SG20251 or SG20251 containing pBR322 (Fig. 3). Thus, we concluded that E. coli SG20251 harboring pMTlacT2 could synthesize the transcript 2 gene FIG. 2. Production of the transcript 2-LacZ fusion protein, and product. This protein also was synthesized in other strains of protein blots reacted with the antibody against transcript 2 determi- E. coli containing pMTlacT2-e.g., MC1061. However, the nants. E. coli TK1046 cells harboring pORF2, pLTt2-6, or yield seemed to be best in strains such as SG20251, which pLTAORF2 were grown at 260C to an Awo of 0.2, shifted to 420C and incubated for 2 hr at 370C. Cells were centrifuged, suspended at an lack the Ion protease (38). A~w of 15 in lysis buffer, and electrophoresed in a NaDodSO4/7.5% Enzymatic Activity of the Transcript 2 Gene Product. Tryp- polyacrylamide gel. (A) Coomassie stain. Lanes contained 25 AlI of tophan is generally regarded as the most common precursor cell lysate. Molecular weight markers are shown 10-3, and the po- for indoleacetic acid biosynthesis in plants and bacteria. The sition of /3galactosidase is marked by a star. (B and Lanes con- three best characterized pathways for the conversion of tained 25 1.d of a 1:500 dilution of the above lysate. Proteins were tryptophan to indoleacetic acid are the indolepyruvic acid electrophoretically transferred to nitrocellulose and treated with pathway (tryptophan -- indolepyruvic acid -* indoleacetal- antiserum against the transcript 2-LacZ fusion protein (B) or treated -* indoleacetic found in with the same antiserum after adsorption with a crude preparation of dehyde acid) plants, Agrobacteri- the OmpF-LacZ hybrid protein from TK1046 harboring um, and Rhizobium (39-42); the tryptamine pathway (tryto- -- -. pLTAORF2(C). Visualization was by incubation with horseradish phan tryptamine indoleacetaldehyde -+ indoleacetic peroxidase-conjugated goat anti-rabbit IgG, followed with hydrogen acid) found in plants (39, 40); and the tryptophan monooxy- peroxide and 4-chloro-1-naphthol. genase pathway (tryptophan -* indoleacetamide -- indole- Downloaded by guest on September 26, 2021 5074 Biochemistry: Thomashow et aL Proc. NatL Acad Sci. USA 81 (1984)

(N4 CN CN C-N u CN u C( (N U _ c-) / D CL Co Era 20- B :m-co 4- co:L Q 205- _ _bow 116- IAM- -_ 97- ....

.4. 4_ 68- IAA ._M Trp 45- wlW

_-,u -,ill-...- IMI., W,'*I'll,`5!1?" 29- ft.

FIG. 3. Detection of the cloned transcript 2 gene product. E. coli FIG. 4. Enzymatic activity of the transcript 2 gene product. SG20251 cells harboring pBR322 tor pMTlacT2 were grown at 370C SG20251 cells harboring pBR322 or pMTlacT2 were grown and ex- to an A6N of0.5. Cells were centrifuged, suspended in lysis buffer at tracts were prepared as described. Reaction mixtures (2.5 ml) con- an A6w of 15, and electrophoresed in a NaDodSO4/10% polyacryl- tained 1-2 mg of extract protein in 20 mM Tris HCl, pH 8.0/50 mM amide gel. Each lane contained 25 p.l of lysate. (A) Coomassie stain. KCI/10 mM MgCl2/5 mM dithiothreitol/10 mM indoleacetamide The M, 49,800 protein is not usually visible in stained gels. Molecu- and were incubated for 3 hr at 30°C. Indoles were extracted into lar weight markers are shown 10-3. (B) Proteins were electropho- diethyl ether, and 20%o of the reaction products were chromato- retically transferred to nitrocellulose and reacted with antiserum graphed on silica gel G plates in n-propanol/methyl acetate/7 M against the OmpF-transcript 2-LacZ protein. The arrow indicates NH40H, 45:35:20 (vol/vol). Standard markers (5 yg) are: IAA, in- the position of the M, 49,800 transcript 2 gene product. doleacetic acid; IAM, indoleacetamide; and Trp, tryptophan. acetic acid) of Pseudomonas savastanoi, a bacterial plant pathogen that causes hyperplasias on oleander and olive the work of White and Braun (18), DeRopp (20), Gautheret trees (35, 43, 44). Data that can be interpreted as evidence (19), and their colleagues. That is, why do crown gall cells for all three of these pathways have been presented for in proliferate in vitro in the absence of added auxin and cytoki- vitro grown crown gall tissue isolated from tumors on sun- nin, whereas normal untransformed cells generally require flower (45). these phytohormones for growth. The results presented here To determine whether the transcript 2 gene product had suggest that the auxin-independent phenotype of crown gall any of the enzymatic activities known to be associated with tissues incited by A. tumefaciens harboring pTiA6 is due in the above pathways, we prepared cell-free extracts of E. coli part to the transfer of an indoleacetic acid biosynthetic gene SG20251 harboring pMTlacT2 and carried out standard en- from bacterium to plant. Specifically, our data indicate that zyme assays; E. coli SG20251 transformed with pBR322 the transcript 2 gene product encoded by the TL-DNA region served as the control. We were unable to detect any differ- of pTiA6 is involved in the conversion of indoleacetamide to ences between the two extracts in assays for tryptophan indoleacetic acid. The simplest interpretation ofthese results transaminase and tryptophan monooxygenase, enzymes is that transcript 2 encodes an indoleacetamide hydrolase. which convert tryptophan to indolepyruvic acid and indole- However, we cannot a priori eliminate the possibility that E. acetamide, respectively. However, the extracts differed dra- coli enzymes may have participated in the observed auxin matically in the activity of indoleacetamide hydrolase, the synthesis. Therefore, formal proof that the transcript 2 poly- second enzyme of the tryptophan monooxygenase pathwvay. peptide is an indoleacetamide hydrolase must await its puri- When pMTlacT2 extracts containing the Mr 49,800 protein fication and characterization. were incubated with indoleacetamide, a compound we iden- Although our results pertain to the induction of auxin inde- tify as indoleacetic acid was synthesized: it comigrated with pendence specifically by pTiA6, it is probable that many if authentic indoleacetic acid in two TLC developing systems not all Ti plasmids bring about the auxin-independent pheno- (data for one system are shown in Fig. 4), and it was extract- type in a similar manner.This prediction follows from hy- ed into diethyl ether only at low pH. The structure of the bridization studies that have shown that most Ti plasmid iso- product was confirmed by combined GC/MS (data not lates have sequences closely related to the TL-DNA region shown). When indoleacetamide was omitted from the reac- of the pTiA6 plasmid family (as reviewed in refs. 1-3). In- tion, indoleacetic acid was not formed. Indoleacetic acid deed, transcriptional and genetic analyses (12, 13, 46, 47) synthesis was not detectable in extracts prepared from plas- suggest that the T-DNA regions of the closely related plas- midless SG20251 or SG20251 harboring pBR322 (Fig. 4). mids pTiT37 and pTiC58 (which share only some 15% over- Taken together, these results indicate that the Mr 49,800 pro- all homology with pTiA6) encode genes equivalent to the tein encoded by the transcript 2 gene has indoleacetamide pTiA6 transcripts 1, 2, and 4 oncogenes as well as a number hydrolase activity. of other pTiA6 TL-DNA genes. A second pTiA6 gene, which encodes transcript 1 of the DISCUSSION TL-DNA, has also been implicated in inducing auxin inde- One of the fundamental questions concerning the molecular pendence in crown gall tissues (11, 12, 14). A likely role for basis of crown gall disease was raised over 30 years ago by this gene product is in the synthesis of indoleacetamide, and Downloaded by guest on September 26, 2021 Biochemistry: Thomashow et aL Proc. NatL. Acad. Sci. USA 81 (1984) 5075

a possible enzymatic activity is suggested by analogy to the 10. Yadav, N. S., Postle, K., Saiki, R. K., Thomashow, M. F. & P. savastanoi indoleacetic acid biosynthetic pathway eluci- Chilton, M. D. (1980) Nature (London) 287, 458-461. dated by Kosuge et al. (43). In this pathway a tryptophan 11. Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., Gordon, M. P. & Nester, E. W. (1981) Cell 27, 143-153. monooxygenase converts tryptophan to indoleacetamide, 12. Ooms, G., Hooykaas, P. J. J., Moolenaar, G. & Schilperoort, and indoleacetamide hydrolase converts indoleacetamide to R. A. (1981) Gene 14, 33-50. indoleacetic acid. Since the pTiA6 transcript 2 gene product 13. Binns, A. N., Sciaky, D. & Wood, H. N. (1982) Cell 31, 605- apparently has indoleacetamide hydrolase activity, it is rea- 612. sonable to hypothesize that the pTiA6 transcript 1 gene may 14. Willmitzer, L., Simons, G. & Schell, J. (1982) EMBO J. 1, 139- encode an enzyme with tryptophan monooxygenase-like ac- 146. tivity. 15. Akiyoshi, D. E., Morris, R. O., Hinz, R., Mischke, B. S., Ko- A final point concerns the apparent complementation of suge, T., Garfinkel, D. J., Gordon, M. P. & Nester, E. W. transcript 2 mutations by some plant species. Binns et al. (1983) Proc. Natl. Acad. Sci. USA 80, 407-411. reasoned that if the 1 and 2 16. Joos, H., Inz6, D., Caplan, A., Sormann, M., Van Montagu, (13) previously transcripts genes M. & Schell, J. (1983) Cell 32, 1057-1067. of pTiA6 were involved in indoleacetic acid biosynthesis, tu- 17. Willmitzer, L., Dhaese, P., Schreier, P. H., Schmalenboch, mors incited by pTiA6 with mutations in these genes should W., Van Montagu, M. & Schell, J. (1983) Cell 32, 1045-1056. be cytokinin-independent but auxin-dependent. Indeed, they 18. White, P. R. & Braun, A. C. (1941) Science 94, 239-241. found that unorganized tumor tissues of Nicotiana tabacum 19. De Ropp, R. S. (1947) Am. J. Bot. 34, 352-362. incited with pTiA66, a spontaneous variant of pTiA6 having 20. Gautheret, R. J. (1947) C. R. Acad. Sci. (Paris) 224, 1728- an insert in the transcript 2 gene, would not grow in vitro 1730. without the addition of auxin to the medium; the tissues grew 21. Schroder, G., Klipp, W., Hillebrand, A., Ehring, R., Koncz, well without cytokinin additions. In contrast, tumor tissues C. & Schroder, J. (1983) EMBO J. 2, 403-409. incited on 22. Sciaky, D. & Thomashow, M. F. (1983) Nucleic Acids Res. 12, by pTiA66 Nicotiana glauca, Nicotiana glutinosa, 1447-1461. or Helianthus annuus exhibited no auxin requirement and 23. Barker, R. F., Idler, K. B., Thompson, D. V. & Kemp, J. D. grew perfectly well as undifferentiated callus on phytohor- (1983) Plant Mol. Biol. 2, 335-350. mone-free medium. The authors concluded that some plant 24. Weinstock, G. M., ap Rhys, C., Berman, M. L., Hampar, B., species can complement the transcript 2 gene mutation of Jackson, D., Silhavy, T. J., Weissman, J. & Zweig, M. (1983) pTiA6, while others do not. Our data suggest that tissues of Proc. Natl. Acad. Sci. USA 80, 4432-4436. N. glauca, N. glutinosa, and H. annuus may have endoge- 25. Guarente, L., Lauer, G., Roberts, T. M. & Ptashne, M. (1980) nous indoleacetamide hydrolase activity. This activity has Cell 20, 543-553. been detected previously in pea, tomato, and broad bean 26. Carpousis, A. J. & Gralla, J. D. (1980) Biochemistry 19, 3245- stem tissues (48). 3253. While this was et 27. Miller, J. (1972) Experiments in Molecular Genetics (Cold manuscript being reviewed, Schroder al. Spring Harbor Laboratory, Cold Spring Harbor, NY). (49) and Inze et al. (50) published data that also strongly sug- 28. Norgard, M. V., Keem, K. & Monahan, J. J. (1978) Gene 3, gests that the transcript 2 gene product has indoleacetamide 279-292. hydrolase activity. 29. Birnboim, H. C. & Doly, J. (1979) Nucleic Acids Res. 6, 1513- 1523. We are grateful to George Weinstock for providing the pORF ve- 30. Hager, D. A. & Burgess, R. R. (1980) Anal. Biochem. 109, 76- hicles prior to publication and for advice concerning their use and to 86. Karl Espelie for the analysis of indoleacetic acid by mass spectros- 31. Laemmli, U. K. (1970) Nature (London) 227, 680-685. copy (Hewlett Packard model 5985). In addition, we thank Rob Jen- 32. Burnette, W. N. (1981) Anal. Biochem. 112, 195-203. sen, Andy Binns, Richard Burgess, Mike Kahn, and Ralph Martinez 33. Hawkes, R., Niday, E. & Gordon, J. (1982) Anal. Biochem. for valuable suggestions; Malcolm Casadaban, Gail Lauer, and Su- 119, 142-147. san Gottesman for bacterial stains and plasmids; Linda Moore and 34. Gelfand, D. H. & Steinberg, R. A. (1977) J. Bacteriol. 130, Wally Buchholz for critical reading of the manuscript; and Jane 429-440. Udell and Diana Devereaux for preparing the manuscript. This work 35. Comai, L. & Kosuge, T. (1980) J. 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