Proc. Natl. Acad. Sci. USAVol. 77, No. 5, pp. 2666-2670, May 1980Biochemistry

Proteins encoded by Agrobacterium tumefaciens Ti plasmid DNA(T-DNA) in crown gall tumors

(messenger RNA/DNA-mRNA hybridization/cell-free protein synthesis/transformation/plasmid)

JOAN C. MCPHERSON*, EUGENE W. NESTERt, AND MILTON P. GORDON*Departments of *Biochemistry and tMicrobiology and Immunology, University of Washington, Seattle, Washington 98195

Communicated by Edwin G. Krebs, March 3, 1980

ABSTRACT In order to detect proteins that may be pro-duced in crown gall tumors as a result of expression of incor-porated Agrobacterium tumefaciens Ti plasmid DNA (T-DNA),we have isolated mRNA complementary to T-DNA and trans-lated this in a protein-synthesizing system derived from wheatgerm. mRNA prepared from cultured El tumor from Nicotianatabacum hybridized with HindIII fragment 1 sequences ofT-DNA immobilized on cellulose nitrate filters. Two proteinsof 30,000 and 16,500 M, were produced when this selected RNAwas released and translated. Other tumor lines from N. tabacumwere investigated, and a protein of slightly less than 30,000 Mrwas encoded by HindIl fragment 1 sequences of 15955/01tumor. No products were observed for 15955/1 tumor line, whichdiffers from El/B6-806 and 15955/01 in that it does not produceoctopine. mRNA species of each of the tumor lines hybridizedto Bst I fragment 8 sequences of T-DNA and produced a com-mon protein of 15,000 Mr. Because this protein is derived fromthe region of the T-DNA that is conserved in octopine- andnopaline-type crown gall tumors, it may play a role in oncoge-nicity.

Crown gall tumors arise in many plant species after woundinfection by Agrobacterium tumefaciens (1). The tumor cellsshow altered properties, such as their ability to grow vigorouslyin culture in the absence of exogenous auxins and cytokinins(2). They also produce unusual amino acid derivatives (3-7)which may be either of the octopine type (N-carboxyethylderivatives of arginine, ornithine, histidine, and lysine) or thenopaline type (N-dicarboxypropyl derivatives of arginine andornithine). The large tumor-inducing (Ti) plasmids present invirulent strains of A. tumefaciens (8, 9) specify which type ofunusual amino acid is produced in the tumor tissue (10, 11).Small portions of these Ti plasmids, termed T-DNA, whichpersist in axenic cultures of crown gall tumors (12), appear tobe integrated into plant DNA at multiple sites (13). Deletionmutants of octopine-type Ti plasmids that lack sequences of theT-DNA mapping to the right of fragment Sma lOc (see Fig. 1)induce tumors that do not produce octopine (14). Recent in-vestigations of the T-DNA present in tumors incited by octo-pine-type Ti plasmids (13) have shown that T-DNA sequencesextending approximately 7 megadaltons to the left of EcoRIfragment 24 (see Fig. 1) are present in each tumor line. Thesecommon regions of T-DNA include sequences that are ho-mologous in octopine- and nopaline-type Ti plasmids (15, 16)and that are considered to be essential for oncogenicity (16). Thepresence of other regions of T-DNA varies among individualtumor lines (13).The means by which the T-DNA affects plant metabolism

is unknown, but transcription studies (17-20) have demon-strated that RNA isolated from several octopine-type crown gall

EcoRILBst I/BamHIL

HindillSma I

2 IAI 7 1241 13 1221 1 $1 1 8 |1 19 1 2

IYXIII 1 5 1 13a 17F64 loc I 3b 1 7

2 x 1 06 daltons

:Coniserved;

FIG. 1. Sequences of the T-DNA of pTi B6-806 used for selectivehybridization of mRNAs of octopine-type tumor lines. The restrictionfragments HindIII.1 and Bst I.8+29, which were immobilized oncellulose nitrate filters, are indicated. The solid box (Bst I.8L) rep-resents the region with which mRNA from each tumor line hybridized(see Results). The open box (HindIII.lR) represents sequences withwhich mRNA from octopine-producing tumors hybridized. Thetranslation products of the selected mRNAs are given in Table 1. Theconserved DNA of octopine- and nopaline-type tumors is accordingto Chilton et al. (15).

tumors hybridizes with regions of the corresponding plasmidT-DNA. Total RNA from E9 tumor (incited by pTi B6-806)hybridized to HindIII fragments 1 and 5 (17), and similar in-vestigations using RNA from El tumor, a sibling clone of E9,demonstrated hybridization with subfragments of the sameHindIII restriction fragments (20). Although transcription tothe left of these fragments was barely detected in El tobaccotumor, a sunflower line incited by pTi 15955 showed a highlytranscribed region of DNA which corresponded to the se-quences of T-DNA that are conserved in both octopine- andnopaline-type tumor lines (20). Polysomal RNA has been iso-lated from El tobacco tumor tissue and the polyadenylylatedfraction has been shown to hybridize strongly with Sma band3b (19). There is no direct evidence that proteins produced inthe tumor tissues are derived from these transcripts. However,octopine synthase, which catalyzes the reductive condensationof arginine with pyruvate to produce octopine, has been isolatedfrom crown gall cultures (21, 22).

In this study we have isolated mRNAs that are complemen-tary to specific regions of T-DNA. These mRNAs directed thesynthesis of specific proteins when translated in a wheat germcell-free system. The translation products for mRNAs isolatedfrom several octopine-type tumor lines are compared.

METHODSCultured Tissues. The established tumor lines were derived

from Nicotiana tabacum var. Xanthi Nc: El was incited by pTiB6-806; tumor lines 15955/1 and 15955/01 were induced bypTi 15955. Tumor tissues were cultured on agar plates con-taining Murashige and Skoog plant salt medium (Flow Labo-

Abbreviations: T-DNA, Ti plasmid DNA in crown gall tumors;poly(A)-RNA, polyadenylylated RNA; HindIII.1, HindIII fragment1; Bst I.8+29, Bst I fragments 8+29.

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The publication costs of this article were defrayed in part by pagecharge payment. This article must therefore be hereby marked "ad-vertisement" in accordance with 18 U. S. C. §1734 solely to indicatethis fact.

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Table 1. Proteins encoded by T-DNA sequences of octopine-typecrown gall tumors

Characteristics Proteins (Mr) encoded byInciting T-DNA regions

Tumor line plasmid Octopine Bst I.8L HindIII.1R

El pTi B6-806 + 15,000 30,000; 16,50015955/01 pTi 15955 + 15,000 0.5-1.0 Ag of polyadenylylated exogenous RNA or 10 ,g ofnonpolyadenylylated mRNA. RNA samples were also translatedin a rabbit reticulocyte lysate cell-free system (29) that wastreated with nuclease by the procedure of Pelham and Jackson(30).The translation products were analyzed by NaDodSO4/

polyacrylamide gel electrophoresis (31). Protein standards(Pharmacia) were used for calibration of molecular weights (see

legend to Fig. 2). The gels were prepared for fluorography bythe method of Bonner and Laskey (32).Cloned Restriction Fragments of Ti Plasmids, B6-806 and

A6. Escherchia coli X1776 and RR1, containing restrictionfragments HindIII.1 (33) and Bst I.8+29 (13) cloned inpBR322, were supplied by D. Garfinkel and S. Gelvin of ourlaboratory. The arrangement of these cloned fragments in theT-DNA is shown in Fig. 1.

Preparation of Plasmid DNA. Plasmid was prepared by amodification (34) of the cleared lysate procedure of Blair et al.(35). The identity of the T-DNA fragments present in the cloneswas determined by restriction endonuclease analysis. All ex-periments involving recombinant DNA molecules were con-ducted under P2-EK2 containment as described by the NIHguidelines (1976) or under P2-EKI containment as describedby the revised guidelines (1978).

Preparation of DNA-Carrying Filters. DNA-carrying filterswere prepared by the method of Young et al. (36).

Hybridization. Cellulose nitrate filter supports (0.6-cmcircles) carrying cloned fragments of T-DNA (e6 ,ug of DNAper filter) were incubated with RNA fractions in 50% (vol/vol)formamide, 0.75 M NaCl, 50mM Tris-HCI (pH 7.5), and 5 mMEDTA at 43°C for 20 hr (37). After hybridization, the filterswere removed, washed extensively (three times for 30 min eachtime) in hybridization buffer followed by two washes in 0.3 MNaCI/0.03 M sodium citrate, and dried. Nonhybridized RNAremaining in the supernatant was precipitated with ethanol andtranslated in intro. RNA hybridizing to individual filters wasreleased with 90% (vol/vol) formamide at 450C for 1 hr. Afterethanol precipitation, the released fraction was translated ina cell-free system.

RESULTSPoly(A)-RNA, isolated by oligo(dT) chromatography, formedapproximately 1-2% of the total polysomal RNA from thetumor tissues and normal callus (normal Xanthi). Totalpoly(A)-RNA was actively translated in mRNA-dependentcell-free systems derived from either rabbit reticulocytes orwheat germ. Incorporation of labeled amino acids gave up to10-fold increases in trichloroacetic acid-precipitable radioac-tivity over endogenous values. The integrity of the translationsystems was monitored by translation of total bovine liverpoly(A)-RNA, followed by immunoprecipitation of prepro-thrombin (Mr 69,000) from the reaction products (data notshown).

Translation products obtained in the wheat germ system withpoly(A)-RNA from tumor lines El (Fig. 2, lane 4), 15955/01(Fig. 3, lane 10), and 15955/1 and from normal Xanthi callus(data not shown) were analyzed by NaDodSO4 gel electro-phoresis. Translation of El poly(A)-RNA in the presence of7-methylguanosine 5'-monophosphate (m7G5'p) gave a 75%reduction of acid-precipitable radioactivity, whereas no inhi-bition was observed in the presence of 7-methylguanosine(m7G). NaDodSO4/polyacrylamide gel electrophoresis of thetranslation products synthesized in the presence of m7G5'pshowed a uniform reduction throughout the profile (data notshown), suggesting that the majority of the polyadenylylatedtranscripts contained 5'-terminal caps (38). The presence of5'-terminal caps, 3'-polyadenylylated tails, and definite proteinproducts (see below) indicates that the RNA has not been de-graded during the isolation procedure.The protein profiles obtained for tumors El, 15955/01, and

15955/1 and for normal Xanthi resemble each other and mustessentially represent products of normal Xanthi cultured tissue.In order to investigate the possibility that any of the proteinproducts from tumor tissue may represent expression of the

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T-DNA sequences, we selected mRNA by hybridization toT-DNA prior to translation. For the hybridization studies wechose cloned fragments (HindIII.1 and Bst 1.8+29) of Tiplasmids B6-806 and A6, respectively, which represent T-DNAoccurring in octopine-type tumor lines (13). The restrictionendonuclease fragmentation patterns of octopine-type Tiplasmids are similar and their T-DNA regions are homologous(13, 39). The cloned fragments are therefore suitable for ex-amining octopine-type tumor lines incited by different Tiplasmids.The fragments HindII.li and Bst I.8+29 overlap slightly and

extend over approximately 10 megadaltons. Their arrangementin the T-DNA region is shown in Fig. 1. Polyadenylylated andnonpolyadenylylated fractions of RNA derived from the tumorlines and normal callus were each hybridized to HindIII.1 andBst 1.8+29 DNA sequences immobilized on cellulose nitratefilters. The specifically hybridized RNA was released and ap-plied to an mRNA-dependent cell-free translation system.Results for El tissue, an octopine-producing tumor incited bypTi B6-806, showed that the poly(A)-RNA, which hybridizedto HindIII.1 filters, could be released and translated in both thewheat germ and reticulocyte lysate systems. The products ob-tained in the wheat germ system, when analyzed by NaDodSO4gel electrophoresis, gave two distinct protein bands of estimatedMr 30,000 and 16,500 (Fig. 2, lane 7). [In the reticulocyte lysatesystem the 16,500 Mr product was detected (profile not shown);however, the high endogenous levels of proteins synthesizedin this system prevented us from detecting additional proteinsin the 30,000 Mr range.] RNA that did not hybridize to thefilters, when precipitated with ethanol and translated, gave aprofile (Fig. 2, lane 5) similar to that of total El poly(A)-RNA(Fig. 2, lane 4). The translational activity of this fraction wasslightly lower than that of the original El poly(A)-RNA andmay be the result of residual formamide carried over from thehybridization procedure. The hybridized and releasedpoly(A)-RNA gave reproducible protein products from one

1 2

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.r_..:_

:_sffi

ss s

t t

-94,000-67.000

- 43.000

-30.000

-20.100

-14,400

I

3 4 5 6 7 8 9 loFIG. 2. Translation products in the wheat germ system of specific

fractions of poly(A)-mRNA of tumor and normal tissues. Poly(A)-mRNA species hybridized toHindIII.1 sequences (see Fig. 1) attachedto cellulose nitrate filters: lane 3, 15955/1; lane 7, El; lane 9, normalXanthi. Lane 4, total El poly(A)-RNA; lane 5, El poly(A)-RNA-thatdid not hybridize to HindIII.1 filters. Poly(A)-mRNA species hy-bridized to Bst I.8+29 sequences, attached to cellulose nitrate filters:lane 2,15955/1; lane 6, El; lane 10, normal Xanthi. Lane 1, no addedRNA. Proteins were analyzed by NaDodSOJ12.5% polyacrylamidegel electrophoresis, and [35S]cysteine-labeled products were detectedby fluorography. The migration values indicated are those of standardproteins (Pharmacia): Mr 94,000, phosphorylase b; Mr 67,000, bovineserum albumin; Mr 43,000, ovalbumin; Mr 30,000, carbonic anhydrase;Mr 20,100, soybean trypsin inhibitor; Mr 14,400, a-lactalbumin.

preparation to another when different batches of RNA andfilters were used (compare Fig. 2, lane 7, and Fig. 3, lane 9). Noproducts were obtained from the hybridization reaction thatinvolved Bst 1.8+29 DNA on filters and El poly(A)-RNA (Fig.2, lane 6). Similar hybridizations using each of the T-DNA fil-ters were performed with poly(A)-RNA from tumor lines in-duced by Ti plasmid 15955. No protein products were detectedfor one of these lines, 15955/1, which did not produce octopine(Fig. 2, lanes 2 and 3). Results obtained for 15955/01 tumor,an octopine producer, gave a diffuse protein band of slightlyless than 30,000 Mr when RNA hybridized to HindIII.l wasreleased and translated in the wheat germ system (Fig. 3, lanes2 and 8). In addition, a minor protein band of approximately40,000 Mr was observed (Fig. 3, lane 8) which can be seenclearly in the overexposed lane (Fig. 3, lane 2). This protein hadidentical mobility to an endogenous component of the wheatgerm system and, although more intense on the autoradiogram,no interpretations were made concerning its specificity.Poly(A)-RNA from 15955/01 tumor, which hybridized to filterBst I.8+29, was released and translated to give a small proteinof estimated Mr 15,000 (Fig. 3, lane 7). Poly(A)-RNA fromnormal Xanthi callus was investigated by using identical pro-cedures for hybridization and translation, and no proteins wereobserved (Fig. 2, lanes 9 and 10).The nonpolyadenylylated RNA fractions from the tumor

lines and normal callus were investigated by the same proce-dures. These RNA fractions stimulated protein synthesis muchless than poly(A)-RNA as a result of nontranslatable (ribosomal)RNA in the nonpolyadenylylated fractions or in these fractions.Selection of RNA by hybridization of each of the nonpoly-adenylylated RNA fractions from El, 15955/01, 15955/1, andnormal Xanthi with HindIII.1 filters resulted in no translatableproducts (data not shown). However, when the nonpolyaden-ylylated RNA fractions of the tumor tissues were hybridized

1"*7FFIF"

-94,000

ki -67,000

-43.000

-30.000

ISR' ^ :4 tX-20,100ii~~~~~~~~~~~~M-14.400

1 2 3 4 5 6 7 8 9 10 11FIG. 3. Translation products in the wheat germ system ofmRNAs

that are complementary to sequences ofT-DNA. mRNA species hy-bridized to HindIII.1 DNA sequences attached to cellulose nitratefilters: lane 9, El poly(A)-RNA; lane 8, and lane 2, overexposed,15955/01 poly(A)-RNA (lane 1, an exposure similar to lane 2 for en-dogenous proteins synthesized). mRNA species hybridized to BstI.8+29 sequences attached to cellulose nitrate filters: lane 4, Elnonpolyadenylylated RNA; lane 5, 15955/1 nonpolyadenylylatedRNA; lane 6, 15955/01 nonpolyadenylylated RNA; lane 11, normalXanthi nonpolyadenylylated RNA; lane 7, 15955/01 poly(A)-RNA.Lane 10, proteins obtained from total 15955/01 poly(A)-RNA; lane3, no added RNA. Proteins were analyzed on NaDodSO4/10% poly-acrylamide gel electrophoresis and their migration values were com-pared to those of standard proteins (see Fig. 2).

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with Bst I.8+29 filters, the selected transcripts gave proteinproducts when translated in the wheat germ system (Fig. 3,lanes 4, 5, and 6). No products were observed for normal callus(Fig. 3, lane 11). The translation products from each tumor lineshowed identical mobilities on NaDodSO4 gel electrophoresis.A protein band of estimated Mr 15,000 was obtained for El,15955/01, and 15955/1 (Fig. 3, lanes 4, 6, and 5, respectively).Translation of the hybridized and released nonpolyadenylylatedRNA of 15955/1 tumor resulted in a higher background thanobtained with similar fractions from other tumor lines (Fig. 3,lane 5; compare with lanes 4 and 6). The nonpolyadenylylatedRNA fraction from 15955/1 contained highly viscous poly-saccharide-like material which was not present in the fractionsfrom the other tissues examined. The higher background mayhave resulted from nonspecific adhesion to these filters. How-ever, the predominant protein species from the hybridized andreleased products of 15955/1 nonpolyadenylylated RNA wasthe common band of Mr 15,000.

As can be observed in Fig. 1, there is an overlapping regionof DNA of approximately 1 megadalton between HindIII. 1 andBst I.8+29. Because none of the protein products were derivedfrom mRNA selected by both filters, it may be assumed thatthe proteins are encoded by more specific regions of the T-DNA, as shown in Fig. 1. Thus, proteins derived from mRNAhybridizing to HindIII.1 filters would be encoded by sequencesmapping to the right of Bst I fragment 29 (referred to as Hind-III.1R). The proteins that are common to each tumor line wouldbe encoded by sequences of fragment Bst I.8 mapping to theleft of HindIII.1 (referred to as Bst I.8L).The T-DNA fragments used for hybridization in this study

are of sufficiently large molecular size to have encoded foradditional protein products. It has been shown (17-20) that onlysmall regions of the T-DNA are transcribed in octopine-typetumor tissues. The significance of the entire T-DNA present intumor tissue is unknown.

DISCUSSIONTumor-derived mRNAs encoded by discrete regions of T-DNAproduce specific proteins in a wheat germ cell-free system(Table 1). The products derived from El transcripts homolo-gous to DNA sequences of HindIII.1R (shown in Fig. 1) areproteins of estimated Mr of 30,000 and 16,500. Transcripts from15955/01 tumor that specifically hybridize to HindIII.Rproduce a diffuse protein band of slightly less than 30,000 Mron NaDodSO4 gel electrophoresis. This molecular weight dif-ference could be the result of variation between individualtumor lines. No products were obtained for 15955/1 tumor,which differed from El and 15955/01 lines in that it did notproduce octopine. Recent investigations of the T-DNA presentin tumors induced by octopine-type Ti plasmids (13) haveshown that those tumors that produce octopine contain theentire restriction fragment Bst 1.19, whereas this fragment iseither absent or has undergone some form of reorganization inthe nonoctopine-producing lines. These findings are consistentwith those obtained with deletion mutants of Ti plasmids, whichindicated that DNA sequences mapping to the right of Sna lOcwere essential for octopine production in the resulting tumors(14). Thus the presence of proteins coded by HindIII.lR (whichextends to the right of Sma 1Oc and includes Bst I.19) may insome way be related to octopine production in tumor lines Eland 15955/01.

It is unknown whether the T-DNA codes for octopine syn-thase or in some way regulates its production from a plant gene.The 30,000 Mr protein product is somewhat smaller than thevalue of 38,000 reported (21) for octopine synthase isolated fromsunflower tumor lines. Differences in molecular weight could

occur because of possible glycosylation of the plant enzyme,which would not occur in vitro, or variation between differenttumor tissues (or both).

Proteins encoded by the T-DNA sequences correspondingto fragment Bst I.8L (shown in Fig. 1) have been produced bytranslation of those mRNAs that were isolated by hybridizationto this region of the DNA. An identical protein band on Na-DodSO4 gel electrophoresis was derived from the nonpoly-adenylylated RNA fractions of each of the tumor lines, El,15955/01, and 15955/1. In addition, a protein band of identicalmobility was derived from poly(A)-RNA of 15955/01, whichalso hybridized to this region of T-DNA. The estimated mo-lecular weight of these products was 15,000. It has been shownby others (see. ref. 40) that some proteins are coded both bypolyadenylylated and nonpolyadenylylated RNA from thesame tissue.

This common protein product derived from the region ofT-DNA that is conserved in both octopine-type and nopaline-type crown gall tumors may be involved in the alteration ofnormal plant regulatory processes. Selection of mRNAs thathybridize to T-DNA, coupled with translation in a eukaryoticcell-free system, represents evidence that proteins are encodedby T-DNA sequences of tumor tissues.We thank Dr. Ted Young and Dr. Richard Palmiter for helpful

discussions We also thank Dr. Ross MacGillivray, who supplied bovineliver poly(A)-RNA and antiprothrombin serum used in monitoring theintegrity of the cell-free translation systems. The investigation wassupported by Grant CA 13015 from the National Cancer Institute.

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