- Email: [email protected]
Physical map of the vitopine Ti plasmid pTiS4
PLASMID
28, 146-156 (1992)
Physical Map of the Vitopine Ti Plasmid pTiS4 JEAN-CLAUDEG&ARD,*JEANCANADAY,* EFCN~SZEGEDI,~ HENRIDELASALLE,*ANDL~ONOTTEN*T' *InsMut de Biologic Mol&ulaire des Plantes du CNRS, UniversitC Louis Pasteur, I2 Rue du G.&n&al Zimmer, 67084 Strasbourg, France, and iResearch Institute of Viticulture and Enology, P.O. Box 25, H-6001 Kecskem& Hungary Received February
14, 1992; revised March 28, 1992
Within the Agrobacterium vitis group the vitopine strains represent a special subclass. Vitopine bacteria carry Ti plasmids with little or no homology with the well-characterized T-DNAs of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The 262-kb Ti plasmid of the vitopine strain S4 was cloned and mapped. Homology studies with the octopine Ti plasmid pTiAch5, the nopaline Ti plasmid pTiC58, and the agropine/mannopine Ri plasmid pRiHR1 identified several regions of homology. The origin of replication was localized to within 2.5 kb. 0 1992 Academic Press, Inc.
The plant diseases crown gall and hairy root disease are caused by soil bacteria of the genus Agrobacterium. These bacteria harbor large plasmids (Ti or Ri)* which play a fundamental role in the disease process. Well-defined fragments of the Ti/Ri plasmids (T-regions or T-DNAs) are transferred to plant cells during infection, become expressed, and lead to plant cell growth and to the production of small molecules, called opines, which the bacterium uses as a carbon, phosphorus, or nitrogen source (reviewed in Zambryski et al., 1989). The many different Agrobacterium strains have been divided into three groups or biovars, according to metabolic and physiological characteristics (Kerr and Panagopoulos, 1977). Most strains of the biovar III group were isolated from galls on Vi/is vinifera (grapevine). On the basis of DNA hybridization data and metabolic studies, the biotype III strains have been found to differ ’ To whom correspondence should be addressed. ’ Abbreviations used: Ti, tumour-inducing; Ri, rootinducing; IS, insertion sequence; Km, kanamycin; Sm, streptomycin; Tc, tetracycline; Ap, ampicillin; Nm, neomycin; Rif, rifampicin; T-DNA, transferred DNA; pTr, tartrate plasmid; o/c, octopine/cucumopine; II’TG, isopropylthiogalactoside; Xgal, 5-bromo-4-chloro-3-indolyl-a-D-galactoside; SSC, standard saline citrate; LB, Luria broth. 0147-619X/92
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights ofreproduction in any form resewed
from all other Agrobacterium strains (i.e., A. tumefaciens, A. rhizogenes, A. rubi, and A. radiobacter), and the biotype III group has therefore been renamed Agrobacterium vitis (Ophel and Kerr, 199 1). The reason(s) for the association of A. vitis with grapevine is still unknown but may be related to the capacity to utilize tartrate as a carbon source (Szegedi, 1985) and to the presence of a specific polygalacturonidase which causes grapevine root decay (Rodriguez-Palenzuela et al., 199 1). A. vitis can be further divided into strains with nopaline, octopine/cucumopine (o/c), and vitopine plasmids (Szegedi et al., 1988; Paulus et al., 1989a). Nopaline and o/c Ti plasmids carry T-DNA genes with strong homology to T-DNA genes of the biovar I octopine and nopaline strains (the so-called “common T-region”). A special class of strains are the vitopine strains, named after the presence of a new, as yet undefined opine in the tumors induced by such strains, which can be detected by paper electrophoresis and silver staining (Szegedi et al., 1988). Preliminary molecular studies of these strains showed that they lack DNA homology to the common Tregion of other Ti plasmids (Paulus et al., 1989a) or to the T-regions of the root-inducing plasmids (this paper). The absence of DNA homology between vitopine strains and 146
PHYSICAL MAP OF pTiS4
known T-DNAs was unsuspected and suggested that the T-region(s) of the vitopine Ti plasmids may contain a novel type of tumor gene. We therefore started a detailed study of these plasmids. Several A. vitis isolates have been identified as vitopine strains: S4, Szl, and Sz2 (Szegediet al., 1988); 268 1 (Paulus et al., 1989a); Bazzi, NW1 1, NW1 13, NW121, and NW 161 (Paulus et al., 1989b). The NW strains were originally classified as “null strains,” i.e., unable to degrade octopine or nopaline (Bien, 1988). Here we report the cloning and physical mapping of the vitopine Ti plasmid pTiS4 and a DNA homology study comparing pTiS4 with the well-known Ti plasmids pTiC58 and pTiAch5 and the Ri plasmid pRiHR1. MATERIALS
AND METHODS
Bacterial media. Media for growth of Agrobacterium and Escherichia coli, and antibiotic concentrations have been described by Leemans et al. (1983). Analytical Agrobacterium plasmid analysis. Agrobacterium plasmid content was analyzed by the method of Currier and Nester ( 1976). Ti plasmid isolation, cloning of Ti plasmid fragments, and mapping. Agrobacterium plasmid DNA was isolated according to Currier and Nester (1976). Purified plasmid DNA was partially digested with Sau3AI or EcoRI, fragments were sized on a sucrose gradient, and 15-kb fragments were ligated to BumHI-digested (for Suu3AI partials) or EcoRI-digested (for EcoRI partials) and dephosphorylated vector DNA. The pBR322derived vector pKC7 (ApR, KmR) was used for Suu3AI partials, and pUCl8 (ApR) for EcoRI partials. pKC7-derived clones can be mobilized to other Agrobacterium strains by the method of van Haute et al. (1983); pUC derivatives cannot. Escherichia coli strain NM522 or DHI (a recA strain) was transformed with the ligation mix and clones with inserts were recovered by selection on LB with IPTG, X-Gal, and ampicillin (100 mg/ liter) or (in the caseof pKC7) kanamycin (25
147
mg/liter). Other cloning procedures were according to Sambrook et al. (1990). Colony hybridization. Colony hybridization was carried out according to Maas (1983) by transfer of bacterial colonies to Whatman 540 paper, denaturation, and hybridization. Southern hybridization. DNAs digested with different enzymes were separatedon agarose, blotted onto Hybond N+ (Amersham), and hybridized overnight at 42°C with probes labeled by oligo priming to a specific activity of 0.5 pCi/ng in 5X SSC, 5X Denhardt’s solution, 0.5% SDS, 100 ggg/ml salmon sperm DNA, and 50% formamide. Filters were washed twice with 2X SSC,0.1% SDS at 42°C. Autoradiography was done at -70°C with Fuji X-ray films, using two intensifying screens. Mobilization of pBR322 derivatives to Agrobacterium. pBR322 derivatives were mobilized from E. coli to Agrobacterium by the helper strain GJ23 (van Haute et al., 1983). The helper plasmids pGJ28 (KmR) and R64drdll (TcR, SmR) were introduced into the strain carrying the pKC7 derivative (ApR, KmR) by conjugation and selection on kanamycin, tetracycline, ampicillin, and streptomycin, and the resulting strain was mated with Agrobacterium. Cointegrates between the Ti plasmid and the pKC7 derivative were selected on minimal A medium with neomycin (400 mg/liter) (Leemans et al., 1983). Conjugation in planta. In preliminary conjugation experiments on agar plates we failed to transfer vitopine plasmids from wild-type strains to a cured A. tumefaciens recipient, UBAPF2. Conjugational transfer may require induction by strain-specific opines (called conjugative opines), as in other Agrobacterium strains (Petit et al., 1978). In the case of vitopine strains, the conjugative opines have not yet been identified. Vitopine may induce conjugation but we did not have sufficient quantities ofthis compound. Therefore, conjugation was carried out in sterile S4 tumor tissue which may produce sufficient opine quantities to induce plasmid
148
GERARD
transfer. Twenty-four-hour old cultures of S4(pTiS4 : : pPM605) (KmR) and UBAPF2 (RifR) (Table 1) were suspended in 10 mM MgSO,, mixed in a 3:l ratio (donor:recipient) and 5 ~1 of this mixture was inoculated onto an approximatively 6 X 6-mm piece of sterile grapevine (cv. “Narancsizti”) tumor tissue induced by S4. After 3 to 4 days of incubation at 25°C 100 mg inoculated tissue was homogenized in 500 ~1 sterile distilled water and 50- to loo-p1 aliquots were plated onto AB minimal medium (Szegedi et al., 1988) with 1% sucrose, 100 mg/liter rifampitin, and 100 mg/liter kanamycin to selectUBAPF2(pTiS4 : : pPM605) exconjugants. Several hundred colonies were obtained. Several of these were repeatedly purified on the same medium. An exconjugant colony which produced 3-ketolactose (a characteristic marker of the biovar I recipient UBAPF2), did not grow on tartrate (tartrate utilization is a characteristic marker of biovar III strains like S4), and contained only pTiS4: :pPM605 was chosen for further study. UBAPF2(pTiS4: : pPM605) was kept on kanamycin-containing medium because pTiS4: :pPM605 was found to be slightly unstable under nonselective conditions. RESULTS
General Characteristicsof Strain S4 Among the 68 virulent grapevine Agrobacterium isolates of our collection, 9 were found to belong to the vitopine type (Paulus et al., 1989a,b). Tumors induced by these strains contain vitopine, an opine with unknown structure (Szegedi et al., 1988). Vitopine strains can easily be identified by the presence in their genome of two or three characteristic restriction fragments which hybridize with the insertion sequenceIS867 and represent individual copies of this sequence called IST4, -5, and -6 (Paulus et al., 1989b). DNA restriction patterns of total DNA of these strains show them to be very similar to each other and different from o/c and nopaline isolates. One of the vitopine strains, S4, has been isolated in Hungary from grapevine
ET AL.
(Szegedi, 1985). S4 carries four plasmids with sizes of about 260, 250, 80, and 60 kb. Of these, the 260-kb plasmid was shown to be the Ti plasmid by transfer to a plasmidless recipient and tumorigenicity tests on grapevine (seebelow). The 250-kb plasmid confers tartrate utilization to S4 and has been called pTrS4 (E. Szegedi, unpublished results). The functions of the two smaller plasmids, p3 and p4, are unknown. p3 carries one IS867 copy (ISTd), whereas pTiS4 carries two copies (IST-4 and IST-5). The other vitopine strains of our collection only carry the pTi-located IS867 copies IST-4 and IST-5.
Cloning and Mapping of pTiS4 Total plasmid DNA of S4 was partially digested with Sau3AI and cloned in pKC7 or partially digested with EcoRI and cloned in pUC 18. pTiS4-derived clones could be distinguished from other clones (carrying fragments from the other three S4 plasmids) by hybridizing 1300 clones to the Ti plasmid of the vitopine strain Sz1, which only carries the Ti plasmid (L. Otten, unpublished). Szl could not be used to prepare pTi clones, since the yields of pure pTi DNA from this strain were very low. Three hundred sixteen S4 clones hybridized to pTiSz1. Partial pTiS4 maps (contigs) were constructed by EcoRI restriction analysis of these clones. The contigs were connected by hybridization of fragments situated at their extremities to restriction digests of various selected clones. pKC7 clones were numbered from pPM601 to pPM917, and pUC clones from pPM9 18 to pPM999. In several cases,the occurrence of repeated elements on pTiS4 and pTiSz1 (in some cases shared with the other S4 plasmids, J-C. Gerard, unpublished) made it difficult to connect the contigs by hybridization to end fragments. These repeated DNAs also led to some doubt as to whether all clones were derived from the Ti plasmid. To simplify the construction of the pTiS4 map, we transferred pTiS4 into the cured biovar I strain UBAPF2, derived from C58 (Hynes et al., 1985). Selection for exconjugants was
PHYSICAL MAP OF pTiS4
149
FIG. la. Map of pTiS4. Twenty-one representative clones are shown (also listed in Table 1). Below the restriction map, the homologies with pTiC58 (stippled), pTiAch5 (crosshatched), and pRiHRI(black) are indicated. ori, origin of replication. a, b, and c indicate fragments of similar size. a represents fragments smaller than 0.5 kb. Hybridization ofEcoR1 fragments 7a, 11, 13a, 8,4, and 6 with pTiC58 and of EcoRI-6 with pRiHR1 was weak.
made possible by the introduction of a selectable marker gene into pTiS4. To this end, an earlier isolated pTiS4 clone (pPM605) which carries a kanamycin resistancegene in its vector part was recombined with pTiS4. pPM605 (Fig. la) was identified as a pTiS4 clone in the course of a search for S4 tumor genes in which a large number of S4 clones FIG. 1b. Localization of origin of replication of pTiS4 on EcoRI map. White bars correspond to pTiS4 clones were recombined with a disarmed Ti vector that replicate in GV3 101; black bars correspond to and tested on plants (J. Canaday, unpubpTiS4 clones that do not replicate in GV3 101. lished). pPM605 is a pBR322 derivative and
150
GERARD ET AL. TABLE 1 STRAINSANDPLASMIDS Characteristics
Strains Agrobacterium s4 C58 UBAPF2 UBAPF2(pTiS4: :PM605) Ach5 HRI GV3101 E. coli NM522 I
DHl GJ23 Plasmids pBR322 pKC7 pUC18 Ti/Ri clones pRiHR1 clones PLJl pLJ40 pLJ85 pLJ72 pLJ16 pLJ56 pLJ154 pLJ46 pLJ80 pLJ44 pTiC58 clones pGv347 pGV348 pGV340 pGV335 pGV342 pGV319 pGV415 pGV329 pGV304 pCV336 pGV3 11 pGV370 pTiAch5 clones pGV207 pGV140 pGV227 pGV206 pGV239 pGV205 pGv222 pGv154 pGV203 pGv211
OligiIl
Biovar III vitopine strain Biovar I nopaline strain RifR cured 08 strain nTiS4 derivative in biovar I strain Biovar I octopine strain Biovar II agropine strain Cured C58 strain (RifR)
Szegedi, 1985 de Vos et al., 1981 Hynes et al., 1985 This work Depicker et al., 1980 Jouanin, 1984 Depicker et al., 1980
Host for pUC and pBR vectors
Murray et al., University of Edinburgh Low, 1968 van Haute et al.. 1983
-
recA host for plasmids and cosmids JC2926(pGJ28)(R64drdl I) mobilizing strain Cloning vector (AmpR) Cloning vector (AmpR, KmR) Cloning vector (AmpR) Fragments BamHI fragments 15-26-27-28-40a-40b-32-8a-30a-5 32-8a-30a-5-3la-lo-30b-16 31a-lo-30b-16-34-20-la 17-8b-37-6-12-39-19b 6- 12-39-19b-4-21 12-39-19b-4-21-38-35-22-25-29-31b 38-35-22-25-29-31b-24-lb-7 7-2-l l-33-23 33-23-9-19a-3-36-13 3-36-13-14-18-15-26-27 Hind111fragments 3-3la-1 l-38-38 38- I7-34-39- 12-9-18 18-14a-27-16 16-37a-7-10 IO-15-14b 14b-19-41-22-31b (19)-4 l-22-3 1b-23-33-(2) 31b-23-33-2-30b-43-26a 1 20-42-24-6-30b-40-28-37b-25a-26b 28-37b-25a-26b-2 la-36-38a-29-2 1b 38a-29-2lb-4 Hind111fragments 33a-13-8-5 (5)-lo-22a-(18a) 25a-2 39a-9a-22b-21a-7- 11 7-l l-12-20 6 15a-9b (34a)-32-24-30a-(15b) 37a-29-30b-27a-19a-33b-38a-22c-28a 39b-26b-3 lb-30c-34b-3-37b
Bolivar et al., 1977 Rao and Rogers, 1979 Messing, 1983 Origin Jouanin, 1984
Depicker et al., 1980
de Vos et al., 198I
151
PHYSICAL MAP OF pTiS4 TABLE I-Continued Oriain
Characteristics pGV204 pGVll0 pGv153 pGV20 1 pGv120 pTiS4 EcoRI fragments pPM982 pPM824 pPM763 pPM724 pPM743 pPM706 pPM620 pPM628 pPM6 17 pPM649 pPM674 pPM716 pPM68 1 pPM737 pPM738 pPM739 pPM7 17 pPM749 pPM769 pPM756 pPM662 pPM705 pPM729 pPM777
37b-21~-19b-34c-27b-39c-21d-17-15c-23a (21d)-17-15c-23a-28b-22d-23b-28c-35-38b-39d-(14) (14)- 18c-22e-38c-36b-(1) 1 (l)-36a-4-31a-(33a) This work 46c-2-42a-30-24-19a (30)-24-19a-41a-46a-42b( 1) ( 19a)-4I a-46a-42b-( 1) (I)-46b-22-33-47a-41 b-32a-(45a) (4 1b)-32a-45a-7a-32b-(38) (32b)-38-1 l-13a-34-(5) (13a)-34-5-44-(9) (5)-44-9-43a-17-(3) (17)-3-36-47b-39-(27a) (3)-36-47b-39-27a-13b-45b-(18) (13b)-45b-18-12a-(16) (12a)-16-IO-28a-(42c) (lo)-28a-42c-14-45c-20-(29) (42c)-14-45c-20-29-(15) (45c)-20-29-15-(41~) (45c)-20-29-15-4lc-40-28b(35) (291-l 5-4 lc-40-28b-35-i9b-42d-(3 I) (15)-41c-40-28b-35-19b-42d-(31) (40)-28b-35-19b-(42d) (19b)-42d-31-45d-23-26-32c-43b-(7b) (32c)-43b-7b-32d-32e-27b-(21) (27b)-21-37-27c-25-28c-(8) (28c)-8-4-(6) (4)-6-12b-46c-(2)
Note. Numbers of clone fragments are from Jouanin, 1984(pRiHR1); Depicker et al., 1980(pTiC58); de Vos et al., 1981 (pTiAch5); and our laboratory (pTiS4). They are listed in the order corresponding to their position on the plasmid. Fragments only partially present are shown within parentheses.
therefore unstable in Agrobacterium. Recombinants between the KmR plasmid pPM605 and the wild-type Ti plasmid pTiS4 could be selected by growth on neomycin. The resulting strain, S4(pTiS4 : : pPM605), was mated with the plasmidless strain UBAPF2 in planta. Colonies were selected on minimal medium with neomycin. All exconjugants were virulent on grapevine. Whereas some contained several plasmids, others contained only one plasmid of 260 kb. One of these, UBAPF2(pTiS4: :pPM605), was used for further studies. The preliminary pTiS4 EcoRI map was checked as follows: representative pTiS4
clones covering the entire plasmid were hybridized to blots with two types of EcoRI-digestedDNA: pTiS4 : : pPM605 and total plasmid DNA of S4. All fragments known from the S4 clone maps were found on EcoRI digestsof pTiS4 and of total S4 plasmid DNA, showing that the maps were correct and that all clones belonged to the Ti plasmid. The pTiS4 map was also checked by constructing maps for SmaI, XbaI, and Not1 by determining the location of these sites with respect to the earlier determined EcoRI map and by comparing the predicted pTi fragment sizes with those obtained by digestion of purified pTiS4: : pPM605 DNA. The
152
GERARD ET AL.
a -
TL-DNA
T&DNA
-
TM1
II sea II
t
TWll
-
I I 31833P13 8 I I
4
~DPM716 -pm705 PpPM662 -pPM756
Tralll
12
20
______
occ.arc
a!w
I I 10 22a,*a 251
, 5
I
I
1%
34a sOa 37a sob 1Bb 1 24 15b , ,
9b
32
19a
29 27a
133b 28a 1 288 22~ 18 25b 2’b
__________________________________ pPM739
b
I
vi&
virA -
...
-pPMBBl
II
11
I
-~~-------------------------------------------------------------------------DPMM? DPM9B2 pPM716
ori, inc
6
II I I -ma 9a 22b21a 7
2
II
-
I
I
virG
31b 34b
37b
3
II
vi& --
virD
1
34~
virE 1
21C14b 1 Id 17
26b 3oc
27b ______
_____ am--y
m
1 D
pFw717
Tral
(L
--
a.
Trail
act
------__________________________________----------------------------------------------_ pPM720 -
act
-
38 b 1
tzs virA _-
a
II 17 34
12
I 18 14. 27 18 37 7
8
II -_-------m--l-----------
Tralll
T-DNA
virE -
virG virCvirD ---
virB
PPM= pPM777 PPFW756 Pm708
I
10
15
14b
~_-__----_--__---------I
not 19
I
2231b
I
I I
23
33
I
2
ori
a I
2h35
0
I
13 I32 I
-m
2%
5
---__----_--__-_ pPMB81 -
pPt.5732 pPM717
I
30b
I I _________________________
,Okb
FIG. 2. Regions of DNA homology between pTiS4 and three standard plasmids. Below each map the homology to pTiS4 is indicated with black bars, each carrying the number of the pTiS4 clone which hybridizes. These pTiS4 clones are also shown in Fig. la. (a) pTiAch5 Hind111map (Engler et al., 1981). Localization of different functions from Engler et al. (198 l), Koekman et al. (1982), Dessaux et al. (1987), and Melchers et al. (1990). Stippled bars, homology with pTiC58 (from Engler et al., 1981); black bars, homology with pTiS4 (this work). (b) Hind111 map of pTiC58 (Depicker et a/., 1980). Localization of different functions from Holsters et al. (1980), Engler et al. (198 l), Schardl and Kado, (1983), Hayman and Farrand (1988) and Rogowsky et al. (1990). Stippled bars: homology with pTiAch5 (Engler et al., 1981); black bars: homology with pTiS4 (this work). (c) BumHI map of pRiHR1 (Jouanin, 1984). Localization of different functions from Jouanin (1984), Nishiguchi et al. (1987) and Hirayama et al. (1988). Stippled bars, homology with pTiC58 (Jouanin, 1984);crosshatchedbars, homology with pTiB6806 (very similar to pTiAch5) (Jouanin, 1984); black bars, homology with pTiS4 (this work). vir genes indicated by virX’ are only localized approximately.
PHYSICAL MAP OF pTiS4
153
C TR-DNA
virB’ -----
virG’virC
13
virD
14
virE
18
TL-DNA
15
I 26 27 28 32
I I 30a I I
8a
TR-DNA
5
I I 31a
10
I I I I 30b 16 34 20
II
I I II .. ., ____________________-------~-----------------------_~__-_____~~-___-_~_--~-------~--------------------pPMm
--pPM743
10kb FIG.
2-Continued
pTiS4 map for EcoRI, SmaI, XbaI, and Not1 is presented in Fig. 1a together with representative clones. The total size of pTiS4 is 262 kb; the left EcoRI site of EcoRI fragment 2 was arbitrarily chosen as coordinate 0.
mined by Southern hybridization between clones of pTiS4 and clones of pTiAch5, pTiC58, and pRiHR1. The clones used in these experiments are listed in Table 1. pTiS4 clones were used to probe blots with digests from heterologous Ti plasmid clones, and Localization of the Origin of Replication those heterologous plasmid clones which hyof pTiS4 bridized to pTiS4 were used to probe blots The origin of replication of pTiS4 was local- with digests from pTiS4 clones. The results of ized by transfer of various pTiS4 clones to the these experiments are summarized in Fig. 2. cured Agrobacterium strain GV3 101 by mo- An example of the hybridization between bilization and selection on neomycin. The pTiS4 and clones of a heterologous Ti plaspTiS4 clones pPM739 and pPM7 17 yielded mid, pTiC58, is given in Fig. 3. The results NmR colonies and GV3 101(pPM739) and can be summarized as follows (pTiS4 coordiGV3 10l(pPM7 17) contained plasmids of the nates are given in parentheses). 1. Origin of replication. A 2.5-kb region in expected size. Experiments with additional EcoRI-15 (170-173) contains the origin of clones (Fig. 1b) delimited the origin ofreplicareplication of pTiS4 (see above). This region tion to a 2.5kb region in the right part of and surrounding sequences(160- 173, repreEcoRI fragment 15 (170- 173). sented by pPM739, -7 17, and -68 1) hybridize Homology Studies with Other Ti/Ri to the ori region of pTiAch5 and to regions Plasmids close to, but not part of, the ori regions of DNA homologies between pTiS4 and the pTiC58 and pRiHR1. earlier described plasmids pTiC58 (Depicker 2. Virulence region. Several regions of et al., 1980), pTiAch5 (de Vos et al., 1981), pTiS4 are homologous to virulence regions of and pRiHR1 (Jouanin, 1984) were deter- the standard plasmids. In the following cases
154
GBRARD ET AL.
fined because no sequence data exist for the standard plasmids. Several pTiS4 fragments (86-94,102-106,118-120,and 193-196)hybridize to BamHI-3 of the pRiHR1 region which contains the virA and virB genes, but not to the corresponding regions of pTiAch5 4.3 and pTiC58. Two separateregions (0- 13 and 194-198) hybridize to HindIII-22d of pTiAch5 which contains the virF gene. 3. Transfer (tra) regions. Several tra re1.6 gions have been defined on pTiC58 and pTiAch5 (Holsters et al., 1980; Engler et al., 1981), here numbered I, II, and III. pTiS4 hybridizes to these regions at the following 0.5 positions: 0- 13 (Ach5 and C58 TraI/II), 7 lFIG. 3. Example of hybridization between pTiS4: 82 (C58 TraI/II), 138- 143 (Ach5 TraI/II and :pPM605 and a heterologous Ti plasmid. A collection of C58 TraII), 160- 174 (Ach5 and C58 TraIII), pTiC58 clones (pGV3XX and pGV4XX series)covering 189-201 (Ach5 and C58 TraI/II), 210-218 88% of the Ti plasmid was used. To detect hybridization (Ach5 TraI/II and C58 TraI), 223-226 (Ach5 to the remaining regions, HindIII-digested pTiC58 was included. pGV415 is an EcoRI clone. Clones were cut TraI/II), and 248-255 (C58 TraI). Some of with HindHI. (1) size marker (0.5 and 1.6 kb); (2) these regions probably correspond to repeats pGV347; (3) pGV348; (4) pGV340; (5) pGV335; (6) located close to the putative pTiS4 tru genes. pGV342; (7) pGV319; (8) pGV415; (9) pGV329; (10) However, region 160- 174 (hybridizing to pGV304; (11) pGV336; (12) pGV311; (13) pGV370; TraIII) corresponds to the region containing (14) pTiC58. In lanes 2 to 7 and 9 to 13, the 4.3-kb band the origin of replication of pTiS4 as shown is the pBR322 vector band. In lane 8, the vector part of pGV4 15 (an EcoRI clone) remains linked to part of above (170-173). HindIII-2 of pTiC58. The weakly hybridizing band in 4. Other regions. Several fragments of lane 2 was found to be irreproducible in later experipTiS4 correspond to regions on pTiAch5, ments and may represent a partial digestion product. pTiC58, and pRiHR1 for which no functions Scale is in kb. have been defined. At the hybridization and washing stringency used here, pTiS4 did not hybridize to (a-c), the homology corresponds to sethe T-DNA regions of pTiAch5, pTiC58, or quenced open reading frames of the heterolopRiHR1, as found earlier (Paulus et al., gous plasmids, strongly suggesting that simi1989a). HindIII-10 of clone pGV335 of lar genesare present on pTiS4: (a) One region pTiC58 hybridized to pTiS4, but further anal(0- 13), covered by pPM982, hybridizes with ysis by hybridization to a HindIII/KpnI douthe virB/G region of pTiC58 (HindIII- 14a) ble digestion of pGV335 showed that this hobut not to the corresponding regions of mology is situated outside the T-region to its pTiAch5 and pRiHR1. (b) A second region left. (58-66, covered by pPM743) has homology with the virD genes of pTiC58 (HindIII-16). pPM743 also hybridizes to BarnHI- 8 of DISCUSSION pRIHR1 which contains part of the virD and virE genes.(c) A third region (70-83, covered The vitopine Ti plasmid of A. vitis strain by pPM706) has homology with the virC and virD genes of pRiHR1 (BumHI- 14) and with S4, pTiS4, represents a new type of Ti plasvir genes of pTiC58: virC (HindIII-27), virB mid. The restriction map of pTiS4 differs from all published Ti or Ri maps. DNA ho(HindIII- 18), and possibly virA (HindIII-9). Other regions of homology are lesswell de- mology studies with clones from three well-
155
PHYSICAL MAP OF pTiS4
known Ti/Ri plasmids-pTiAch5, pTiC58, and pRiHRI-also show an unusual pattern. A remarkable feature is the absence of DNA homology with known T-DNA genes under standard hybridization conditions. Recent results in our laboratory (J. Canaday, unpublished) have shown that pTiS4 carries sequenceswith low (60%) nucleotide homology to known T-DNA genes. Studies are in progress to characterize these regions. The pTiS4 map presented here and the collection of mobilizable pTiS4 clones should allow identification of the tumor genesand a study of their functional properties. Our hybridization studies have revealed a pTiS4 region (between 50 and 75) with homology to the virD genes of pTiC58 and pRiHR1, but not to the virD genes of pTiAch5. This region may be a starting point for further studies of the vir genes of pTiS4. Our studies show that the vir region of pTiS4 is lesscompact than on other Ti plasmids and covers at least 60 kb. More precise mapping studies which discriminate between the different geneswithin a vir operon are required. Whereas some sequencesof pTiS4 hybridize to vir genes of pRiHR1 and pTiC58 but not to those of pTiAch5, the S4 origin of replication hybridizes to the ori region of pTiAch5 but not to those of pRiHR1 and pTiC58. This and the fact that pTiS4 regions with homology to known Ti plasmids are interspersed with nonhomologous regions show that pTiS4 has a mosaic structure. Such a mosaic pattern was already noted for pTiC58 and pTiAch5 (by Engler et al., 1981) and has also been found for other Ti plasmids (for a recent review seeOtten et al., 1992). Preliminary experiments (J-C. Gerard, unpublished results) have shown that pTiS4 contains many different repeated sequences, some of which are also present on other Ti plasmids. These elements may have contributed to the formation of the vitopine Ti plasmids by providing recombination hot spots. The pTiS4 repeats can now be localized and their influence on the stability of pTiS4 can be studied. Subclones containing the repeats will enable us to study their distribution in
the chromosomes of vitopine strains and in the genomes of other Agrobacterium strains. Several vitopine isolates have been identified in our laboratory. pTiS4 can serve as a model for vitopine Ti plasmids and allow characterization of other vitopine Ti plasmids. Finally, sequencing of pTiS4 regions with homology to known Ti plasmids may tell us something about the way in which the different Ti plasmids were assembled. ACKNOWLEDGMENTS We thank A. Depicker, J. Schell, and M. van Montagu for C58 and Ach5 clones and L. Jouanin for HRI clones. J-C. Gtrard received a grant from the French Ministry of Research and Technology, and H. de la Salle received a grant from Transghne, Strasbourg.
REFERENCES BIEN, E. (1988). “Isolierung und Characterisierung von Agrubacterium tumefaciens biotype III, dem Erreger der Mauke an Reben.” Ph.D. thesis, University of Kaiserslautern, Germany. BOLIVAR, F., RODRIGUEZ,R. L., GREENE,P. J., BETLACH, M. C., HEYNECKER,H. L., BOYER, H. W., CROSA,J. H., AND FALKOW,S. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95-114. CURRIER,T. C., AND NESTER,E. W. (1976). Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biuchem. 76,43 l-44 1. DEPICKER,A., DE WILDE, M., DE Vos, G., DE Vos, R., VAN MONTAGU, M., AND SCHELL,J. (1980). Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Plusmid 3, 193-2 11. DESSAUX,Y., TEMPI?,J., AND FARRAND, S. K. ( 1987). Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955. Mol. Gen. Genet. 208, 301-308. DE Vos, G., DE BEUCKELEER,M., VAN MONTAGU, M., AND SCHELL, J. (1981). Restriction endonuclease mapping of the octopine tumor-inducing plasmid pTiAch5 of Agrobacterium tumefaciens. Plasmid 13, 99-105. ENGLER, G., DEPICKER, A., MAENHAUT, R., VILLARROEL,R., VAN MONTAGU, M., AND SCHELL,J. (1981). Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens. J. Mol. Biol. 152, 183-208. HAYMAN, G. T., AND FARRAND,S. K. (1988). Characterization and mapping of the agrocinopine-agrocin 84
156
GERARD ET AL.
GTTEN, L., CANADAY, J., GERARD,J. C., CROUZET,P., AND PAULUS, F. (1992). Evolution of Agrobacteria and their Ti plasmids. A review. Mol. Plant Microbe HIRAYAMA, T., MURANAKA, T., OHKAWA, H., AND Interact., in press. OKA, A. (1988). Organization and characterization of PAULUS, F., Huss, B., BONNARD,G., RID$ M., SZEthe virCD genesfrom Agrobacterium rhizogenes. Mol. GEDI, E., TEMPI?, J., PETIT, A., AND OTTEN, L. Gen. Genet. 213,229-237. (1989a). Molecular systematics of biotype III Ti plasHOLSTERS,M., SILVA, B., VAN VLIET, F., GENETELLO, mids of Agrobacterium tumefaciens. Mol. Plant MiC., DE BLOCK, M., DHAESE,P., DEPICKER,A., 1~~6, crobe Interact. 2, 64-74. D., ENGLER, G., VILLARROEL, R., VAN MONTAGU, M., AND SCHELL,J. (1980). The functional organiza- PAULUS,F., RID& M., AND OTTEN, L. (1989b). Distribution of two Agrobacterium tumefaciens insertion eltion of the nopaline Agrobacterium tumefaciens plasements in natural isolates: Evidence for stable associamid pTiC58. Plasmid 3, 212-230. tion between Ti plasmids and their bacterial hosts. HYNES,M. F., SIMON,R., AND POHLER,A. (1985). The Mol. Gen. Genet. 219, 145-152. development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhi- PETIT, A., TEMPT, J., HOLSTERS,M., VAN MONTAGU, M., AND SCHELL, J. (1978). Substrate induction of zobium meliloti plasmid to eliminate pAtC58. Plasconjugative activity of Agrobacterium tumefaciens Ti mid 13,99-105. plasmids. Nature 271, 570-57 1. JOUANIN,L. ( 1984).Restriction map of an agropine-type RAO, R. N., AND ROGERS,S. G. (1979). Plasmid pKC7: Ri plasmid and its homology with Ti plasmids. PlasA vector containing ten restriction endonuclease sites mid 12,91-102. suitable for cloning DNA segments. Gene 7,79-82. KERR, A., AND PANAGOPOULOS, C. G. (I 977). Biovars of RODRIGUEZ-PALENZUELA, P., BURR,T., AND COLLMER, Agrobacterium radiobacter var. tumefaciens and their A. (199 1). Polygalacturonidase is a virulence factor in biological control. Phytopathol. Z. 90, 172-179. Agrobacterium tumefaciens biovar 3. J. Bacterial. 173, KOEKMAN, B. P., HOOYKAAS,P. J. J., AND SCHILPER6547-6552. OORT,R. A. ( 1982).A functional map ofthe replicator ROGOWSKY,P. M., POWELL,B. S., SHIRASU,K., LIN, region of the octopine Ti plasmid. Plasmid 7, 119T. S., MOREL,P., ZYPRIAN,E. M., STECK, T. R., AND 132. KADO, C. I. (1990). Molecular characterization of the LEEMANS,J., HERNALSTEENS, J., DEBLAERE,J. P., DE vir regulon of Agrobacterium tumefaciens: Complete GREEVE,H., THIA-TOONG, L., VAN MONTAGU, M., nucleotide sequence and gene organization of the AND SCHELL,J. (1983). Genetic analysis of T-DNA 28.63-kbp regulon cloned as a single unit. Plasmid 23, 85-106. and regeneration of transformed plants. In “Molecular Genetics of the Bacteria-Plant Interaction” (A. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1990). “Molecular Cloning. A Laboratory Manual.” Ptihler, Ed.), pp. 322-330. Springer-Verlag, Berlin. Cold Spring Harbor Laboratory, Cold Spring Harbor, Low, B. (1968). Formation of merodipldids in matings NY. with a classof Ret- recipient strains of Escherichia coli SCHARDL,C. L., AND KADO, C. I. (1983). A functional K12. Proc. Natl. Acad. Sci. USA 60, 160- 164. map of the nopaline catabolism genes on the Ti plasMAAS, R. (1983). An improved colony hybridization mid of Agrobacterium tumefaciens C58. Mol. Gen. method with significantly increased sensitivity for deGenet. 191, 10-16. tection of single genes.Plasmid 10, 296-298. MELCHERS,L. S., MARONEY,M. J., DENDULK-RAS, A., SZEGEDI,E. (1985). Host range and specific L(+)-tartrate utilization of biotype III of Agrobacterium tumefaTHOMPSON,D. V., VAN VUUREN, H. A. J., SCHILPERciens. Acta Phytopathol. Acad. Scient. Hung. 20, 17OORT,R. A., AND HOOYKAAS,P. J. J. (1990). &to22. pine and nopaline strains of Agrobacterium tumefaSZEGEDI,E., CZAKO, M., OTTEN, L., AND KONCZ, Cs. ciens differ in virulence: Molecular characterization of (1988). Opines in crown gall tumours induced by biothe virF locus. Plant Mol. Biol. 14, 249-259. type III isolates of Agrobacterium tumefaciens. PhysMESSING,J. (1983). New M 13 vectors for cloning. In iol. Mol. Plant Pathol. 32, 237-247. “Methods in Enzymology” (R. Wu, L. Grossman, and VAN HAUTE, E., Joos, H., MAES, M., WARREN,G., VAN K. Moldave, Eds.), Vol. 101, pp. 20-78. Academic MONTAGU, M., AND SCHELL,J. (1983). Intergeneric Press,San Diego, CA. transfer and exchange recombination of restriction NISHIGUCHI,R., TAKANAMI, M., AND OKA, A. (1987). fragments cloned in pBR322: A novel strategy for the Characterization and sequence determination of the reversed genetics of the Ti plasmids of Agrobacterium replicator region in the hairy-root-inducing plasmid tumefaciens. EMBO J. 2, 4 1l-4 17. pRiA4b. Mol. Gen. Genet. 206, l-8. ZAMBRYSKI, P., TEMP~$J., AND SCHELL, J. (1989). OPHEL,K., AND KERR, A. (199 1).Agrobacterium vitisTransfer and function of T-DNA genes from AgroNew species for strains of Agrobacterium biotype III bacterium Ti and Ri plasmids in plants. Cell 56, 193from grapevine. Int. J. Syst. Bacterial. 40,236-24 1. 201. locus on the nopaline Ti plasmid pTiC58. J. Bacterial. 170, 1759-1767.
28, 146-156 (1992)
Physical Map of the Vitopine Ti Plasmid pTiS4 JEAN-CLAUDEG&ARD,*JEANCANADAY,* EFCN~SZEGEDI,~ HENRIDELASALLE,*ANDL~ONOTTEN*T' *InsMut de Biologic Mol&ulaire des Plantes du CNRS, UniversitC Louis Pasteur, I2 Rue du G.&n&al Zimmer, 67084 Strasbourg, France, and iResearch Institute of Viticulture and Enology, P.O. Box 25, H-6001 Kecskem& Hungary Received February
14, 1992; revised March 28, 1992
Within the Agrobacterium vitis group the vitopine strains represent a special subclass. Vitopine bacteria carry Ti plasmids with little or no homology with the well-characterized T-DNAs of Agrobacterium tumefaciens or Agrobacterium rhizogenes. The 262-kb Ti plasmid of the vitopine strain S4 was cloned and mapped. Homology studies with the octopine Ti plasmid pTiAch5, the nopaline Ti plasmid pTiC58, and the agropine/mannopine Ri plasmid pRiHR1 identified several regions of homology. The origin of replication was localized to within 2.5 kb. 0 1992 Academic Press, Inc.
The plant diseases crown gall and hairy root disease are caused by soil bacteria of the genus Agrobacterium. These bacteria harbor large plasmids (Ti or Ri)* which play a fundamental role in the disease process. Well-defined fragments of the Ti/Ri plasmids (T-regions or T-DNAs) are transferred to plant cells during infection, become expressed, and lead to plant cell growth and to the production of small molecules, called opines, which the bacterium uses as a carbon, phosphorus, or nitrogen source (reviewed in Zambryski et al., 1989). The many different Agrobacterium strains have been divided into three groups or biovars, according to metabolic and physiological characteristics (Kerr and Panagopoulos, 1977). Most strains of the biovar III group were isolated from galls on Vi/is vinifera (grapevine). On the basis of DNA hybridization data and metabolic studies, the biotype III strains have been found to differ ’ To whom correspondence should be addressed. ’ Abbreviations used: Ti, tumour-inducing; Ri, rootinducing; IS, insertion sequence; Km, kanamycin; Sm, streptomycin; Tc, tetracycline; Ap, ampicillin; Nm, neomycin; Rif, rifampicin; T-DNA, transferred DNA; pTr, tartrate plasmid; o/c, octopine/cucumopine; II’TG, isopropylthiogalactoside; Xgal, 5-bromo-4-chloro-3-indolyl-a-D-galactoside; SSC, standard saline citrate; LB, Luria broth. 0147-619X/92
$5.00
Copyright 0 1992 by Academic Press, Inc. All rights ofreproduction in any form resewed
from all other Agrobacterium strains (i.e., A. tumefaciens, A. rhizogenes, A. rubi, and A. radiobacter), and the biotype III group has therefore been renamed Agrobacterium vitis (Ophel and Kerr, 199 1). The reason(s) for the association of A. vitis with grapevine is still unknown but may be related to the capacity to utilize tartrate as a carbon source (Szegedi, 1985) and to the presence of a specific polygalacturonidase which causes grapevine root decay (Rodriguez-Palenzuela et al., 199 1). A. vitis can be further divided into strains with nopaline, octopine/cucumopine (o/c), and vitopine plasmids (Szegedi et al., 1988; Paulus et al., 1989a). Nopaline and o/c Ti plasmids carry T-DNA genes with strong homology to T-DNA genes of the biovar I octopine and nopaline strains (the so-called “common T-region”). A special class of strains are the vitopine strains, named after the presence of a new, as yet undefined opine in the tumors induced by such strains, which can be detected by paper electrophoresis and silver staining (Szegedi et al., 1988). Preliminary molecular studies of these strains showed that they lack DNA homology to the common Tregion of other Ti plasmids (Paulus et al., 1989a) or to the T-regions of the root-inducing plasmids (this paper). The absence of DNA homology between vitopine strains and 146
PHYSICAL MAP OF pTiS4
known T-DNAs was unsuspected and suggested that the T-region(s) of the vitopine Ti plasmids may contain a novel type of tumor gene. We therefore started a detailed study of these plasmids. Several A. vitis isolates have been identified as vitopine strains: S4, Szl, and Sz2 (Szegediet al., 1988); 268 1 (Paulus et al., 1989a); Bazzi, NW1 1, NW1 13, NW121, and NW 161 (Paulus et al., 1989b). The NW strains were originally classified as “null strains,” i.e., unable to degrade octopine or nopaline (Bien, 1988). Here we report the cloning and physical mapping of the vitopine Ti plasmid pTiS4 and a DNA homology study comparing pTiS4 with the well-known Ti plasmids pTiC58 and pTiAch5 and the Ri plasmid pRiHR1. MATERIALS
AND METHODS
Bacterial media. Media for growth of Agrobacterium and Escherichia coli, and antibiotic concentrations have been described by Leemans et al. (1983). Analytical Agrobacterium plasmid analysis. Agrobacterium plasmid content was analyzed by the method of Currier and Nester ( 1976). Ti plasmid isolation, cloning of Ti plasmid fragments, and mapping. Agrobacterium plasmid DNA was isolated according to Currier and Nester (1976). Purified plasmid DNA was partially digested with Sau3AI or EcoRI, fragments were sized on a sucrose gradient, and 15-kb fragments were ligated to BumHI-digested (for Suu3AI partials) or EcoRI-digested (for EcoRI partials) and dephosphorylated vector DNA. The pBR322derived vector pKC7 (ApR, KmR) was used for Suu3AI partials, and pUCl8 (ApR) for EcoRI partials. pKC7-derived clones can be mobilized to other Agrobacterium strains by the method of van Haute et al. (1983); pUC derivatives cannot. Escherichia coli strain NM522 or DHI (a recA strain) was transformed with the ligation mix and clones with inserts were recovered by selection on LB with IPTG, X-Gal, and ampicillin (100 mg/ liter) or (in the caseof pKC7) kanamycin (25
147
mg/liter). Other cloning procedures were according to Sambrook et al. (1990). Colony hybridization. Colony hybridization was carried out according to Maas (1983) by transfer of bacterial colonies to Whatman 540 paper, denaturation, and hybridization. Southern hybridization. DNAs digested with different enzymes were separatedon agarose, blotted onto Hybond N+ (Amersham), and hybridized overnight at 42°C with probes labeled by oligo priming to a specific activity of 0.5 pCi/ng in 5X SSC, 5X Denhardt’s solution, 0.5% SDS, 100 ggg/ml salmon sperm DNA, and 50% formamide. Filters were washed twice with 2X SSC,0.1% SDS at 42°C. Autoradiography was done at -70°C with Fuji X-ray films, using two intensifying screens. Mobilization of pBR322 derivatives to Agrobacterium. pBR322 derivatives were mobilized from E. coli to Agrobacterium by the helper strain GJ23 (van Haute et al., 1983). The helper plasmids pGJ28 (KmR) and R64drdll (TcR, SmR) were introduced into the strain carrying the pKC7 derivative (ApR, KmR) by conjugation and selection on kanamycin, tetracycline, ampicillin, and streptomycin, and the resulting strain was mated with Agrobacterium. Cointegrates between the Ti plasmid and the pKC7 derivative were selected on minimal A medium with neomycin (400 mg/liter) (Leemans et al., 1983). Conjugation in planta. In preliminary conjugation experiments on agar plates we failed to transfer vitopine plasmids from wild-type strains to a cured A. tumefaciens recipient, UBAPF2. Conjugational transfer may require induction by strain-specific opines (called conjugative opines), as in other Agrobacterium strains (Petit et al., 1978). In the case of vitopine strains, the conjugative opines have not yet been identified. Vitopine may induce conjugation but we did not have sufficient quantities ofthis compound. Therefore, conjugation was carried out in sterile S4 tumor tissue which may produce sufficient opine quantities to induce plasmid
148
GERARD
transfer. Twenty-four-hour old cultures of S4(pTiS4 : : pPM605) (KmR) and UBAPF2 (RifR) (Table 1) were suspended in 10 mM MgSO,, mixed in a 3:l ratio (donor:recipient) and 5 ~1 of this mixture was inoculated onto an approximatively 6 X 6-mm piece of sterile grapevine (cv. “Narancsizti”) tumor tissue induced by S4. After 3 to 4 days of incubation at 25°C 100 mg inoculated tissue was homogenized in 500 ~1 sterile distilled water and 50- to loo-p1 aliquots were plated onto AB minimal medium (Szegedi et al., 1988) with 1% sucrose, 100 mg/liter rifampitin, and 100 mg/liter kanamycin to selectUBAPF2(pTiS4 : : pPM605) exconjugants. Several hundred colonies were obtained. Several of these were repeatedly purified on the same medium. An exconjugant colony which produced 3-ketolactose (a characteristic marker of the biovar I recipient UBAPF2), did not grow on tartrate (tartrate utilization is a characteristic marker of biovar III strains like S4), and contained only pTiS4: :pPM605 was chosen for further study. UBAPF2(pTiS4: : pPM605) was kept on kanamycin-containing medium because pTiS4: :pPM605 was found to be slightly unstable under nonselective conditions. RESULTS
General Characteristicsof Strain S4 Among the 68 virulent grapevine Agrobacterium isolates of our collection, 9 were found to belong to the vitopine type (Paulus et al., 1989a,b). Tumors induced by these strains contain vitopine, an opine with unknown structure (Szegedi et al., 1988). Vitopine strains can easily be identified by the presence in their genome of two or three characteristic restriction fragments which hybridize with the insertion sequenceIS867 and represent individual copies of this sequence called IST4, -5, and -6 (Paulus et al., 1989b). DNA restriction patterns of total DNA of these strains show them to be very similar to each other and different from o/c and nopaline isolates. One of the vitopine strains, S4, has been isolated in Hungary from grapevine
ET AL.
(Szegedi, 1985). S4 carries four plasmids with sizes of about 260, 250, 80, and 60 kb. Of these, the 260-kb plasmid was shown to be the Ti plasmid by transfer to a plasmidless recipient and tumorigenicity tests on grapevine (seebelow). The 250-kb plasmid confers tartrate utilization to S4 and has been called pTrS4 (E. Szegedi, unpublished results). The functions of the two smaller plasmids, p3 and p4, are unknown. p3 carries one IS867 copy (ISTd), whereas pTiS4 carries two copies (IST-4 and IST-5). The other vitopine strains of our collection only carry the pTi-located IS867 copies IST-4 and IST-5.
Cloning and Mapping of pTiS4 Total plasmid DNA of S4 was partially digested with Sau3AI and cloned in pKC7 or partially digested with EcoRI and cloned in pUC 18. pTiS4-derived clones could be distinguished from other clones (carrying fragments from the other three S4 plasmids) by hybridizing 1300 clones to the Ti plasmid of the vitopine strain Sz1, which only carries the Ti plasmid (L. Otten, unpublished). Szl could not be used to prepare pTi clones, since the yields of pure pTi DNA from this strain were very low. Three hundred sixteen S4 clones hybridized to pTiSz1. Partial pTiS4 maps (contigs) were constructed by EcoRI restriction analysis of these clones. The contigs were connected by hybridization of fragments situated at their extremities to restriction digests of various selected clones. pKC7 clones were numbered from pPM601 to pPM917, and pUC clones from pPM9 18 to pPM999. In several cases,the occurrence of repeated elements on pTiS4 and pTiSz1 (in some cases shared with the other S4 plasmids, J-C. Gerard, unpublished) made it difficult to connect the contigs by hybridization to end fragments. These repeated DNAs also led to some doubt as to whether all clones were derived from the Ti plasmid. To simplify the construction of the pTiS4 map, we transferred pTiS4 into the cured biovar I strain UBAPF2, derived from C58 (Hynes et al., 1985). Selection for exconjugants was
PHYSICAL MAP OF pTiS4
149
FIG. la. Map of pTiS4. Twenty-one representative clones are shown (also listed in Table 1). Below the restriction map, the homologies with pTiC58 (stippled), pTiAch5 (crosshatched), and pRiHRI(black) are indicated. ori, origin of replication. a, b, and c indicate fragments of similar size. a represents fragments smaller than 0.5 kb. Hybridization ofEcoR1 fragments 7a, 11, 13a, 8,4, and 6 with pTiC58 and of EcoRI-6 with pRiHR1 was weak.
made possible by the introduction of a selectable marker gene into pTiS4. To this end, an earlier isolated pTiS4 clone (pPM605) which carries a kanamycin resistancegene in its vector part was recombined with pTiS4. pPM605 (Fig. la) was identified as a pTiS4 clone in the course of a search for S4 tumor genes in which a large number of S4 clones FIG. 1b. Localization of origin of replication of pTiS4 on EcoRI map. White bars correspond to pTiS4 clones were recombined with a disarmed Ti vector that replicate in GV3 101; black bars correspond to and tested on plants (J. Canaday, unpubpTiS4 clones that do not replicate in GV3 101. lished). pPM605 is a pBR322 derivative and
150
GERARD ET AL. TABLE 1 STRAINSANDPLASMIDS Characteristics
Strains Agrobacterium s4 C58 UBAPF2 UBAPF2(pTiS4: :PM605) Ach5 HRI GV3101 E. coli NM522 I
DHl GJ23 Plasmids pBR322 pKC7 pUC18 Ti/Ri clones pRiHR1 clones PLJl pLJ40 pLJ85 pLJ72 pLJ16 pLJ56 pLJ154 pLJ46 pLJ80 pLJ44 pTiC58 clones pGv347 pGV348 pGV340 pGV335 pGV342 pGV319 pGV415 pGV329 pGV304 pCV336 pGV3 11 pGV370 pTiAch5 clones pGV207 pGV140 pGV227 pGV206 pGV239 pGV205 pGv222 pGv154 pGV203 pGv211
OligiIl
Biovar III vitopine strain Biovar I nopaline strain RifR cured 08 strain nTiS4 derivative in biovar I strain Biovar I octopine strain Biovar II agropine strain Cured C58 strain (RifR)
Szegedi, 1985 de Vos et al., 1981 Hynes et al., 1985 This work Depicker et al., 1980 Jouanin, 1984 Depicker et al., 1980
Host for pUC and pBR vectors
Murray et al., University of Edinburgh Low, 1968 van Haute et al.. 1983
-
recA host for plasmids and cosmids JC2926(pGJ28)(R64drdl I) mobilizing strain Cloning vector (AmpR) Cloning vector (AmpR, KmR) Cloning vector (AmpR) Fragments BamHI fragments 15-26-27-28-40a-40b-32-8a-30a-5 32-8a-30a-5-3la-lo-30b-16 31a-lo-30b-16-34-20-la 17-8b-37-6-12-39-19b 6- 12-39-19b-4-21 12-39-19b-4-21-38-35-22-25-29-31b 38-35-22-25-29-31b-24-lb-7 7-2-l l-33-23 33-23-9-19a-3-36-13 3-36-13-14-18-15-26-27 Hind111fragments 3-3la-1 l-38-38 38- I7-34-39- 12-9-18 18-14a-27-16 16-37a-7-10 IO-15-14b 14b-19-41-22-31b (19)-4 l-22-3 1b-23-33-(2) 31b-23-33-2-30b-43-26a 1 20-42-24-6-30b-40-28-37b-25a-26b 28-37b-25a-26b-2 la-36-38a-29-2 1b 38a-29-2lb-4 Hind111fragments 33a-13-8-5 (5)-lo-22a-(18a) 25a-2 39a-9a-22b-21a-7- 11 7-l l-12-20 6 15a-9b (34a)-32-24-30a-(15b) 37a-29-30b-27a-19a-33b-38a-22c-28a 39b-26b-3 lb-30c-34b-3-37b
Bolivar et al., 1977 Rao and Rogers, 1979 Messing, 1983 Origin Jouanin, 1984
Depicker et al., 1980
de Vos et al., 198I
151
PHYSICAL MAP OF pTiS4 TABLE I-Continued Oriain
Characteristics pGV204 pGVll0 pGv153 pGV20 1 pGv120 pTiS4 EcoRI fragments pPM982 pPM824 pPM763 pPM724 pPM743 pPM706 pPM620 pPM628 pPM6 17 pPM649 pPM674 pPM716 pPM68 1 pPM737 pPM738 pPM739 pPM7 17 pPM749 pPM769 pPM756 pPM662 pPM705 pPM729 pPM777
37b-21~-19b-34c-27b-39c-21d-17-15c-23a (21d)-17-15c-23a-28b-22d-23b-28c-35-38b-39d-(14) (14)- 18c-22e-38c-36b-(1) 1 (l)-36a-4-31a-(33a) This work 46c-2-42a-30-24-19a (30)-24-19a-41a-46a-42b( 1) ( 19a)-4I a-46a-42b-( 1) (I)-46b-22-33-47a-41 b-32a-(45a) (4 1b)-32a-45a-7a-32b-(38) (32b)-38-1 l-13a-34-(5) (13a)-34-5-44-(9) (5)-44-9-43a-17-(3) (17)-3-36-47b-39-(27a) (3)-36-47b-39-27a-13b-45b-(18) (13b)-45b-18-12a-(16) (12a)-16-IO-28a-(42c) (lo)-28a-42c-14-45c-20-(29) (42c)-14-45c-20-29-(15) (45c)-20-29-15-(41~) (45c)-20-29-15-4lc-40-28b(35) (291-l 5-4 lc-40-28b-35-i9b-42d-(3 I) (15)-41c-40-28b-35-19b-42d-(31) (40)-28b-35-19b-(42d) (19b)-42d-31-45d-23-26-32c-43b-(7b) (32c)-43b-7b-32d-32e-27b-(21) (27b)-21-37-27c-25-28c-(8) (28c)-8-4-(6) (4)-6-12b-46c-(2)
Note. Numbers of clone fragments are from Jouanin, 1984(pRiHR1); Depicker et al., 1980(pTiC58); de Vos et al., 1981 (pTiAch5); and our laboratory (pTiS4). They are listed in the order corresponding to their position on the plasmid. Fragments only partially present are shown within parentheses.
therefore unstable in Agrobacterium. Recombinants between the KmR plasmid pPM605 and the wild-type Ti plasmid pTiS4 could be selected by growth on neomycin. The resulting strain, S4(pTiS4 : : pPM605), was mated with the plasmidless strain UBAPF2 in planta. Colonies were selected on minimal medium with neomycin. All exconjugants were virulent on grapevine. Whereas some contained several plasmids, others contained only one plasmid of 260 kb. One of these, UBAPF2(pTiS4: :pPM605), was used for further studies. The preliminary pTiS4 EcoRI map was checked as follows: representative pTiS4
clones covering the entire plasmid were hybridized to blots with two types of EcoRI-digestedDNA: pTiS4 : : pPM605 and total plasmid DNA of S4. All fragments known from the S4 clone maps were found on EcoRI digestsof pTiS4 and of total S4 plasmid DNA, showing that the maps were correct and that all clones belonged to the Ti plasmid. The pTiS4 map was also checked by constructing maps for SmaI, XbaI, and Not1 by determining the location of these sites with respect to the earlier determined EcoRI map and by comparing the predicted pTi fragment sizes with those obtained by digestion of purified pTiS4: : pPM605 DNA. The
152
GERARD ET AL.
a -
TL-DNA
T&DNA
-
TM1
II sea II
t
TWll
-
I I 31833P13 8 I I
4
~DPM716 -pm705 PpPM662 -pPM756
Tralll
12
20
______
occ.arc
a!w
I I 10 22a,*a 251
, 5
I
I
1%
34a sOa 37a sob 1Bb 1 24 15b , ,
9b
32
19a
29 27a
133b 28a 1 288 22~ 18 25b 2’b
__________________________________ pPM739
b
I
vi&
virA -
...
-pPMBBl
II
11
I
-~~-------------------------------------------------------------------------DPMM? DPM9B2 pPM716
ori, inc
6
II I I -ma 9a 22b21a 7
2
II
-
I
I
virG
31b 34b
37b
3
II
vi& --
virD
1
34~
virE 1
21C14b 1 Id 17
26b 3oc
27b ______
_____ am--y
m
1 D
pFw717
Tral
(L
--
a.
Trail
act
------__________________________________----------------------------------------------_ pPM720 -
act
-
38 b 1
tzs virA _-
a
II 17 34
12
I 18 14. 27 18 37 7
8
II -_-------m--l-----------
Tralll
T-DNA
virE -
virG virCvirD ---
virB
PPM= pPM777 PPFW756 Pm708
I
10
15
14b
~_-__----_--__---------I
not 19
I
2231b
I
I I
23
33
I
2
ori
a I
2h35
0
I
13 I32 I
-m
2%
5
---__----_--__-_ pPMB81 -
pPt.5732 pPM717
I
30b
I I _________________________
,Okb
FIG. 2. Regions of DNA homology between pTiS4 and three standard plasmids. Below each map the homology to pTiS4 is indicated with black bars, each carrying the number of the pTiS4 clone which hybridizes. These pTiS4 clones are also shown in Fig. la. (a) pTiAch5 Hind111map (Engler et al., 1981). Localization of different functions from Engler et al. (198 l), Koekman et al. (1982), Dessaux et al. (1987), and Melchers et al. (1990). Stippled bars, homology with pTiC58 (from Engler et al., 1981); black bars, homology with pTiS4 (this work). (b) Hind111 map of pTiC58 (Depicker et a/., 1980). Localization of different functions from Holsters et al. (1980), Engler et al. (198 l), Schardl and Kado, (1983), Hayman and Farrand (1988) and Rogowsky et al. (1990). Stippled bars: homology with pTiAch5 (Engler et al., 1981); black bars: homology with pTiS4 (this work). (c) BumHI map of pRiHR1 (Jouanin, 1984). Localization of different functions from Jouanin (1984), Nishiguchi et al. (1987) and Hirayama et al. (1988). Stippled bars, homology with pTiC58 (Jouanin, 1984);crosshatchedbars, homology with pTiB6806 (very similar to pTiAch5) (Jouanin, 1984); black bars, homology with pTiS4 (this work). vir genes indicated by virX’ are only localized approximately.
PHYSICAL MAP OF pTiS4
153
C TR-DNA
virB’ -----
virG’virC
13
virD
14
virE
18
TL-DNA
15
I 26 27 28 32
I I 30a I I
8a
TR-DNA
5
I I 31a
10
I I I I 30b 16 34 20
II
I I II .. ., ____________________-------~-----------------------_~__-_____~~-___-_~_--~-------~--------------------pPMm
--pPM743
10kb FIG.
2-Continued
pTiS4 map for EcoRI, SmaI, XbaI, and Not1 is presented in Fig. 1a together with representative clones. The total size of pTiS4 is 262 kb; the left EcoRI site of EcoRI fragment 2 was arbitrarily chosen as coordinate 0.
mined by Southern hybridization between clones of pTiS4 and clones of pTiAch5, pTiC58, and pRiHR1. The clones used in these experiments are listed in Table 1. pTiS4 clones were used to probe blots with digests from heterologous Ti plasmid clones, and Localization of the Origin of Replication those heterologous plasmid clones which hyof pTiS4 bridized to pTiS4 were used to probe blots The origin of replication of pTiS4 was local- with digests from pTiS4 clones. The results of ized by transfer of various pTiS4 clones to the these experiments are summarized in Fig. 2. cured Agrobacterium strain GV3 101 by mo- An example of the hybridization between bilization and selection on neomycin. The pTiS4 and clones of a heterologous Ti plaspTiS4 clones pPM739 and pPM7 17 yielded mid, pTiC58, is given in Fig. 3. The results NmR colonies and GV3 101(pPM739) and can be summarized as follows (pTiS4 coordiGV3 10l(pPM7 17) contained plasmids of the nates are given in parentheses). 1. Origin of replication. A 2.5-kb region in expected size. Experiments with additional EcoRI-15 (170-173) contains the origin of clones (Fig. 1b) delimited the origin ofreplicareplication of pTiS4 (see above). This region tion to a 2.5kb region in the right part of and surrounding sequences(160- 173, repreEcoRI fragment 15 (170- 173). sented by pPM739, -7 17, and -68 1) hybridize Homology Studies with Other Ti/Ri to the ori region of pTiAch5 and to regions Plasmids close to, but not part of, the ori regions of DNA homologies between pTiS4 and the pTiC58 and pRiHR1. earlier described plasmids pTiC58 (Depicker 2. Virulence region. Several regions of et al., 1980), pTiAch5 (de Vos et al., 1981), pTiS4 are homologous to virulence regions of and pRiHR1 (Jouanin, 1984) were deter- the standard plasmids. In the following cases
154
GBRARD ET AL.
fined because no sequence data exist for the standard plasmids. Several pTiS4 fragments (86-94,102-106,118-120,and 193-196)hybridize to BamHI-3 of the pRiHR1 region which contains the virA and virB genes, but not to the corresponding regions of pTiAch5 4.3 and pTiC58. Two separateregions (0- 13 and 194-198) hybridize to HindIII-22d of pTiAch5 which contains the virF gene. 3. Transfer (tra) regions. Several tra re1.6 gions have been defined on pTiC58 and pTiAch5 (Holsters et al., 1980; Engler et al., 1981), here numbered I, II, and III. pTiS4 hybridizes to these regions at the following 0.5 positions: 0- 13 (Ach5 and C58 TraI/II), 7 lFIG. 3. Example of hybridization between pTiS4: 82 (C58 TraI/II), 138- 143 (Ach5 TraI/II and :pPM605 and a heterologous Ti plasmid. A collection of C58 TraII), 160- 174 (Ach5 and C58 TraIII), pTiC58 clones (pGV3XX and pGV4XX series)covering 189-201 (Ach5 and C58 TraI/II), 210-218 88% of the Ti plasmid was used. To detect hybridization (Ach5 TraI/II and C58 TraI), 223-226 (Ach5 to the remaining regions, HindIII-digested pTiC58 was included. pGV415 is an EcoRI clone. Clones were cut TraI/II), and 248-255 (C58 TraI). Some of with HindHI. (1) size marker (0.5 and 1.6 kb); (2) these regions probably correspond to repeats pGV347; (3) pGV348; (4) pGV340; (5) pGV335; (6) located close to the putative pTiS4 tru genes. pGV342; (7) pGV319; (8) pGV415; (9) pGV329; (10) However, region 160- 174 (hybridizing to pGV304; (11) pGV336; (12) pGV311; (13) pGV370; TraIII) corresponds to the region containing (14) pTiC58. In lanes 2 to 7 and 9 to 13, the 4.3-kb band the origin of replication of pTiS4 as shown is the pBR322 vector band. In lane 8, the vector part of pGV4 15 (an EcoRI clone) remains linked to part of above (170-173). HindIII-2 of pTiC58. The weakly hybridizing band in 4. Other regions. Several fragments of lane 2 was found to be irreproducible in later experipTiS4 correspond to regions on pTiAch5, ments and may represent a partial digestion product. pTiC58, and pRiHR1 for which no functions Scale is in kb. have been defined. At the hybridization and washing stringency used here, pTiS4 did not hybridize to (a-c), the homology corresponds to sethe T-DNA regions of pTiAch5, pTiC58, or quenced open reading frames of the heterolopRiHR1, as found earlier (Paulus et al., gous plasmids, strongly suggesting that simi1989a). HindIII-10 of clone pGV335 of lar genesare present on pTiS4: (a) One region pTiC58 hybridized to pTiS4, but further anal(0- 13), covered by pPM982, hybridizes with ysis by hybridization to a HindIII/KpnI douthe virB/G region of pTiC58 (HindIII- 14a) ble digestion of pGV335 showed that this hobut not to the corresponding regions of mology is situated outside the T-region to its pTiAch5 and pRiHR1. (b) A second region left. (58-66, covered by pPM743) has homology with the virD genes of pTiC58 (HindIII-16). pPM743 also hybridizes to BarnHI- 8 of DISCUSSION pRIHR1 which contains part of the virD and virE genes.(c) A third region (70-83, covered The vitopine Ti plasmid of A. vitis strain by pPM706) has homology with the virC and virD genes of pRiHR1 (BumHI- 14) and with S4, pTiS4, represents a new type of Ti plasvir genes of pTiC58: virC (HindIII-27), virB mid. The restriction map of pTiS4 differs from all published Ti or Ri maps. DNA ho(HindIII- 18), and possibly virA (HindIII-9). Other regions of homology are lesswell de- mology studies with clones from three well-
155
PHYSICAL MAP OF pTiS4
known Ti/Ri plasmids-pTiAch5, pTiC58, and pRiHRI-also show an unusual pattern. A remarkable feature is the absence of DNA homology with known T-DNA genes under standard hybridization conditions. Recent results in our laboratory (J. Canaday, unpublished) have shown that pTiS4 carries sequenceswith low (60%) nucleotide homology to known T-DNA genes. Studies are in progress to characterize these regions. The pTiS4 map presented here and the collection of mobilizable pTiS4 clones should allow identification of the tumor genesand a study of their functional properties. Our hybridization studies have revealed a pTiS4 region (between 50 and 75) with homology to the virD genes of pTiC58 and pRiHR1, but not to the virD genes of pTiAch5. This region may be a starting point for further studies of the vir genes of pTiS4. Our studies show that the vir region of pTiS4 is lesscompact than on other Ti plasmids and covers at least 60 kb. More precise mapping studies which discriminate between the different geneswithin a vir operon are required. Whereas some sequencesof pTiS4 hybridize to vir genes of pRiHR1 and pTiC58 but not to those of pTiAch5, the S4 origin of replication hybridizes to the ori region of pTiAch5 but not to those of pRiHR1 and pTiC58. This and the fact that pTiS4 regions with homology to known Ti plasmids are interspersed with nonhomologous regions show that pTiS4 has a mosaic structure. Such a mosaic pattern was already noted for pTiC58 and pTiAch5 (by Engler et al., 1981) and has also been found for other Ti plasmids (for a recent review seeOtten et al., 1992). Preliminary experiments (J-C. Gerard, unpublished results) have shown that pTiS4 contains many different repeated sequences, some of which are also present on other Ti plasmids. These elements may have contributed to the formation of the vitopine Ti plasmids by providing recombination hot spots. The pTiS4 repeats can now be localized and their influence on the stability of pTiS4 can be studied. Subclones containing the repeats will enable us to study their distribution in
the chromosomes of vitopine strains and in the genomes of other Agrobacterium strains. Several vitopine isolates have been identified in our laboratory. pTiS4 can serve as a model for vitopine Ti plasmids and allow characterization of other vitopine Ti plasmids. Finally, sequencing of pTiS4 regions with homology to known Ti plasmids may tell us something about the way in which the different Ti plasmids were assembled. ACKNOWLEDGMENTS We thank A. Depicker, J. Schell, and M. van Montagu for C58 and Ach5 clones and L. Jouanin for HRI clones. J-C. Gtrard received a grant from the French Ministry of Research and Technology, and H. de la Salle received a grant from Transghne, Strasbourg.
REFERENCES BIEN, E. (1988). “Isolierung und Characterisierung von Agrubacterium tumefaciens biotype III, dem Erreger der Mauke an Reben.” Ph.D. thesis, University of Kaiserslautern, Germany. BOLIVAR, F., RODRIGUEZ,R. L., GREENE,P. J., BETLACH, M. C., HEYNECKER,H. L., BOYER, H. W., CROSA,J. H., AND FALKOW,S. (1977). Construction and characterization of new cloning vehicles. II. A multipurpose cloning system. Gene 2, 95-114. CURRIER,T. C., AND NESTER,E. W. (1976). Isolation of covalently closed circular DNA of high molecular weight from bacteria. Anal. Biuchem. 76,43 l-44 1. DEPICKER,A., DE WILDE, M., DE Vos, G., DE Vos, R., VAN MONTAGU, M., AND SCHELL,J. (1980). Molecular cloning of overlapping segments of the nopaline Ti-plasmid pTiC58 as a means to restriction endonuclease mapping. Plusmid 3, 193-2 11. DESSAUX,Y., TEMPI?,J., AND FARRAND, S. K. ( 1987). Genetic analysis of mannityl opine catabolism in octopine-type Agrobacterium tumefaciens strain 15955. Mol. Gen. Genet. 208, 301-308. DE Vos, G., DE BEUCKELEER,M., VAN MONTAGU, M., AND SCHELL, J. (1981). Restriction endonuclease mapping of the octopine tumor-inducing plasmid pTiAch5 of Agrobacterium tumefaciens. Plasmid 13, 99-105. ENGLER, G., DEPICKER, A., MAENHAUT, R., VILLARROEL,R., VAN MONTAGU, M., AND SCHELL,J. (1981). Physical mapping of DNA base sequence homologies between an octopine and a nopaline Ti plasmid of Agrobacterium tumefaciens. J. Mol. Biol. 152, 183-208. HAYMAN, G. T., AND FARRAND,S. K. (1988). Characterization and mapping of the agrocinopine-agrocin 84
156
GERARD ET AL.
GTTEN, L., CANADAY, J., GERARD,J. C., CROUZET,P., AND PAULUS, F. (1992). Evolution of Agrobacteria and their Ti plasmids. A review. Mol. Plant Microbe HIRAYAMA, T., MURANAKA, T., OHKAWA, H., AND Interact., in press. OKA, A. (1988). Organization and characterization of PAULUS, F., Huss, B., BONNARD,G., RID$ M., SZEthe virCD genesfrom Agrobacterium rhizogenes. Mol. GEDI, E., TEMPI?, J., PETIT, A., AND OTTEN, L. Gen. Genet. 213,229-237. (1989a). Molecular systematics of biotype III Ti plasHOLSTERS,M., SILVA, B., VAN VLIET, F., GENETELLO, mids of Agrobacterium tumefaciens. Mol. Plant MiC., DE BLOCK, M., DHAESE,P., DEPICKER,A., 1~~6, crobe Interact. 2, 64-74. D., ENGLER, G., VILLARROEL, R., VAN MONTAGU, M., AND SCHELL,J. (1980). The functional organiza- PAULUS,F., RID& M., AND OTTEN, L. (1989b). Distribution of two Agrobacterium tumefaciens insertion eltion of the nopaline Agrobacterium tumefaciens plasements in natural isolates: Evidence for stable associamid pTiC58. Plasmid 3, 212-230. tion between Ti plasmids and their bacterial hosts. HYNES,M. F., SIMON,R., AND POHLER,A. (1985). The Mol. Gen. Genet. 219, 145-152. development of plasmid-free strains of Agrobacterium tumefaciens by using incompatibility with a Rhi- PETIT, A., TEMPT, J., HOLSTERS,M., VAN MONTAGU, M., AND SCHELL, J. (1978). Substrate induction of zobium meliloti plasmid to eliminate pAtC58. Plasconjugative activity of Agrobacterium tumefaciens Ti mid 13,99-105. plasmids. Nature 271, 570-57 1. JOUANIN,L. ( 1984).Restriction map of an agropine-type RAO, R. N., AND ROGERS,S. G. (1979). Plasmid pKC7: Ri plasmid and its homology with Ti plasmids. PlasA vector containing ten restriction endonuclease sites mid 12,91-102. suitable for cloning DNA segments. Gene 7,79-82. KERR, A., AND PANAGOPOULOS, C. G. (I 977). Biovars of RODRIGUEZ-PALENZUELA, P., BURR,T., AND COLLMER, Agrobacterium radiobacter var. tumefaciens and their A. (199 1). Polygalacturonidase is a virulence factor in biological control. Phytopathol. Z. 90, 172-179. Agrobacterium tumefaciens biovar 3. J. Bacterial. 173, KOEKMAN, B. P., HOOYKAAS,P. J. J., AND SCHILPER6547-6552. OORT,R. A. ( 1982).A functional map ofthe replicator ROGOWSKY,P. M., POWELL,B. S., SHIRASU,K., LIN, region of the octopine Ti plasmid. Plasmid 7, 119T. S., MOREL,P., ZYPRIAN,E. M., STECK, T. R., AND 132. KADO, C. I. (1990). Molecular characterization of the LEEMANS,J., HERNALSTEENS, J., DEBLAERE,J. P., DE vir regulon of Agrobacterium tumefaciens: Complete GREEVE,H., THIA-TOONG, L., VAN MONTAGU, M., nucleotide sequence and gene organization of the AND SCHELL,J. (1983). Genetic analysis of T-DNA 28.63-kbp regulon cloned as a single unit. Plasmid 23, 85-106. and regeneration of transformed plants. In “Molecular Genetics of the Bacteria-Plant Interaction” (A. SAMBROOK, J., FRITSCH, E. F., AND MANIATIS, T. (1990). “Molecular Cloning. A Laboratory Manual.” Ptihler, Ed.), pp. 322-330. Springer-Verlag, Berlin. Cold Spring Harbor Laboratory, Cold Spring Harbor, Low, B. (1968). Formation of merodipldids in matings NY. with a classof Ret- recipient strains of Escherichia coli SCHARDL,C. L., AND KADO, C. I. (1983). A functional K12. Proc. Natl. Acad. Sci. USA 60, 160- 164. map of the nopaline catabolism genes on the Ti plasMAAS, R. (1983). An improved colony hybridization mid of Agrobacterium tumefaciens C58. Mol. Gen. method with significantly increased sensitivity for deGenet. 191, 10-16. tection of single genes.Plasmid 10, 296-298. MELCHERS,L. S., MARONEY,M. J., DENDULK-RAS, A., SZEGEDI,E. (1985). Host range and specific L(+)-tartrate utilization of biotype III of Agrobacterium tumefaTHOMPSON,D. V., VAN VUUREN, H. A. J., SCHILPERciens. Acta Phytopathol. Acad. Scient. Hung. 20, 17OORT,R. A., AND HOOYKAAS,P. J. J. (1990). &to22. pine and nopaline strains of Agrobacterium tumefaSZEGEDI,E., CZAKO, M., OTTEN, L., AND KONCZ, Cs. ciens differ in virulence: Molecular characterization of (1988). Opines in crown gall tumours induced by biothe virF locus. Plant Mol. Biol. 14, 249-259. type III isolates of Agrobacterium tumefaciens. PhysMESSING,J. (1983). New M 13 vectors for cloning. In iol. Mol. Plant Pathol. 32, 237-247. “Methods in Enzymology” (R. Wu, L. Grossman, and VAN HAUTE, E., Joos, H., MAES, M., WARREN,G., VAN K. Moldave, Eds.), Vol. 101, pp. 20-78. Academic MONTAGU, M., AND SCHELL,J. (1983). Intergeneric Press,San Diego, CA. transfer and exchange recombination of restriction NISHIGUCHI,R., TAKANAMI, M., AND OKA, A. (1987). fragments cloned in pBR322: A novel strategy for the Characterization and sequence determination of the reversed genetics of the Ti plasmids of Agrobacterium replicator region in the hairy-root-inducing plasmid tumefaciens. EMBO J. 2, 4 1l-4 17. pRiA4b. Mol. Gen. Genet. 206, l-8. ZAMBRYSKI, P., TEMP~$J., AND SCHELL, J. (1989). OPHEL,K., AND KERR, A. (199 1).Agrobacterium vitisTransfer and function of T-DNA genes from AgroNew species for strains of Agrobacterium biotype III bacterium Ti and Ri plasmids in plants. Cell 56, 193from grapevine. Int. J. Syst. Bacterial. 40,236-24 1. 201. locus on the nopaline Ti plasmid pTiC58. J. Bacterial. 170, 1759-1767.