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JOURNAL OF BACTERIOLOGY, Mar. 1980, P.

1134-1141

Vol. 141, No. 3

0021-9193/80/03-1134/08$02.00/0

Hairy Root: Plasmid Encodes Virulence TrqKts in Agrobacterium rhizogenes


FRANK F. WHITE AND EUGENE W. NESTER Department of Microbiology and Immunology, University of Washington, Seattle, Washington 98195

Agrobacterium rhizogenes strain 15834, which incites hairy root disease in plants, harbors three large plasmids: pAr15834a (107 x 10' daltons), pAr15834b (154 x 10' daltons), and pArl5834c (258 x 10" daltons). Kanamycin-resistant transconjugants were selected in a cross of a kanamycin-resistant derivative of strain 15834 and an avirulent recipient. The transconjugants belonging to one class were virulent and contained all three donor plasmids. These transconjugants also acquired sensitivity to the bacteriocin agrocin 84. The loss of plaids from virulent transconjugants during growth at 37C indicated that virulence genes reside on pArl5834b, whereas agrocin 84 sensitivity genes reside on pArl5834a. The pathology induced by the virulent transconjugants containing only pAr15834b was identical to that produced by the wild-type strain ofA. rhizogenes. Restriction endonuclease fragment analysis of plasmids from the transconjugants and the donor revealed that pArl5834c is a cointegrate of pAr15834a and pArl5834b. Kanamycin-resistant transconjugants belonging to a second class were avirulent and contained an altered form of pArl5834b. Strain 15834 can utilize octopine. However, this trait was not detected in any of the transconjugants. Octopine is not synthesized by infected plant tissue.
Members of the genus Agrobacterium incite growth on susceptible plants when introduced into wound sites. In the case of Agrobacterium tumefaciens, oncogenicity is encoded on a large plasmid (Ti) (41, 43). Although the exact mechanism of transformation remains unknown, DNA sequences homologous to the Ti plasmid are found in transformed DNA but not normal plant DNA (9; F. Yang et al., Mol. Gen. Genet., in press). Other traits localized on Ti plasmids include genes that specify octopine or nopaline synthesis (by the tumors) and utilization (by the bacteria) (6, 27, 32) and sensitivity to agrocin 84, a bacteriocin produced by Agrobacterium radiobacter strain K84 (14). Agrobacterium rhizogenes is closely related to members of the biotype 2 group of A. tumefaciens by physiological and DNA homology criteria (13, 19, 23). A. rhizogenes is distinguished from A. tumefaciens because it induces hairy root disease (36), a disease characterized by extensive proliferation of roots from an infected plant wound. A. rhizogenes strains also contain large plasmids (11, 39). Albinger and Beiderbeck (1) isolated a virulent transconjugant from an in planta cross (20). Whereas virulence and agrocin 84 sensitivity were transferred from an A. rhizogenes donor, physical evidence of plasmid transfer was not presented. We now present evidence that associates virulence with an A. rhizogenes plasmid. (A preliminary presentation of these results was made at the 79th Annual Meeting of the

American Society for Microbiology, Honolulu, Hawaii, May 1979.)

MATERIALS AND METHODS Bacterial strains and culture media. The bacterial strains used in this work are described in Table 1. Agrobacterium strains were maintained on nutrient agar (Difco Laboratories) at 4C. Escherichia coli strain 1830 was maintained on L agar (31) containing 50 ltg of kanamycin (Sigma Chemical Co.) per ml.

Nutrient liquid medium consisted of a 1:1 mixture of L broth and mannitol-glutamate (37). The defined media used were AB minimal medium (8) and modified Schroth medium (R. Hamilton, personal communication). Modified Schroth medium consists of (per liter 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 FeSOV 7H20, 0.009 M Na2-EDTA). The pH of the medium was adjusted to 7.0 with 6 N NaOH. For solid defined media, 1.5% agar (Difco) was included. Conjugation conditions. The transposon Tn5, which encodes kanamycin resistance (3), was introduced into A. rhizogenes 15834 by conjugation with E. coli 1830(pJB4J1). Plasmid pJB4J1 is a conjugative R factor carrying phage Mu and Tn5 insertions (4). This plasmid is not maintained in Agrobacterium (42), and drug-resistant transconjugants are recovered when Tn5 tranposes to the recipient genome. Strains 1830 and 15834 were grown overnight in L broth and nutrient liquid medium, respectively, mixed 1:1, and 0.1 ml of the mixture was placed on a membrane filter

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TABLE 1. Bacterial strans
Strain
1830

VIRULENCE PLASMID AND A. RHIZOGENES


Relevant phenotype/geno-

1135

Plasmid

typea

Derivation

pJB4J1

Knr, gentami- E. coli from Bercin resistant, inger et al. (4)

pro met
15834

pArl5834a

VirUlentb
Ag84

pAr15834b pArl5834c
F5

Virulent pArl5834a pArl5834b::Tn5 Ag84' Knr pArl5834c

Wild type A. rhizogenes from J. L Lippincott This study

A136 FC2

pAt-C58c

Avirulent Rifr Nar

Virulent pAt-C58 Ag84 pAr15834a pArl5834b::Tn5 Kn' Rif' Nalr pAr15834c

Agrobacterium from Watson et al. (43) This study

bacteriocin agrocin 84, which was produced by strain K84 (21), by replica plating colonies to modified Schroth agar overlaid with 5 ml of modified Schroth agar containing 20 id of partially purified agrocin 84 (38). Colony growth was compared with growth on modified Schroth agar without agrocin 84. Strains were tested for virulence on carrot disks. A sample of a strain grown in modified Schroth medium was spread across the surface of each carrot disk. The disks were scored after 2 weeks. Plasmid isolation. Cells were grown in modified Schroth medium (1-liter cultures) for 14 to 16 h at 280C with shaking. Plasmids were isolated by a modified procedure of Currier and Nester (12). Sodium acetate (4.08 g/100 ml of lysate) was added in place of MgCI2 and sodium phosphate in the alcohol precipitation step, according to the procedure of Casse et al. Plasmid contour length measurements of electron photomicrographs were made by the methods of Kleinschmidt et al. (25) and Lang (26), using an electronic graphics calculator (Numonics Corp.). Plasmid sreening. A method that combines the lysis procedure of Casse et al. (7) and the isolation procedure of Hansen and Olsen (16) was devised. A 100-ml culture (modified Schroth medium) was harvested after shaking for 12 to 14 h at 28C. The cells were suspended in 25 ml of lysis buffer (0.05 M Tris, pH 12.3, 0.020 M EDTA, 1% [wt/vol] sodium dodecyl sulfate) and incubated for 20 min at 37C. The lysate was neutralized with 1.5 ml of 2 M Tris, pH 7.0. The sodium dodecyl sulfate content was increased to 4% with the addition of 3.6 ml of 25% (wt/vol) sodium dodecyl sulfate; 7.0 ml of 5 M NaCl was immediately added, and the lysate was placed on ice for 4 h. It was then centrifuged for 40 min at 17,000 x g in a Sorvall SS34 rotor. To the supernatant 8.2 ml of 50% (wt/vol) polyethylene glycol 6000 was added (final polyethylene glycol concentration, approximately 10%), and the lysate was stored overnight at 4C. The precipitate was collected by low-speed centrifugation (5 min, 1,000 x 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, a 50-pl sample was subjected to agarose gel electropho-

(7).

FHT6 pAt-C58 pArl5834a

Avirlent

Ag84'

This study

Rifr Nalr

Virulent FHT14 pAt-C58 pArl5834b::Tn5 Kn Rifr Nar


FC15

This study

Avirulent This study pAt-C58 pArl5834b'::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-resistant cells were selected on AB agar containing biotin (2 ug/ ml) and kanamycin (400 ,ug/ml). Transconjugants were screened for resistance to gentamicin (10 jug/ml) to insure that transposition had occurred. Kanamycinresistant transconjugants were recovered at a frequency of 0.5 x 10-5 to 1.0 x 10-5 (per recipient plated), whereas the recovery of gentamicin resistance was less than 10'. Five virulent clones were tested as donors during in planta crosses, and one (F5) transferred kanamycin resistance at a frequency of 4 x 10'4 per recipient recovered from the plant tissue. This strain contains a Tn5 insertion in pArl5834b (White and Nester, manuscript in preparation). For in planta crosses, carrot slices were inoculated with the donor strain (27). After 1 week the slices were inoculated with the avirulent recipient. After 4 weeks the callus tissue was excised and ground in a sterile mortar with 3 ml of AB medium. Plant debris was removed by low-speed centrifugation. The tissue extract was diluted and plated onto AB agar containing rifampicin (5 yig/ml; Sigma), nalidixic acid (50 pg/mi; Sigma), and kanamycin (100 pg/ml). After purification, transconjugants were maintained on nutrient agar containing 100 jg of kanamycin per ml. Agrocin 84 sensitivity and virulence assay. Transconjugants were tested for sensitivity to the

resin.

Vertical gel electrophoresis. Plasmid preparations were analyzed by the procedure of Meyers et al. (30). Electrophoresis was carried out at 80 to 100 V for 3 to 6 h in a solution containing 89 mM Tris, 2.5 mM Na2EDTA, and 8.9 mM boric acid in 0.7% agarose. Size estimates of plasmids were obtained by comparing the log of a relative migration with the logs of the relative migrations of plasmid standards. Different gels were normalized to the migration of pTi-T37 in each gel. We did not find the discrepancies reported by Hansen and Olsen (16) between such estimates and the sizes as determined by electron microscopy when extrapolating to pArl5834c, which was considerably larger than any of the standards. The plasmids used as 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 (125 Mdal) (40), and pTi-K27 (153 Mdal) (11). Endonuclease digestion. Restriction endonuclease BstI was generously provided by R. Meager. A 1.0-

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pg sample of plasmid DNA was digested in a 25-pi reaction volume of 0.1 M Tris-(pH 7.6)-0.006 M MgClr-O.l M NaCl at 370C for 2 h. SmaI (Bethesda Research 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 dodecyl sulfate, 0.05% [wt/vol] bromophenol blue). Restriction endonuclease digests were subjected to electrophoresis on horizontal slab gels (30 cm by 18.3 cm by 4 mm) of 0.7% agarose (SeaKem ME) in 0.08 M Tris-0.04 M sodium acetate-0.004 M EDTA, pH 8.0 (adjusted with glacial acetic acid). Samples (15 to 30 pl) containing approximately 0.5 pig of DNA each were loaded into welLs (0.5 by 0.1 cm). Electrophoresis was carried out at 1.3 V/cm for 36 h. After the vertical and horizontal agarose gels were stained in a solution of 0.5 pg of ethidium bromide (Sigma) per ml, they were illuminated with a UV light box (model C-61; Ultra-Violet Products, Inc.) and photographed 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 as described by Montoya et al. (32). Analysis of octopine and nopaline production in hairy root tissue. Callus and roots induced by strain 15834 were analyzed for octopine and nopaline by the method of Montoya et al. (32). The pH 3.5 buffer system previously described was used (43).

o\

<C

LO

pAr15834c
-z

pAr 15834 b pArl5834 a

If

RESULTS

Plasmids of A. rhizogenes 15834. We first determined that virulent A. rhizogenes strain 15834 contained three large plasmids (Fig. 1). From their relative electrophoretic mobilities, the sizes of the plasmids were estimated to be 107 5, 163 9, and 271 12 Mdal. Contour length measurements of electron micrographs gave estimates of 107 3, 154 3, and 258 6 Mdal (eight, seven, and five molecules measured, respectively). Transfer of virulence. To determine whether virulence was associated with the plasmids of A. rhizogenes, strain F5 was crossed with avirulent strain A136 in planta. Selection for kanamycin resistance allowed the direct selection of potentially virulent transconjugants. Two classes of kanamycin-resistant transconjugants which had the properties of the recipient cells were obtained (Table 2). One class (class I), which comprised 20% of the kanamycin-resistant transconjugants, was virulent on carrots and Kalanchoe daigremontiana. A total of 10 virulent transconjugants from two independent crosses were screened for plasmids. These strains contained all three of the donor plasmids (Fig. 2, lanes b through d) in addition to the large endogenous plasmid of the recipient. The amount of the largest donor plasmid,

FIG. 1. Agarose gel electrophoresis of plasmid8 from A. rhizogenes 15834. Approximately 1 pg of plasmid DNA, which was isolated by the preparative technique, was loaded into each well. A208 contains ph-T37, a 122-Mdal A. tumefaciens plasmid, and is shown for comparison. Electrophoresis was in a vertical gel (thickness, 6 mm) at 100 V for 3 h. DNA migrated from top to bottom. The lowest bands visible in this gel and subsequent vertical gels are linear DNA fragments (7f) (30).

TABLE 2. Characterization of transconjugants Tranconjugant F5 (do- A136 (reclass Trait(s) Trait(s) nor) cipient) 1 (20%) II (80%) + + Virulence + + Agrocin84 sensitivity + + + Kanamycin resistance + + + Ketolactose formationa + + + Rifampicin and nalidixic acid resistance + Growth on
+ + + Growth at 370C 'Tested by the method of Bernaerts and DeLey

erythritolb

(5). b

Medium of New and Kerr (35).

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a b c de

-if

FIG. 2. Agarose gel electrophoresis of pkasmiw,s from virulent transconjugants. Plasmids were i80 lated by the screeningprocedure described i n the text. F5 plasmid was prepared as described in the legend to Fig. 1. Lysates loaded into the wells ivere from recipient strain A136 (lane a), virulent troansconjugant strains FC2, FC3, and FC4 (lanes b, c, and d, respectively) and donor F5 (lane e). Electr'ophore8is was for 6 h at 80 V. If, Linear DNA fragme.nts.

virulence was a plasmid-coded trait. However, in all cases virulent strains contained three donor plasnids. We therefore isolated strains which contained only one A. rhizogenes plasmid in order to determine whether the virulence genes and the agrocin 84 sensitivity genes resided on one or several plamids. Such strains were obtained by growing virulent transconjugants at an elevated temperature (370C) in the absence of kanamycin and then screening colonies for kanamycin resistance and agrocin 84 sensitivity. After 12 h of growth at 370C, 3% had lost either kanamycin resistance or agrocin 84 sensitivity. The kanamycin-sensitive isolates contained pAr15834a (Fig. 3, lanes c and f) and were avirulent. The cells which lost agrocin 84 sensitivity yet retained kanamycin resistance pArl5834b::Tn5 (Fig. 3, lanes a and d). These strains were virulent and incited hairy root symptoms identical to the symptoms induced by the multiple-plaid transconjugants and the donor A. rhizogenes strain. These results indicate that the virulence genes reside solely on pAr15834b: :Tn5. The strains that retained agrocin 84 sensitivity always contained pArl5834a, indicating that agrocin 84 sensitivity is encoded by this plasmid. Endonuclease digestion of A. rhizogenes plasmids. The physical relationships between the plasmids recovered from the crosses and subsequent heat treatments were established by

pAr15834c, varied considerably relatiire to the amounts of pAr15834a and pAr15834b ::Tn5, as indicated by the intensities of the banids in the vertical gel analysis (Fig. 2). In fact, a p)articular virulent transconjugant may contain eiither predominately pAr15834c or pA415&834a and pA415834b: :Tn5, depending on the con(ditions of growth and storage of the strain. This inverse relationship between the relative amtounts of pAri5834c compared with pArl5834a and pArl5834b::Tn5 suggests that pArl5tB34c is a cointegrate of pAr15834a and pArl15&34b: :Tn5. Strains 15834 and F5 are both serisitive to agrocin 84. This trait, whenever prese]nt in v'i.ulentA. tumefaciens strains, is always aissociated with the Ti plasmid (14, 43). There,fore, the transconjugants were also screened fotr agrocin 84 sensitivity. All virulent transconjugemnts were agrocin 84 sensitive. Avirulent kanamy(cin transconjugants (class II) (Table 2), as we'fl as 400 kanamycin-sensitive recipient coloinies recovered from crosses, were all agrocin 84 resistant. Identification of the virulence :plasmid. The concomitant transfer of virulencEe and the A. rhizogenes plasmids indicated thsEt indeed

FIG. 3. Plasmids of heat-treated transconjugants. Lysates were prepared from representative strains derived from the virulent transconjugants FC2 and FC31 (lanes b and e) and had lost either kanamycin resistance (FHT6 and FHT7 [lanes c and f, respectively]) or agrocin 84 sensitivity (FHT14 and FHT4 [lanes a and d, respectively]). Lysate from recipient A136 was loaded in lane g. Electrophoresis was for 6 h at 80 V. If, Linear DNA fragments.

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FIG. 4. Restriction endonuclease digestion patof A. rhizogenes plasmids. Each well was loaded with plasmid DNA digested with BstI, with the exception of pTi-B6806, which was digested with SmaI and used for size standards (10). Lane a, pTiB6806 from strain A277; lane b, pAr15834a, pAr15834b::Th5, and pAr15834c from donor strain F5; lane c, pAr15834a from strain FHT6; lane d, pAr15834b::Tn5 from strain FHT14; lane e, pArl5834b'::Tn5 from strain FC15. The arrowheads designate the variant fragments of pArl5834b::Tn5
terns

analyzing restriction endonuclease fragment patterns. The fragments generated from the plasmids of heat-treated strains, which contained pAr15834a and pArl5834b::Tn5 (Fig. 4, lanes c and d), corresponded to the fragments generated by the digestion of donor F5 plasmid DNA, which consisted of pAr15834a, pArl5834b: :Tn5, and pAr15834c (Fig. 4, lane b). Identical results were obtained with plasmids of virulent transconjugants from two independent crosses (data not shown). These results confirm that the plasmids of the virulent transconjugants originated in the donor A. rhizogenes strain. Furthennore, the fragments from the two plasmids (pAr15834a and pArl5834b::Tn5) accounted for all of the fragments of the donor plasmids despite the fact that pArl5834c was the predominant plasmid in the plasmid DNA preparation from F5 (Fig. 2, lane e). Since there was no unique set of fragments attributable to pArl5834c, it is likely that pArl5834c is a cointegrate of pAr15834a and pArl5834b. This interpretation is supported by the fact that the combined sizes of the two smaller plasmids (261 Mdal) equal the size of the large plasmid (258 Mdal), as well as by the observation of an inverse relationship between the amount of pAr15834c relative to the amount of pAr15834a and pArl5834b::Tn5 recovered from the virulent transconjugants. Plasmids of avirulent transconjugants. A total of 80% of the kanamycin-resistant cells from the in planta crosses were avirulent, yet these strains contained a plasmid similar in size to pArl5834b::Tn5, which carried all of the genes required for virulence (Fig. 5, lanes c through e). Fragmentation analysis of plasmids from four avirulent strains revealed that all had identical fragment patterns (data not shown). However, this pattem could be distinguished from the pattem of pArl5834b: :Tn5 present in virulent strains. We refer to the plasmid from the avirulent strain as pArl5834b': :Tn5. For example, the plasmid from the avirulent strain FC15 (Fig. 4, lane e) contained at least three BstI fragments which were not present in pArl5834b::Tn5 from virulent strains (an 8.8Mdal fragment, a 4.8-Mdal fragment present as a doublet, and a 2.8-Mdal fragment). This plasmid lacked at least four BstI fragments present in pArl5834b: :Tn5 (an 11.2-Mdal fragment present as a doublet, a 7.0-Mdal fragment, a 2.8Mdal fragment, and a 2.0-Mdal fragment present as a doublet). This analysis only accounts for fragments which are larger than 0.6 Mdal, so
and pArl5834b'::Tn5. Electrophoresis was for 36 h at 1.33 V/cm. The size calibrations on left are in

megadaltons.

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TABLE 3. Utilization of octopine and nopaline by Agrobacterium and production by tumor tissue Utilization of:a Production by tumor Octopine Nopaline + Octopine A277b + Nopaline A208C NTV A136 + Neither F5 NT FC2 NT FC3 a Determined radiometrically as previously described (32). b A277 contains pTi-B6806, an octopine-type plasmid. 'A208 contains pTi-T37, a nopaline-type plasmid. d NT, Not tested.

dence that chromosomal genes code for octopine utilization in biotype 2 Agrobacterium strains. Additionally, neither callus nor roots induced by 1-lf A. rhizogenes 15834 synthesized octopine or nopaline (Table 3). DISCUSSION The data presented here indicate that the FIG. 5. Plasmids of avirulent transconjutgants. Lysates were prepared from representative avirulent induction of hairy root disease by A. rhizogenes kanamycin-resistant transconjugants. Lane a, A136; is associated with a large plasmid. This finding lane b, F5 donor; lanes c, d, and e, FC12, JFC14, and extends the work of Albinger and Beiderbeck FC15, respectively. Electrophoresis was foi r 6 h at 80 (1), who found that virulence and agrocin 84 V. If, Linear DNA fragments. sensitivity could be transferred during in planta crosses ofstrain 15834 and an avirulent recipient. The findings were not definitive, however, since additional differences may exist. The variant fragments present in pJ kr15834b: they recovered only one transconjugant and pre:Tn5 represent a total of 23.0 Mdal of DNA sented no physical evidence for the transfer of a compared with a total of 16.4 Mda;1 for the plasmid. In the case of F5, a derivative of 15834, viruvariant fragments in pArl5834b'::Tn5, resulting in a difference of 6.6 Mdal. Thus, the two plas- lence was transferred simultaneously with the mids remain approximnately the same size. We transfer of three donor plasmids, although viruconclude that the plasmids from avirulent lence could be localized to the 154-Mdal plasmid strains are generated by a mechanis nm which pArl5834b::Tn5. This finding differs from a results in a specific alteration of plh ismid se- study reported by Moore et al. (34). These auquences. These alterations probably ac count for thors concluded that virulence was associated with a single 110-Mdal plasmid which they identhe differences in virulence. Octopine and nopaline utilizatihDn. Viru- tified in A. rhizogenes strain A4. We isolated lent biotype 2 strains of A. tumefaciens utilize plasmid DNA from strain A4 (received from L. both octopine and nopaline, but tumo:rs incited Moore) and found three plasmids similar in size by these strains synthesize only nop?aline. In to the three plasmids in strain 15834. Restriction these strains only nopaline utilization vvas trans- analysis of A4 plasmid DNA suggests that these ferred by conjugation and transformn ation (22, plasmids are very similar to the plasmids in 33). Montoya et al. (33) concluded that octopine 15834 (White and Nester, manuscript in prepautilization is coded by chromosomal genes in ration). The results in this paper reinforce the general biotype 2 Agrobacterium strains. A. ri iizogenes 15834, a biotype 2 strain, utilizes only octopine model of plasmid-associated virulence in the (28). In a cross of F5 and A136 the totad comple- genus Agrobacterium. On the basis of physiologment of plasmids was transferred, yet tthe trans- ical and DNA homology criteria, A. rhizogenes conjugants did not acquire the ability to utilize strain 15834 is a member of the group of Agrooctopine (Table 3). This result is fui rther evi- bacterium strains designated by Keane et al.

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(19) as biotype 2. Strain 15834 differs from A. tumefaciens strains of biotype 2 only in morphology of plant growth at the infected wound site and plasmid content. The simultaneous transfer of pasmid and virulence from a biotype 2 A. rhizogenes strain (15834) to a biotype 1 strain (A136) is analogous to the transfer of oncogenicity from the biotype 2 crown gall formers (A. tumefaciens strains 223 and 27) to biotype 1 stains (41, 43). Therefore, it is reasonable to conclude that virulence in A. rhizogenes is coded by plasinid genes. In addition, our results support the conclusions of others that the classification of Agrobacterium should not be based on pathogenicity, which is predominately a property of a plasmid. The distinction between A. rhizogenes and A. tumefiaciens is further obscured by reports which indicate that the morphologies of the growths which they induce depend both on the particular host plants and on the plasmids which they contain (15, 18). Indeed, investigators have reported that some strains of A. rhizogenes incite galls on certain host plants (2). The possibility exists that hairy root induction results from the transfer and incorporation of plaid DNA into plant cell DNA. Plasmid sequences have been detected in DNAs from both unorganized tumor and teratoma tissues (9; Yang et al., in press). Hairy root may represent another tumor type. In an effort to determine whether plasmid DNA is transferred to plant cell DNA, we are attempting to establish axenic hairy root tissue in culture. Although cointegrates of R factors and Agrobacterium plasnids have been constructed (17), this is the first reported instance of the cointegration of endogenous Agrobacterium plasmids. The factors controlling this arrangement and the significance of the cointegrate form to the biology of A. rhizogenes are not clear. It is possible that pAr15834a and pAr15834b are transferred as the cointegrate. This would explain the presence of all three donor plasmids in the viruent transconjugants. Since we were unable to isolate transconjugants with only pAr15834a, it appears that pAr15834a is not transferred independently but is mobilized by pAr15834b. Interestingly, most Agrobacterium strains harbor several plasmids (7, 11, 29), and a question arises as to whether cointegration of endogenous Agrobacterium plasmids is a general phenomenon. How avirulent transconjugants are generated remains unexplained. The plasmid in avirulent strains (pArl5834'::Tn5) is similar in size to the plasmid in virulent strains but differs in several restriction endonuclease fragments. Transposa-

ble elements are known to generate mutations by either insertion or imprecise excision (24), and such effects may be responsible for the two plasmid forms. However, we have evidence that Tn5 deletions resulting form Tn5 excision are not responsible for the alterations in pAr15834b': :Tn5. Alternatively, pArl5834b': :Tn5 may result from the improper dissociation or interrupted transfer of the cointegrate plasmid, pAr15834c.
ACKNOWLEDGMENTS We 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 predoctoral fellowship and by National Researh Service Award GM07270.

LTERATURE CTED
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