A. Rhizogenes

JOURNAL OF BACTERIOLOGY, Mar. 1980, P. 1134-1141 Vol. 141, No. 30021-9193/80/03-1134/08$02.00/0

Hairy Root: Plasmid Encodes Virulence TrqKts inAgrobacterium rhizogenes

FRANK F. WHITE AND EUGENE W. NESTER

Department ofMicrobiology and Immunology, University of Washington, Seattle, Washington 98195

Agrobacterium rhizogenes strain 15834, which incites hairy root disease inplants, harbors three large plasmids: pAr15834a (107 x 10' daltons), pAr15834b(154 x 10' daltons), and pArl5834c (258 x 10" daltons). Kanamycin-resistanttransconjugants were selected in a cross of a kanamycin-resistant derivative ofstrain 15834 and an avirulent recipient. The transconjugants belonging to oneclass were virulent and contained all three donor plasmids. These transconjugantsalso acquired sensitivity to the bacteriocin agrocin 84. The loss of plaids fromvirulent transconjugants during growth at 37°C indicated that virulence genesreside on pArl5834b, whereas agrocin 84 sensitivity genes reside on pArl5834a.The pathology induced by the virulent transconjugants containing onlypAr15834b was identical to that produced by the wild-type strain ofA. rhizogenes.Restriction endonuclease fragment analysis of plasmids from the transconjugantsand the donor revealed that pArl5834c is a cointegrate of pAr15834a andpArl5834b. Kanamycin-resistant transconjugants belonging to a second classwere avirulent and contained an altered form of pArl5834b. Strain 15834 canutilize octopine. However, this trait was not detected in any of the transconju-gants. Octopine is not synthesized by infected plant tissue.

Members of the genus Agrobacterium incitegrowth on susceptible plants when introducedinto wound sites. In the case of Agrobacteriumtumefaciens, oncogenicity is encoded on a largeplasmid (Ti) (41, 43). Although the exact mech-anism of transformation remains unknown,DNA sequences homologous to the Ti plasmidare found in transformed DNA but not normalplant DNA (9; F. Yang et al., Mol. Gen. Genet.,in press). Other traits localized on Ti plasmidsinclude genes that specify octopine or nopalinesynthesis (by the tumors) and utilization (by thebacteria) (6, 27, 32) and sensitivity to agrocin 84,a bacteriocin produced by Agrobacterium radi-obacter strain K84 (14).Agrobacterium rhizogenes is closely related

to members of the biotype 2 group of A. tume-faciens by physiological and DNA homologycriteria (13, 19, 23). A. rhizogenes is distin-guished from A. tumefaciens because it induceshairy root disease (36), a disease characterizedby extensive proliferation of roots from an in-fected plant wound. A. rhizogenes strains alsocontain large plasmids (11, 39). Albinger andBeiderbeck (1) isolated a virulent transconju-gant from an in planta cross (20). Whereas vir-ulence and agrocin 84 sensitivity were trans-ferred from an A. rhizogenes donor, physicalevidence of plasmid transfer was not presented.We now present evidence that associates viru-lence with an A. rhizogenes plasmid.

(A preliminary presentation of these resultswas made at the 79th Annual Meeting of theAmerican Society for Microbiology, Honolulu,Hawaii, May 1979.)

MATERIALS AND METHODSBacterial strains and culture media. The bac-

terial strains used in this work are described in Table1. Agrobacterium strains were maintained on nutrientagar (Difco Laboratories) at 4°C. Escherichia colistrain 1830 was maintained on L agar (31) containing50 ltg of kanamycin (Sigma Chemical Co.) per ml.Nutrient liquid medium consisted of a 1:1 mixture ofL broth and mannitol-glutamate (37). The definedmedia used were AB minimal medium (8) and modi-fied Schroth medium (R. Hamilton, personal commu-nication). Modified Schroth medium consists of (perliter of water): mannitol, 10 g; L-glutamate, 2 g;KH2PO4, 0.3 g; NaCl, 0.2 g; MgSO4, 0.1 g; biotin, 5 ,ug;and Fe-EDTA, 5 ml of stock solution (0.02 M FeSOV7H20, 0.009 M Na2-EDTA). The pH of the mediumwas adjusted to 7.0 with 6 N NaOH. For solid definedmedia, 1.5% agar (Difco) was included.

Conjugation conditions. The transposon Tn5,which encodes kanamycin resistance (3), was intro-duced into A. rhizogenes 15834 by conjugation withE. coli 1830(pJB4J1). Plasmid pJB4J1 is a conjugativeR factor carrying phage Mu and Tn5 insertions (4).This plasmid is not maintained in Agrobacterium (42),and drug-resistant transconjugants are recoveredwhen Tn5 tranposes to the recipient genome. Strains1830 and 15834 were grown overnight in L broth andnutrient liquid medium, respectively, mixed 1:1, and0.1 ml of the mixture was placed on a membrane filter

1134

VIRULENCE PLASMID AND A. RHIZOGENES

TABLE 1. Bacterial stransRelevant phe-

Strain Plasmid notype/geno- Derivationtypea

1830 pJB4J1 Knr, gentami- E. coli from Ber-cin resistant, inger et al. (4)pro met

15834 pArl5834a VirUlentb Wild type A. rhi-pAr15834b Ag84 zogenes frompArl5834c J. L Lippin-

cottF5 pArl5834a Virulent This study

pArl5834b::Tn5 Ag84'pArl5834c Knr

A136 pAt-C58c Avirulent AgrobacteriumRifr Nar from Watson

et al. (43)FC2 pAt-C58 Virulent This study

pAr15834a Ag84pArl5834b::Tn5 Kn' Rif' NalrpAr15834c

FHT6 pAt-C58 Avirlent This studypArl5834a Ag84'

Rifr Nalr

FHT14 pAt-C58 Virulent This studypArl5834b::Tn5 Kn Rifr Nar

FC15 pAt-C58 Avirulent This studypArl5834b'::Tn5 Kr Rifr Nar

'Kn, Kanamycin; Ag84, agrocin 84; Rif, rifampin; Nal,nalidixic acid.

bVirulent strains all incite roots upon infection of plant.'The detection of pAt-C58 was initially reported by Came

et al. (7). The molecular weight has not yet been reported.

(pore size, 0.45 pm; Millipore Corp.) on nutrient agar.After incubation at 280C for 24 h, kanamycin-resistantcells were selected on AB agar containing biotin (2 ug/ml) and kanamycin (400 ,ug/ml). Transconjugants werescreened for resistance to gentamicin (10 jug/ml) toinsure that transposition had occurred. Kanamycin-resistant transconjugants were recovered at a fre-quency of 0.5 x 10-5 to 1.0 x 10-5 (per recipientplated), whereas the recovery of gentamicin resistancewas less than 10'. Five virulent clones were tested asdonors during in planta crosses, and one (F5) trans-ferred kanamycin resistance at a frequency of 4 x 10'4per recipient recovered from the plant tissue. Thisstrain contains a Tn5 insertion in pArl5834b (Whiteand Nester, manuscript in preparation).

For in planta crosses, carrot slices were inoculatedwith the donor strain (27). After 1 week the slices wereinoculated with the avirulent recipient. After 4 weeksthe callus tissue was excised and ground in a sterilemortar with 3 ml of AB medium. Plant debris wasremoved by low-speed centrifugation. The tissue ex-tract was diluted and plated onto AB agar containingrifampicin (5 yig/ml; Sigma), nalidixic acid (50 pg/mi;Sigma), and kanamycin (100 pg/ml). After purifica-tion, transconjugants were maintained on nutrientagar containing 100 jg of kanamycin per ml.Agrocin 84 sensitivity and virulence assay.

Transconjugants were tested for sensitivity to the

bacteriocin agrocin 84, which was produced by strainK84 (21), by replica plating colonies to modifiedSchroth agar overlaid with 5 ml of modified Schrothagar containing 20 id of partially purified agrocin 84(38). Colony growth was compared with growth onmodified Schroth agar without agrocin 84.

Strains were tested for virulence on carrot disks. Asample of a strain grown in modified Schroth mediumwas spread across the surface of each carrot disk. Thedisks were scored after 2 weeks.Plasmid isolation. Cells were grown in modified

Schroth medium (1-liter cultures) for 14 to 16 h at280C with shaking. Plasmids were isolated by a mod-ified procedure of Currier and Nester (12). Sodiumacetate (4.08 g/100 ml of lysate) was added in place ofMgCI2 and sodium phosphate in the alcohol precipi-tation step, according to the procedure of Casse et al.(7).

Plasmid contour length measurements of electronphotomicrographs were made by the methods ofKleinschmidt et al. (25) and Lang (26), using an elec-tronic graphics calculator (Numonics Corp.).Plasmid sreening. A method that combines the

lysis procedure of Casse et al. (7) and the isolationprocedure of Hansen and Olsen (16) was devised. A100-ml culture (modified Schroth medium) was har-vested after shaking for 12 to 14 h at 28°C. The cellswere suspended in 25 ml of lysis buffer (0.05 M Tris,pH 12.3, 0.020 M EDTA, 1% [wt/vol] sodium dodecylsulfate) and incubated for 20 min at 37°C. The lysatewas neutralized with 1.5 ml of 2 M Tris, pH 7.0. Thesodium dodecyl sulfate content was increased to 4%with the addition of 3.6 ml of 25% (wt/vol) sodiumdodecyl sulfate; 7.0 ml of 5 M NaCl was immediatelyadded, and the lysate was placed on ice for 4 h. It wasthen centrifuged for 40 min at 17,000 x g in a SorvallSS34 rotor. To the supernatant 8.2 ml of 50% (wt/vol)polyethylene glycol 6000 was added (final polyethyl-ene glycol concentration, approximately 10%), and thelysate was stored overnight at 4°C. The precipitatewas collected by low-speed centrifugation (5 min, 1,000x g) and dissolved in 0.2 to 0.5 ml of TES (0.5 M Tris,pH 8.0, 0.005 M NaCl, 0.005 M EDTA). Generally, a50-pl sample was subjected to agarose gel electropho-resin.

Vertical gel electrophoresis. Plasmid prepara-tions were analyzed by the procedure of Meyers et al.(30). Electrophoresis was carried out at 80 to 100 V for3 to 6 h in a solution containing 89 mM Tris, 2.5 mMNa2EDTA, and 8.9 mM boric acid in 0.7% agarose.

Size estimates of plasmids were obtained by com-paring the log of a relative migration with the logs ofthe relative migrations of plasmid standards. Differentgels were normalized to the migration of pTi-T37 ineach gel. We did not find the discrepancies reportedby Hansen and Olsen (16) between such estimates andthe sizes as determined by electron microscopy whenextrapolating to pArl5834c, which was considerablylarger than any of the standards. The plasmids usedas standards were pAt-Ab2/73 (91 megadaltons[Mdal]) (29), pAr-TR7a (98 Mdal) (11), pAr-TR7b(140 Mdal) (11), pTi-T37 (122 Mdal) (40), pTi-223 (125Mdal) (40), and pTi-K27 (153 Mdal) (11).Endonuclease digestion. Restriction endonucle-

ase BstI was generously provided by R. Meager. A 1.0-

1135VOL. 141, 1980

1136 WHITE AND NESTER

pg sample of plasmid DNA was digested in a 25-pireaction volume of 0.1 M Tris-(pH 7.6)-0.006 MMgClr-O.l M NaCl at 370C for 2 h. SmaI (BethesdaResearch Laboratories) digestions were in 0.15 M Tris(pH 8.0)-0.006 M MgClr.0.015 M KCI at 30° for 2 h.Reactions were stopped by adding 5 pl of stop mixture(20% [wt/vol] Ficoll, 0.2% [wt/vol] sodium dodecylsulfate, 0.05% [wt/vol] bromophenol blue).

Restriction endonuclease digests were subjected toelectrophoresis on horizontal slab gels (30 cm by 18.3cm by 4 mm) of 0.7% agarose (SeaKem ME) in 0.08MTris-0.04 M sodium acetate-0.004 M EDTA, pH 8.0(adjusted with glacial acetic acid). Samples (15 to 30pl) containing approximately 0.5 pig ofDNA each wereloaded into welLs (0.5 by 0.1 cm). Electrophoresis wascarried out at 1.3 V/cm for 36 h.

After the vertical and horizontal agarose gels werestained in a solution of 0.5 pg of ethidium bromide(Sigma) per ml, they were illuminated with a UV lightbox (model C-61; Ultra-Violet Products, Inc.) and pho-tographed with an MP-4 Land camera (Polaroid)equipped with orange and UV filters.Measurement of uptake of labeled octopine

and nopaline. Incorporation of [3H]octopine and[3H]nopaline by bacteria was assayed essentially asdescribed by Montoya et al. (32).

Analysis of octopine and nopaline productionin hairy root tissue. Callus and roots induced bystrain 15834 were analyzed for octopine and nopalineby the method of Montoya et al. (32). The pH 3.5buffer system previously described was used (43).

RESULTS

Plasmids of A. rhizogenes 15834. We firstdetermined that virulent A. rhizogenes strain15834 contained three large plasmids (Fig. 1).From their relative electrophoretic mobilities,the sizes of the plasmids were estimated to be107 ± 5, 163 ± 9, and 271 ± 12 Mdal. Contourlength measurements of electron micrographsgave estimates of 107 ± 3, 154 ± 3, and 258 ± 6Mdal (eight, seven, and five molecules measured,respectively).Transfer of virulence. To determine

whether virulence was associated with the plas-mids of A. rhizogenes, strain F5 was crossedwith avirulent strain A136 in planta. Selectionfor kanamycin resistance allowed the direct se-lection of potentially virulent transconjugants.Two classes of kanamycin-resistant transconju-gants which had the properties of the recipientcells were obtained (Table 2). One class (classI), which comprised 20% of the kanamycin-re-sistant transconjugants, was virulent on carrotsand Kalanchoe daigremontiana. A total of 10virulent transconjugants from two independentcrosses were screened for plasmids. Thesestrains contained all three of the donor plasmids(Fig. 2, lanes b through d) in addition to thelarge endogenous plasmid of the recipient.The amount of the largest donor plasmid,

J. BACTERIOL.

0

o\ O<C LO

-z

pAr15834c

pAr 15834 bpArl5834 a

If

FIG. 1. Agarose gel electrophoresis of plasmid8from A. rhizogenes 15834. Approximately 1 pg ofplasmid DNA, which was isolated by thepreparativetechnique, was loaded into each well. A208 containsph-T37, a 122-Mdal A. tumefaciens plasmid, and isshown for comparison. Electrophoresis was in a ver-tical gel (thickness, 6 mm) at 100 V for 3 h. DNAmigratedfrom top to bottom. The lowest bands visiblein this gel and subsequent vertical gels are linearDNA fragments (7f) (30).

TABLE 2. Characterization of transconjugantsTranconjugant

Trait(s) F5 (do- A136 (re- classTrait(s) nor) cipient)1 (20%) II (80%)

Virulence + - +Agrocin84 sen- + - +

sitivityKanamycin re- + - + +

sistanceKetolactose for- - + + +mationa

Rifampicin and - + + +nalidixic acidresistance

Growth on +erythritolb

Growth at 370C - + + +

'Tested by the method of Bernaerts and DeLey(5).

b Medium of New and Kerr (35).

VIRULENCE PLASMID AND A. RHIZOGENES 1137

a b c de

FIG. 2. Agarose gel electrophoresis offrom virulent transconjugants. Plasmidslated by the screeningprocedure described iF5plasmid was prepared as described in tto Fig. 1. Lysates loaded into the wells irecipient strain A136 (lane a), virulent trogant strains FC2, FC3, and FC4 (lanes b,respectively) and donor F5 (lane e). Electrwas for 6 h at 80 V. If, Linear DNA fragme.

pAr15834c, varied considerably relatiiamounts of pAr15834a and pAr15834bindicated by the intensities of the banvertical gel analysis (Fig. 2). In fact, a pvirulent transconjugant may contain eidominately pAr15834c or pA415&8pA415834b: :Tn5, depending on the con(growth and storage of the strain. Thirelationship between the relative ampAri5834c compared with pArl58pArl5834b::Tn5 suggests that pArl5tcointegrate of pAr15834a and pArl15&

Strains 15834 and F5 are both seragrocin 84. This trait, whenever prese]ulentA. tumefaciens strains, is always aiwith the Ti plasmid (14, 43). Theretransconjugants were also screened fo84 sensitivity. All virulent transconjugeagrocin 84 sensitive. Avirulent kanamy(conjugants (class II) (Table 2), as wekanamycin-sensitive recipient coloicovered from crosses, were all agrocinant.

Identification of the virulence :The concomitant transfer of virulencEA. rhizogenes plasmids indicated ths

virulence was a plasmid-coded trait. However,in all cases virulent strains contained three do-nor plasnids. We therefore isolated strainswhich contained only one A. rhizogenes plasmidin order to determine whether the virulencegenes and the agrocin 84 sensitivity genes re-sided on one or several plamids. Such strainswere obtained by growing virulent transconju-gants at an elevated temperature (370C) in theabsence of kanamycin and then screening colo-nies for kanamycin resistance and agrocin 84sensitivity. After 12 h of growth at 370C, 3% hadlost either kanamycin resistance or agrocin 84sensitivity. The kanamycin-sensitive isolatescontained pAr15834a (Fig. 3, lanes c and f) andwere avirulent. The cells which lost agrocin 84sensitivity yet retained kanamycin resistancepArl5834b::Tn5 (Fig. 3, lanes a and d). These

-if strains were virulent and incited hairy rootsymptoms identical to the symptoms induced bythe multiple-plaid transconjugants and thedonor A. rhizogenes strain. These results indi-cate that the virulence genes reside solely on

pkasmiw,s pAr15834b: :Tn5. The strains that retained agro-were i80 cin 84 sensitivity always contained pArl5834a,n the text. indicating that agrocin 84 sensitivity is encodedhe legend by this plasmid.vere from Endonuclease digestion of A. rhizogenesansconju- plasmids. The physical relationships betweenc, and d, the plasmids recovered from the crosses and'ophore8is subsequent heat treatments were established bynts.

re to the::Tn5, asids in the)articularither pre-34a andditions ofs inversetounts of34a andB34c is a34b: :Tn5.isitive tont in v'i.- 1

ssociated,fore, thetr agrocinmnts werecin trans-'fl as 400nies re-84 resist-

plasmid.e and theEt indeed

FIG. 3. Plasmids of heat-treated transconjugants.Lysates were prepared from representative strainsderived from the virulent transconjugants FC2 andFC31 (lanes b and e) and had lost either kanamycinresistance (FHT6 and FHT7 [lanes c and f, respec-tively]) or agrocin 84 sensitivity (FHT14 and FHT4[lanes a and d, respectively]). Lysate from recipientA136 was loaded in lane g. Electrophoresis was for6 h at 80 V. If, Linear DNA fragments.

VOL. 141, 1980


FIG. 4. Restriction endonuclease digestion pat-terns of A. rhizogenes plasmids. Each well wasloaded with plasmid DNA digested with BstI, withthe exception ofpTi-B6806, which was digested withSmaI and used for size standards (10). Lane a, pTi-B6806 from strain A277; lane b, pAr15834a,pAr15834b::Th5, and pAr15834c from donor strainF5; lane c, pAr15834a from strain FHT6; lane d,pAr15834b::Tn5 from strain FHT14; lane e,pArl5834b'::Tn5 from strain FC15. The arrowheadsdesignate the variant fragments ofpArl5834b::Tn5

analyzing restriction endonuclease fragment pat-terns. The fragments generated from the plas-mids of heat-treated strains, which containedpAr15834a and pArl5834b::Tn5 (Fig. 4, lanes cand d), corresponded to the fragments generatedby the digestion of donor F5 plasmid DNA,which consisted of pAr15834a, pArl5834b: :Tn5,and pAr15834c (Fig. 4, lane b). Identical resultswere obtained with plasmids of virulent trans-conjugants from two independent crosses (datanot shown). These results confirm that the plas-mids of the virulent transconjugants originatedin the donor A. rhizogenes strain. Furthennore,the fragments from the two plasmids (pAr15834aand pArl5834b::Tn5) accounted for all of thefragments of the donor plasmids despite the factthat pArl5834c was the predominant plasmid inthe plasmid DNA preparation from F5 (Fig. 2,lane e). Since there was no unique set of frag-ments attributable to pArl5834c, it is likely thatpArl5834c is a cointegrate of pAr15834a andpArl5834b. This interpretation is supported bythe fact that the combined sizes of the twosmaller plasmids (261 Mdal) equal the size ofthe large plasmid (258 Mdal), as well as by theobservation of an inverse relationship betweenthe amount of pAr15834c relative to the amountof pAr15834a and pArl5834b::Tn5 recoveredfrom the virulent transconjugants.Plasmids of avirulent transconjugants. A

total of 80% of the kanamycin-resistant cellsfrom the in planta crosses were avirulent, yetthese strains contained a plasmid similar in sizeto pArl5834b::Tn5, which carried all of thegenes required for virulence (Fig. 5, lanes cthrough e). Fragmentation analysis of plasmidsfrom four avirulent strains revealed that all hadidentical fragment patterns (data not shown).However, this pattem could be distinguishedfrom the pattem of pArl5834b: :Tn5 present invirulent strains. We refer to the plasmid fromthe avirulent strain as pArl5834b': :Tn5. For ex-ample, the plasmid from the avirulent strainFC15 (Fig. 4, lane e) contained at least threeBstI fragments which were not present inpArl5834b::Tn5 from virulent strains (an 8.8-Mdal fragment, a 4.8-Mdal fragment present asa doublet, and a 2.8-Mdal fragment). This plas-mid lacked at least four BstI fragments presentin pArl5834b: :Tn5 (an 11.2-Mdal fragment pres-ent as a doublet, a 7.0-Mdal fragment, a 2.8-Mdal fragment, and a 2.0-Mdal fragment presentas a doublet). This analysis only accounts forfragments which are larger than 0.6 Mdal, so

and pArl5834b'::Tn5. Electrophoresis was for 36 hat 1.33 V/cm. The size calibrations on left are inmegadaltons.

J. BACTERIOL.


a b c d e

FIG. 5. Plasmids ofavirulent transconjusates were prepared from representativekanamycin-resistant transconjugants. Lanlane b, F5 donor; lanes c, d, and e, FC12, JFC15, respectively. Electrophoresis was foiV. If, Linear DNA fragments.

additional differences may exist.The variant fragments present in pJ

:Tn5 represent a total of 23.0 Mdalcompared with a total of 16.4 Mda;variant fragments in pArl5834b'::Tn5,in a difference of 6.6 Mdal. Thus, themids remain approximnately the sameconclude that the plasmids fromstrains are generated by a mechanisresults in a specific alteration of plhquences. These alterations probably acthe differences in virulence.Octopine and nopaline utilizatih

lent biotype 2 strains of A. tumefacieboth octopine and nopaline, but tumo:by these strains synthesize only nopthese strains only nopaline utilization v

ferred by conjugation and transformn33). Montoya et al. (33) concluded thatutilization is coded by chromosomalbiotype 2 Agrobacterium strains. A. ri

15834, a biotype 2 strain, utilizes only(28). In a cross of F5 and A136 the totament of plasmids was transferred, yet tconjugants did not acquire the abilityoctopine (Table 3). This result is fui

TABLE 3. Utilization of octopine and nopaline byAgrobacterium andproduction by tumor tissue

Utilization of:a Production

Octopine Nopaline by tumor

A277b + - OctopineA208C - + NopalineA136 - - NTVF5 + - NeitherFC2 - - NTFC3 - - NT

a Determined radiometrically as previously de-scribed (32).

b A277 contains pTi-B6806, an octopine-type plas-mid.

'A208 contains pTi-T37, a nopaline-type plasmid.d NT, Not tested.

dence that chromosomal genes code for octopineutilization in biotype 2 Agrobacterium strains.

1-lf Additionally, neither callus nor roots induced byA. rhizogenes 15834 synthesized octopine or no-paline (Table 3).

DISCUSSION

tgants. Ly- The data presented here indicate that theavirulent induction of hairy root disease by A. rhizogenese a, A136; is associated with a large plasmid. This findingFC14, and extends the work of Albinger and Beiderbeckr 6 h at 80 (1), who found that virulence and agrocin 84

sensitivity could be transferred during in plantacrosses ofstrain 15834 and an avirulent recipient.The findings were not definitive, however, since

kr15834b: they recovered only one transconjugant and pre-of DNA sented no physical evidence for the transfer of a

1 for the plasmid.resulting In the case of F5, a derivative of 15834, viru-two plas- lence was transferred simultaneously with thesize. We transfer of three donor plasmids, although viru-avirulent lence could be localized to the 154-Mdal plasmidnm which pArl5834b::Tn5. This finding differs from aismid se- study reported by Moore et al. (34). These au-count for thors concluded that virulence was associated

with a single 110-Mdal plasmid which they iden-Dn. Viru- tified in A. rhizogenes strain A4. We isolatedns utilize plasmid DNA from strain A4 (received from L.rs incited Moore) and found three plasmids similar in size?aline. In to the three plasmids in strain 15834. Restrictionvas trans- analysis of A4 plasmid DNA suggests that theseation (22, plasmids are very similar to the plasmids inoctopine 15834 (White and Nester, manuscript in prepa-genes in ration).iizogenes The results in this paper reinforce the generaloctopine model of plasmid-associated virulence in thed comple- genus Agrobacterium. On the basis of physiolog-the trans- ical and DNA homology criteria, A. rhizogenesto utilize strain 15834 is a member of the group of Agro-rther evi- bacterium strains designated by Keane et al.

VOL. 141, 1980


(19) as biotype 2. Strain 15834 differs from A.tumefaciens strains of biotype 2 only in mor-phology of plant growth at the infected woundsite and plasmid content. The simultaneoustransfer ofpasmid and virulence from a biotype2 A. rhizogenes strain (15834) to a biotype 1strain (A136) is analogous to the transfer ofoncogenicity from the biotype 2 crown gall form-ers (A. tumefaciens strains 223 and 27) to bio-type 1 stains (41, 43). Therefore, it is reasonableto conclude that virulence in A. rhizogenes iscoded by plasinid genes. In addition, our resultssupport the conclusions of others that the clas-sification ofAgrobacterium should not be basedon pathogenicity, which is predominately aproperty of a plasmid.The distinction between A. rhizogenes and A.

tumefiaciens is further obscured by reportswhich indicate that the morphologies of thegrowths which they induce depend both on theparticular host plants and on the plasmids whichthey contain (15, 18). Indeed, investigators havereported that some strains of A. rhizogenes in-cite galls on certain host plants (2). The possi-bility exists that hairy root induction resultsfrom the transfer and incorporation of plaidDNA into plant cell DNA. Plasmid sequenceshave been detected in DNAs from both unor-ganized tumor and teratoma tissues (9; Yang etal., in press). Hairy root may represent anothertumor type. In an effort to determine whetherplasmid DNA is transferred to plant cell DNA,we are attempting to establish axenic hairy roottissue in culture.Although cointegrates of R factors and Agro-

bacterium plasnids have been constructed (17),this is the first reported instance of the cointe-gration of endogenous Agrobacterium plasmids.The factors controlling this arrangement andthe significance of the cointegrate form to thebiology of A. rhizogenes are not clear.

It is possible that pAr15834a and pAr15834bare transferred as the cointegrate. This wouldexplain the presence of all three donor plasmidsin the viruent transconjugants. Since we wereunable to isolate transconjugants with onlypAr15834a, it appears that pAr15834a is nottransferred independently but is mobilized bypAr15834b. Interestingly, most Agrobacteriumstrains harbor several plasmids (7, 11, 29), anda question arises as to whether cointegration ofendogenous Agrobacterium plasmids is a gen-eral phenomenon.How avirulent transconjugants are generated

remains unexplained. The plasmid in avirulentstrains (pArl5834'::Tn5) is similar in size to theplasmid in virulent strains but differs in severalrestriction endonuclease fragments. Transposa-

ble elements are known to generate mutationsby either insertion or imprecise excision (24),and such effects may be responsible for the twoplasmid forms. However, we have evidence thatTn5 deletions resulting form Tn5 excision arenot responsible for the alterations in pAr15834b'::Tn5. Alternatively, pArl5834b': :Tn5may resultfrom the improper dissociation or interruptedtransfer of the cointegrate plasmid, pAr15834c.

ACKNOWLEDGMENTSWe thank John Beringer for generously providing strain

1830 and K. Spangler for typing the manuscpt.This work was supported in part by Public Health Service

grant CA13015 from the National Institutes of Health. F.F.W.was supported by a National Science Foundation predoctoralfellowship and by National Researh Service Award GM07270.

LTERATURE CTED1. Albinger, G., and R. Beiderbeck. i977. tlbertragung

der Fihigkeit zur Wurzelinduktion von Agrobacteriumrhizogenes aufA. tumefaciens. Phytopatho. Z. 90:306-310.

2. Anderson, A. R., and L W. Moore. 1979. Host specific-ity in the genus Agrobacterium. Phytopathology 69:320-323.

3. Berg, D., J. Davies, B. Allot, and J.-D. Rochaiz. 1975.Transposition of R factor genes to bacteriophage A.Proc. Natl. Acad. Sci. U.S.A. 72:3628-3632.

4. Beringer, J. E., J. L Beynon, A. V. Buchanan-Wol-laston, and A. W. B. Johnson. 1978. Transfer of thedrug-resistance transposon Tn5 to Rhizobium. Nature(London) 276:633-634.

5. Bernaerts, AL J., and J. DeLey. 1963. A biochemicaltest for crown gall bacteria. Nature (London) 197:406-407.

6. Bomhoff, G., P. M. Klapwijk, H. C. M. Kester, R. A.Schilperoort, J. P. Hernalsteens, and J. Schell.1976. Octopine and nopaline synthesis and breakdowngenetically controlled by a plasmid of Agrobacteriumtwnefaciens. Mol. Gen. Genet. 145:177-181.

7. Casm, F., C. Boucher, J. S. Jufliot, KL Michal, and J.D6narie. 1978. Identification and characterization oflarge plasmids in Rhizobium meliloti using agarose gelelectrophoresis. J. Gen. Microbiol. 113:229-242.

8. Chilton, M.-D., T. C. Currier, S. K. Farrand, A. J.Bendich, M. P. Gordon, and E. W. Nester. 1974.Agrobacterium tumefaciens DNA and PS8 bacterio-phage DNA not detected in crown gall tumor Proc.Natl. Acad. Sci. U.S.A. 71:3672-3676.

9. Chilton, M.-D., K. H. Drummond, D. J. Merlo, D.Sciaky, A. L. Montoya, K P. Gordon, and E. W.Nestr. 1977. Stable incorporation of plasmid DNAinto higher plant cells: the molecular basis of crown galltumorigenesis. Cell 11:263-271.

10. Chilton, M.-D., A. L Montoya, D. J. Merlo, M. H.Drummond, R. Nutter, KL P. Gordon, and E. W.Nester. 1978. Restriction endonucleas mapping of aplasmid that confers oncogenicity upon Agrobacterinumtumefaciens strain B6-806. Plasmid 1:254-269.

11. Currier, T. C., and E. W. Nester. 1976. Evidence fordiverse types of large plasmids in tumor-inducingstrains of Agrobacterium. J. Bacteriol. 126:157-165.

12. Currier, T. C., and E. W. Nester. 1976. Isolation ofcovalently closed circular DNA of high molecularweight from bacteria. Anal. Biochem. 66:431-441.

13. DeLey, J., R. Tijtgat, J. De Smedt, and KL Michiels.1973. Thermal stability of DNA:DNA hybrids within

J. BACTERIOL.


the genus Agrobacterium. J. Gen. Microbiol. 78:241-252.

14. Engler, G., M. Holsters, M. Van Montagu, J. Schell,J. P. Hernalsteens, and R. Schilperoort 1975. Agro-cin 84 sensitivity: a plasmid determined property inAgrobacterium tumefaciens. Mol. Gen. Genet. 138:345-349.

15. Gresshoff, P. M., M. L Skotnicki, and B. G. Rolfe.1979. Crown gall teratoma formation is plasmid andplant controlled. J. Bacteriol. 137:1020-1021.

16. Hansen, J. B., and R. H. Olsen. 1978. Isolation of largebacterial plasmids and characterization ofthe P2 incom-patibility group plasmids pMG1 and pMG5. J. Bacte-riol. 135:227-238.

17. Holsters, M., B. Silva, C. Gentello, G. Engler, F. vanVliet, M. deBlock, R. Villarroel, MI van Montagu,and J. Schell. 1978. Spontaneous formation of cointe-grates of the oncogenic Ti-plasmid and the wide hostrange P-plasmid RP4. Plasmid 1:456-467.

18. Hooykaas, P. J. J., P. M. Klapwijk, M. P. Nuti, R. A.Schilperoort, and A. Rdrsch. 1977. Transfer of theAgrobacterium tumefaciens Ti-plasmid to avirulentagrobactera and Rhizobium ex planta. J. Gen. Micro-biol. 98:477-484.

19. Keane, P. J., A. Kerr, and P. B. New. 1970. Crown gallof stone fruit. fI. Identification and nomenclature ofAgrobacterium isolates. Aust. J. Biol. Sci 23:585-595.

20. Kerr, A. 1969. Transfer of virulence between strains ofAgrobacterium. Nature (London) 223:1175-1176.

21. Kerr, A., and K. Hatay. 1974. Biological control ofcrown-gall through bacteriocin production. Physiol.Plant Pathol. 4:37-44.

22. Kerr, A., and W. P. Roberts. 1976. Agrobacterium:correlations between and transfer of pathogenicity, oc-topine and nopaline metabolism and bacteriocin 84sensitivity. Physiol. Plant Pathol. 9:205-221.

23. Kersters, K., J. DeLey, P. H. A. Sneath, and M.Suckin. 1973. Numerical taxonomic analysis of Agro-bacterium. J. Gen. Microbiol. 78:227-239.

24. KIeckner, N., J. Roth, and D. Botatein. 1977. Geneticengineenng in vivo using translocatable drug-resistanceelements. New methods in bacterial genetics. J. Mol.Biol. 116:125-159.

25. Kleinschmidt, A. K., D. Lang, D. Jaeherts, and R. K.Zahn. 1962. Preparation and length measurements ofthe total deoxyribonucleic acid content of T2 bacterio-phages. Biochim. Biophys. Acta 61:857467.

26. Lang, D. 1970. Molecular weights of coliphages and coli-phage DNA. HI. Contour length and molecular weightof DNA from bacteriophages T4, T5 and T7, and frombovine papilloma virus. J. Mol. Biol. 54:557-65.

27. Liao, C. H., and G. T. Heberlein. 1978. A method forthe transfer of tumorigenicity between strains ofAgro-bacterium tumefaciens in carrot root disks. Phytopath-ology 68:135-137.

28. Lippincott, J. A., R. Beiderbeck, and B. B. Lippin-cott. 1973. Utilization of octopine and nopaline byAgrobacterium. J. Bacteriol. 116:378-383.

29. Merlo, D. J., and E. W. Nester. 1977. Plasmids inavirulent strains of Agrobacterium. J. Bacteriol. 129:

76-80.30. Meyers, J. A., D. Sanchez, L. P. Elwell, and S. Fal-

kow. 1976. Simple agarose gel electrophoretic methodfor the identification and characterization of plasmiddeoxyribonucleic acid. J. Bacteriol. 127:1529-1537.

31. Miller, J. H. 1972. Experiments in molecular genetics.Cold Spring Harbor Laboratory, Cold Spring Harbor,N. Y.

32. Montoya, A. L., M.-D. Chilton, M. P. Gordon, D.Sciaky, and E. W. Nester. 1977. Octopine and nopa-line metabolism in Agrobacteruum tumefaciens andcrown gall tumor cells: role of plasmid genes. J. Bacte-riol. 129:101-107.

33. Montoya, A. L, L W. Moore, MI P. Gordon, and E.W. Nester. 1978. Multiple genes coding for octopine-degrading enzymes inAgrobacterium. J. Bacteriol. 136:909-915.

34. Moore, L., G. Warren, and G. Strobel. 1979. Involve-ment of a plasmid in the hair root disease of plantscaused by Agrobacterium rhizogenes. Plasmid 2:617-626.

35. New, P. B., and A. Kerr. 1971. A selective medium forAgrobacterium radiobacter biotype 2. J. Appl. Bacte-riol. 34:233-236.

36. Riker, A. J., W. M. Banfield, W. H. Wright, G. W.Keitt, and H. E. Sagen. 1930. Studies on infectioushairy-root of nursery apple trees. J. Agric. Res. (Wash-ington, D.C.) 41:507-540.

37. Roberts, W. P., and A. Kerr. 1974. Crown gall induction:seriological reactions, isozyme patterns and sensitivityto mitomycin C and to bacteriocin of pathogenic andnonpathogenic strains of Agrobacterium radiobacter.Physiol. Plant Pathol. 4:81-91.

38. Roberts, W. P., M. E. Tate, and A. Kerr. 1977. Agrocin84 is a 6-N-phosphoramidate of an adenine nucleotideanalogue. Nature (London) 265:379-381.

39. Schell J., ML Van Montagu, A. DePicker, D. De-Waele, G. Engler, C. Geneteflo, J. 0. Hernalsteens,M. Holsters, E. Messens, B. Silva, S. Van den El-sacken, N. Van Larabeke, and I. Zaenen. 1976.Agrobacterium tumefaciens: what segment of the plas-mid is responsible for the induction ofcrown gall tissue?,p. 329-342. In L. Bogorad and J. H. Weil (ed.), Nucleicacids and protein synthesis in plants. Plenum Press,New York.

40. Sciaky, D., A. L. Montoya, and ML-D. Chilton. 1977.Fingerprints ofAgrobacterium Ti plasmids. Plasmid 1:238-253.

41. Van Larabeke, N., G. Engler, M. Holsters, S. Vanden Elsacker, L. Zaenen, R. A. Schilperoort, and J.Schell. 1974. Large plasmid in Agrobacterium tumefa-ciens essential for crown gall-inducing ability. Nature(London) 252:169-170.

42. Van Vliet, F., B. Silva, M. Van Montagu, and J.Schell. 1978. Transfer of RP4::Mu plasmids to Agro-bacterium tumefaciens. Plasmid 1:446 455.

43. Watson, B., T. C. Currier, M P. Gordon, M.-D. Chil-ton, and E. W. Nester. 1975. Plasmid required forvirulence of Agrobacterium tumefaciens. J. Bacteriol.123:255-264.

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A. Rhizogenes