Proc. Natl. Acad. Sci. USA Vol. 92, pp. 230-234, January 1995 Plant

Intracellular can transfer DNA to the of the host plant JESUS ESCUDERO*, GUNTHER NEUHAUSt, AND BARBARA HOHN*t *Friedrich Miescher-Institut, Postfach 2543, CH-4002 Basel, Switzerland; and tlnstitut fuir Pflanzenwissenschaften, Eidgenossiche Technische Hochschule- Zentrum, Universitatstrasse 2, CH-8092 Zurich, Switzerland Communicated by Frederick M. Ausubel, Massachusetts General Hospital, Boston, MA, September 29, 1994 (received for review June 23, 1994)

ABSTRACT Agrobacterium tumefaciens is a Gram- On the basis of homology between Agrobacteiiwn virulence negative, soil-borne bacterium responsible for the crown gall and the transfer genes ofseveral conjugative plasmids, it now disease ofplants. The galls result from genetic transformation appears that T-DNA is transferred to plant cells by a mechanism ofplant cells by the bacteria. Genes located on the transferred analogous to bacterial conjugation (10), in which the recipient DNA (T-DNA), which is part of the large tumor-inducing (Ti) bacterial cell is replaced by a eukaryotic cell. The current model for plasmid of Agrobacterium, are integrated into host plant plant infection by Agrobacteriwn involves several steps. Among chromosomes and expressed. This transfer requires virulence them, chemotaxis to plant wounds and attachment to the plant cell (vir) genes that map outside the T-DNA on the and surface are the earliest critical events allowing the cell-cell contact that encode a series ofelaborate functions that appear similar needed for DNA transfer. Transcriptional induction of the vir transfer. It remains a major by phenolic compounds and sugars (11, 12), released from to those ofinterbacterial plasmid wounded plant cells, results in the processing of T-DNA molecules challenge to understand how T-DNA moves from Agrobacte- (the so-called T strands), and it has been suggested that the proteins rium into the plant cell nucleus, in view of the complexity of encoded by the virB provide the structural apparatus that obstacles presented by the eukaryotic host cell. Specific allows the export of the T-DNA complex through the bacterial anchoring of bacteria to the outer surface of the plant cell envelope (1-4). DNA delivered from the bacteria has been recently seems to be an important prelude to the mobilization of the shown to reach the plant-cell nucleus in single-stranded form (13), T-DNA/protein complex from the bacterial cell to the plant in analogy to bacterial conjugation. However, the mechanism by cell. However, the precise mode of infection is not clear, which this T-DNA complex passes from the bacterium to the although a requirement of wounded cells has been docu- plant-cell nucleus remains enigmatic, especially when the eukary- mented. By using a microinjection approach, we show here otic cell barriers that the T-DNA has to cross are considered. that the process of T-DNA transfer from the bacteria to the After early microscopic observations (for review, see ref. 14) eukaryotic nucleus can occur entirely inside the plant cell. and some microinjection studies using a tumorigenesis assay Such transfer is absolutely dependent on induction ofvir genes (15), Agrobacterium has been described as an obligately ex- and a functional virB operon. Thus, A. tumefaciens can func- tracellular pathogen. However, in these experiments bacteria tion as an intracellular infectious agent in plants. cultured in standard broth medium were used, and the neces- sity for transcriptional induction of bacterial virulence genes Crown gall is a neoplastic disease caused by Agrobacterium by plant-released compounds was unknown at that time. Here tumefaciens in dicotyledonous plants. The engineering of this we addressed the question whether the early stage of bacterial genetic change in the eukaryotic host cell represents a highly binding to plant cells is required for T-DNA transfer. For this evolved case of microbe-plant interaction (for review, see refs. purpose we analyzed the behavior of intracellular agrobacte- 1-4). For the plant cell, the major consequence of this natural ria. With the help of a very sensitive assay for transient interkingdom DNA transfer is its transformation into a tu- expression of a chimeric reporter (16), we studied the morigenic state in which, as is the case in animal tumorigen- transfer of T-DNA from Agrobacterium cells deposited within esis, new mechanisms govern growth and differentiation (5). plant cells by microinjection. Our results suggest that transfer as T-DNA for of T-DNA does occur under these intracellular conditions and DNA transferred to the host plant cell, known that it is absolutely dependent on both vir gene activation and transferred DNA, represents a defined segment on a large (200 VirB functions. In addition, we show that most likely bacteria kb) extrachromosomal element originally isolated from viru- delivered into a plant cell by microinjection release T-DNA lent agrobacteria and named pTi for tumor-inducing plasmid copies into the nucleus of the very same plant cell. (6). Transformation occurs by a process in which bacteria are required for the establishment of neoplastic development, but their continued presence in tumor cells is not needed (7). Upon MATERIALS AND METHODS integration into the plant-cell T-DNA overproduces Bacteria and Plasmids. A. tumefaciens strains used are plant growth hormones ( and ), which results in listed in Table 1. Strain C58 is a wild-type virulent bacterium the cancerous phenotype, as well as other compounds () that harbors the corresponding nopaline Ti plasmid (17). believed to serve as nutrients for the infecting bacteria (8, 9). Bacterial strains A136 and A348 (18), as well as LBA1100 and Besides T-DNA, the Ti plasmid also contains an essential LBA1143 (19), are all C58 derivatives cured of their original genetic component (vir) known as virulence region. vir genes pTi. A136 contains no pTi plasmid and consequently is avir- are designed to provide the cellular machinery that T-DNA ulent; A348 contains a wild-type octopine pTiA6 conferring needs both in bacteria and in the plant. In addition to the vir virulence; LBA1100 contains the virulent vir-helper plasmid genes located on the Ti plasmid, Agrobacterium requires some pAL1100, an octopine pTiB6 in which the T-DNA, conjuga- chromosomal genes for efficient plant colonization (1-4). tion transfer, and octopine catabolism regions have been

The publication costs of this article were defrayed in part by page charge Abbreviations: T-DNA, transferred DNA; GUS, ,3-glucuronidase; AS, payment. This article must therefore be hereby marked "advertisement" in ; CaMV, cauliflower mosaic virus. accordance with 18 U.S.C. §1734 solely to indicate this fact. tTo whom reprint requests should be addressed. 230 Downloaded by guest on October 4, 2021 Plant Biology: Escudero et al. Proc NatL Acad Sci USA 92 (1995) 231 Table 1. Agrobacterium strains and plasmids pGUS23 was injected as supercoiled pure DNA in water (105 Strain Ref. Characteristics GUS plasmid* molecules per injection). Alternatively to the cell injection, seedlings were infiltrated (24) by application of moderate vacuum (=0.4 C58 17 Wild-type, nopaline pTiC58 pBG5 atmosphere, 5 min) with bacterial suspensions identical to those A136 18 pTi cured of C58 pBG5 used for injection. Injected and infiltrated tobacco seedlings were A348 18 A136, octopine pTiA6 pCG5, pC-90G cultivated for 3 days on MS medium (36)/agar plates under LBA1100 19 Octopine pTiB6 derivative pCG5 standard-growth chamber conditions. GUS activity was then as- LBA1143 19 pTiB6(virB4::Tn3) derivative pCG5 sayed by a histochemical procedure using 2 mM 5-bromo-4-chloro- A6.1h 20 Attachment defective, pTiA6 pCG5 3-indolyl (3-D-glucuronic acid (X-Gluc; Biosynth, Staad, Switzer- *Two different plasmid vectors (binaries) containing either nopaline land) in 100 mM phosphate buffer, pH 7/10 mM Na2 EDTA/0.1% (pBG5) or octopine (pCG5, pC-90G) T-DNA border sequences were NaN3, supplemented with 3 mM K3 [Fe(CN)6] to avoid product used, depending on the kind of native Ti plasmid in the bacterial strain. diffusion (37). Seedlings were dipped and infiltrated under mod- erate vacuum in the staining solution for 10 min, and the reaction deleted; LBA1143 contains pAL1100 with the transposon was continued for 2.5-16 hr at 37C in the dark. The plant tissuewas Tn3Hohol inserted in the virB4 gene, creating a polar and then fixed overnight at room temperature with 4% (vol/vol) avirulent mutation (21). Strain A6.1h is derived from the formaldehyde/0.8% NaCl/0.1% NaN3 in 100 mM phosphate octopine type A6 and contains a TnS insertion in the pscA buffer. Bleaching with ethanol and transfer to phosphate-buffered (exoC) chromosomal locus, resulting in the inability of the saline facilitated scoring for GUS-expressing cells under the ste- bacteria to attach to plant cells and in loss of virulence (20). reomicroscope. AZeiss Axiophot microscope was used for detailed Both nopaline TiC58 and octopine TiA6 or TiB6-derived examination and photography. All GUS plasmids were tested for plasmids were tested in combination with the appropriate induction ofGUS activity inAgrobacterium under conditions similar binary vector plasmid carrying a T-DNA encoding 3-glucuron- to the transient assays in plants, with negative results. idase (GUS; ref. 22). pBG5 (nopaline borders) and pCG5 (octopine borders) contain the N-terminal start of cauliflower mosaic virus (CaMV) gene V fused to the GUS coding RESULTS sequence (23) and driven by a complete CaMV 35S promoter. Agrobacterium Cells Injected into Single Tobacco Cells This GUS fusion has been previously reported as a T-DNA Mediate T-DNA Transfer. To study Agrobacterium-plant in- marker in plants (24, 25). pC-90G was constructed using the teraction at the unicellular level in the plant, single mesophyll EcoRI-BamHI fragment from plasmid X-GUS-90 (26), con- cells of otherwise intact tobacco seedlings were injected with taining the domain A (-90 to +8) of the CaMV 35S promoter a low number (10 on average) of bacterial cells and tested for (here named "Mini" promoter), the GUS coding sequence, the expression of a T-DNA-encoded GUS gene, which had and the 3' end from the small subunit of the ribulose bisphos- been modified to avoid expression in bacteria (23). As a phate carboxylase 3C gene of pea. This fragment was cloned control, similar tobacco seedlings were infiltrated with iden- into the polylinker of pCGN1589 (27). Plasmid pGUS23 (28) tical bacterial suspensions, a process that introduces bacteria contains the same CaMV-GUS fusion as pBG5 and pCG5. For into intercellular spaces. Both injected and infiltrated tobacco standard techniques Escherichia coli DH5a and cells exhibited GUS activity that was strictly dependent on the NM522 strains were used (29), and purified plasmids were presence of the bacterial Ti plasmid. Plant-cell injection with introduced into Agrobacterium by (30). A136(pBG5), a bacterial strain that lacks the Ti plasmid and vir-Gene Induction Conditions.Agrobacterium was grown at therefore the whole vir gene region, did not result in T-DNA 28°C in YEB medium (31) with appropriate antibiotics (ri- gene expression (Table 2). With microinjection, a positive fampicin, 20 mg/liter; kanamycin, 50 mg/liter; neomycin, 40 result was strictly dependent on conditions favoring transcrip- mg/liter; gentamycin, 20 mg/liter). Overnight shaking (250 tional induction of bacterial virulence genes-i.e., preinduc- rpm) cultures were washed and diluted in liquid M9 minimal tion in AS-containing medium at acidic pH. Infiltration ob- medium (29) to an OD600 of 0.1. Two kinds of M9 bacterial viated this requirement because the manipulation of the cultures were prepared: (i) Preinduced, in the presence of 0.2 seedlings caused wounding of plant cells and thereby vir gene mM acetosyringone (AS) at pH 5.5; (ii) nonpreinduced, induction. Failure to detect T-DNA transfer from nonprein- without AS at pH 7. After 12 hr at 28°C, 250 rpm, bacteria were duced bacteria after microinjection was probably a conse- collected, washed with 10 mM MgSO4, and adjusted to OD6wo quence of the minimal injury of the plant cell brought about = 1 (=109 colony-forming units per ml) in 10 mM MgSO4, by injection, which apparently produced insufficient com- before use. Induction conditions were assayed by using the pounds to activate bacterial virulence genes. These results bacterial strain A348(pSM219) carrying a virH::lacZ fusion represent a positive correlation between bacterial culture and measuring 13-galactosidase activity as described (32). conditions that induce vir genes on the Ti plasmid and T-DNA Injection of Plant Cells and GUS Assay. Seeds of Nicotiana activity in plant cells injected with agrobacteria. tabacum cv. Petit Havana, SR1 line, were germinated in plastic GUS gene expression after injection with preinduced Agrobac- dishes with moist filter paper under sterile conditions in a teriwn could result from bacteria transferring T-DNA within an growth chamber. Cotyledons of 7- to 10-day-old tobacco intact plant cell (Fig. 1, route A). However, at least two alternative seedlings were used in all injection experiments. Normally, explanations are possible. (i) The "bacterial lysate" hypothesis: the three single-cell injections were performed per cotyledon. The injected bacterial suspension might contain active T-DNA com- mesophyll layer immediately underneath the epidermis was plexes resulting from preinduction during culture and lysis during chosen as target to facilitate microscopic observation. The manipulation (Fig. 1, route B). (u) The "neighboring cell" hypoth- injection capillary (0.7-1 ,Lm in diameter) was coupled to an esis: injected bacteria might escape one plant cell, attach to another Eppendorf 5242 microinjector, and the pressure was adjusted plant cell in thevicinity, and only then transfer T-DNA (Fig. 1, route to allow delivery of 10 pl. Cytoplasmic microinjections were C). optimized by using fluorescent dyes (lucifer yellow) as has been Agrobacterium Cells Remain Metabolically Active After reported (33, 34). Injection into Plant Cells. When plant cells were injected with TGA-la, a transcription factor specifically binding to a 21-bp bacteria cultured under noninducing conditions, reproducibly, sequence in domain A of the CaMV 35S promoter (35), was T-DNA transfer was undetectable (see Table 2). As a test for overproduced in E. coli and purified by DNA-affinity column the bacterial lysate hypothesis, we studied this time the be- chromatography. The protein was mixed withAgrobacmrium in 10 havior of nonpreinduced wild-type agrobacteria coinjected mM MgSO4 before injection (-3 x 104 molecules per injected cell). with 0.2 mM AS. Our expectation was to restore functionality Downloaded by guest on October 4, 2021 232 Plant Biology: Escudero et al. Proc Natt Acad Sci USA 92 (1995) Table 2. T-DNA transfer in cells of tobacco seedlings injected withAgrobacterium Bacteria not preinduced* Bacteria preinduced* Injected plant GUS-positive Injected plant GUS-positive Injected strain cells, no. plant cells, no. Efficiency,t % cells, no. plant cells, no. Efficiency,t % A348(pCG5) 60 0 <1 130 18 14 A348(pCG5)t 60 6 10 NA C58(pBG5) 60 0 <1 173 19 11 C58(pBG5)t 80 7 9 NA LBA1100(pCG5) ND 200 15 7 LBA1143(pCG5) ND 480 0 <1 A6.lh(pCG5) ND 144 20 14 A136(pBG5) 60 0 <1 140 0 <1 NA, not applicable; ND, not determined. Data represent three independent experiments. *Bacteria were cultured under either noninducing or inducing conditions before injection. tEfficiency was calculated as the number of tobacco cells showing GUS activity (positives) per total number of injected cells after histochemical staining. tBacteria cultured under noninducing conditions coinjected with 0.2 mM of AS. in the bacteria due to. the vir gene activation by the inducer DNA across bacterial membranes, even in the absence of after injection. A high frequency of GUS-positive plant cells bacterial attachment to the external surface of the plant cell. was obtained. Thus, bacteria responded in planta to AS with Thus, the absence of T-DNA expression in the injected plant efficient T-DNA transfer [Table 2, compare the A348(pCG5) cells with the mutant strain LBA1143 suggests again that the data and also the C58(pBG5) data]. Because of the short time bacterial lysate hypothesis is unlikely. (a few minutes) between addition of the vir-inducer AS to the Upon Injection into Plant Cells Attachment-Defective bacteria and injection of this suspension (pH of -7) into Agrobacterium Very Efficiently Transfers T-DNA. To test the tobacco cells, we conclude that the Agrobacterium cells were neighboring cell hypothesis, we used an Agrobacterium pscA- metabolically active after injection and that de novo gene strain (A6.1h, see Table 1) defective in attachment to plant expression steps in the bacteria led to DNA transfer inside the cells and severely attenuated in virulence. In plant-inoculation assays strain A6.1h was tested repeatedly for tumorigenesis, plant cells. Because the combined action of AS and acidic pH both in turnip and in tobacco plants, and failed (data not seems essential for efficient activation of virulence genes (38), shown). When tested by infiltration into young tobacco seed- the bacteria might be exposed to low pH intracellularly. lings, the highest numbef of GUS positives recorded from Intracellular Agrobacterium in the Plant Needs the VirB A6.lh(pCG5) was 3% of the value seen for wild-type A348 Proteins for Transfer of T-DNA. To test a relevant function for (data not shown), in agreement with the tumorigenesis test. intraplant cellular T-DNA transfer, we chose to inject a However, after injection of the mutant into mesophyll cells, preinduced Agrobacterium strain mutated in the virB operon, T-DNA transfer occurred at wild-type levels (Table 2). This which is essential for T-DNA transfer. This mutant strain result implies that attachment per se is not required for (LBA1143) has been previously described as DNA-transfer Agrobacterium T-DNA transfer, and it is, therefore, unneces- deficient but proficient in T-DNA processing (19, 39). Plant sary to postulate invasion of neighboring plant cells by the cells injected with virB-deficient agrobacteria had undetect- injected bacteria. Because GUS expression was generally able levels of GUS expression, whereas its wild-type progen- detected only in single plant cells after injection of bacteria itor (LBA1100) was transfer proficient [Table 2, compare (see Fig. 2a) T-DNA transfer was limited to injected cotyledon LBA1100(pCG5) and LBA1143(pCG5) data]. This result cells. agrees with the proposed role of the VirB proteins to transport Is the Injected Plant Cell Expressing T-DNA Genes? A further experiment was devised to test whether injected plant cells could express the T-DNA marker. For this purpose, we used a bacterial strain containing a defective GUS gene, the expression of which absolutely depends on the presence of an activator protein in the same plant cell. As T-DNAwe used the plasmid pC-90G, carrying the GUS coding sequence fused to the A domain ("Mini") of the CaMV 35S promoter. Strain A348 carrying this T-DNA was coinjected into tobacco cells with TGA-la protein, the cognate plant transcriptional acti- vator (35). Because of its large size (40 kDa), this protein is not %,,~~~~~~~..,,...... believed to move from cell to cell. It has indeed been shown V 4V E -b to function only within the injected cell in transgenic tobacco carrying the corresponding binding sequence (34). In addition, TGA-la protein has been shown to possess nuclear targeting functions in- plants (40). When strain A348(pC-90G) was I-Ncomplex injected together with purified TGA-la, single 6ells histo- chemically stained blue were observed. Therefore, nuclei of _Agrobacferlum _ plant cells injected with Agrobacterium are direct T-DNA T-DNA complex recipients (Table 3). Expression of the defective gene was on with the TGA-la activator FIG. 1. Schematic representation of the plant-cell microinjection strictly dependent coinjection and three simple ways to explain the GUS activity arising from protein. The efficiency of T-DNA transfer under these con- Agrobacterium. Route A, bacteria injected into the plant cell deliver ditions was equivalent to that obtained with the strain carrying T-DNA, which is targeted to the plant cell nucleus; route B, bacteria the T-DNA construct coupled to a complete CaMV 35S are injected as lysate; route C, bacteria injected into the plant cell promoter (pCG5) whose activity is TGA-la independent. attach and deliver T-DNA to juxtaposed cell. Tobacco cells injected with 105 plasmid pGUS23 DNA Downloaded by guest on October 4, 2021 Plant Biology: Escudero et al. Proa Nati Acad Sci USA 92 (1995) 233

FIG. 2. Representative tobacco mesophyll cells showing GUS activity after microinjection with a few Agrobacterium cells (a) or with plasmid pGUS23 DNA (-105 molecules) encoding the GUS marker gene (b). Note the indigo blue precipitate that was observed in single cells after 5-bromo-4-chloro-3-indolyl )3-D-glucuronic acid staining (arrows). (Bars = 10 Am.)

molecules carrying the complete 35S promoter-GUS reporter plant cells in a natural infection. However, the expression of also resulted in single-cell GUS activity (Fig. 2b), although at T-DNA in single plant cells injected with agrobacteria does not a lower efficiency. support this cell-to-cell T-DNA movement in the plant. Agrobacterium anchorage to the surface of plant cells is most DISCUSSION probably a prerequisite for crown gall disease. Nevertheless, we present definitive evidence here that at least the specific F'rom the results reported above we conclude that individual steps responsible for T-DNA transfer from the bacterium to plant cells injected with Agrobacterium can act as primary the eukaryotic nucleus can occur without external binding- T-DNA recipients. With our experimental set-up we cannot i.e., from inside the plant cell. Our results also point to the exclude that trafficking of T-DNA complexes occurs between requirement of the VirB apparatus for the transfer of T-DNA Table 3. Recovery of T-DNA gene expression by coinjected transcriptional activator CaMV 35S promoter Injected plant GUS-positive Injected specimen* driving GUS cells, no. plant cells, no. Efficiency, % A348(pCG5) Full 76 15 20 A348(pC-90G) Mini 75 0 <1 A348(pC-90G) + TGA-lat Mini 72 18 25 pGUS 23 DNAt Full 88 4 4 *Agrobacteria were preinduced before use. tCoinjected (-3 x 104 molecules) into the plant cell with Agrobacterium. tA 5-5-kb-long plasmid encoding the CaMV gene V-GUS fusion used in pBG5 and pCG5 (_105 molecules per injected plant cell). Downloaded by guest on October 4, 2021 234 Plant Biology: Escudero et al. Proa Natl Acad Sci USA 92 (1995) from Agrobacterium within the plant cell. Although the virB 6. Zaenen, I., Van Larebeke, N., Teuchy, H., Van Montagu, M. & genes have been repeatedly reported to be critical forvirulence Schell, J. (1974) J. Mol. Biol. 86, 109-127. (for recent data, see ref. 41 and references therein), our studies 7. Braun, A. (1982) inMolecular Biology ofPlant Tumors, eds. Kahl, reveal that VirB proteins likely facilitate the translocation of G. & Schell, J. S. (Academic, New York), pp. 155-210. the T-DNA complex out of the bacterial cell. Thus, the 8. Willmitzer, L., Debeuckeleer, M., Lemmers, M., Van Montagu, mechanism by which the bacteria transfer T-DNA from within M. & Schell, J. (1980) Nature (London) 287, 359-361. remains unclear but 9. Tempe, J. & Goldmann, A. (1982) in ofPlant triggering of the transfer by the nuclear Tumors, eds. Kahl, G. & Schell, J. S. (Academic, New York), pp. membrane might be one possibility. 427-449. Our evidence of infection within the plant cell clearly 10. Lessl, M. & Lanka, E. (1994) Cell 77, 1-4. conflicts with the conclusions of Hildebrand (15), although his 11. Stachel, S. E., Nester, E. W. & Zambryski, P. C. (1986) Proc. results agree with the present study in that nonpreinduced Natl. Acad. Sci. USA 83, 379-383. agrobacteria are deficient in T-DNA transfer when injected 12. Cangelosi, G. A., Ankenbauer, R. G. & Nester, E. W. (1990) into plant cells. Agrobacterium, therefore, may have the po- Proc. Natl. Acad. Sci. USA 87, 6708-6712. tential to be an intracellular plant parasite, as suggested by 13. Tinland, B., Hohn, B. & Puchta, H. (1994) Proc. Natl. Acad. Sci. Smith (14). This property may be related to the evolutionary USA 91, 8000-8004. origin of Agrobacterium, in agreement with the homologies 14. Rodgers, A. D. (1952) Erwin F. Smith, A Story ofNorthAmerican found for the 16S rRNAs among agrobacteria, rhizobacteria, Plant Pathology (Am. Philos. Soc., Philadelphia), pp. 565-577. rickettsias, and plant mitochondria (42). 15. Hildebrand, E. M. (1942) J. Agric. Res. 65, 45-59. We do not know or 16. Rossi, L., Escudero, J., Hohn, B. & Tinland, B. (1993) Plant Mol. whetherAgrobacterium invades becomes Biol. Rep. 11, 220-229. internalized in plant cells as part of its repertoire of natural 17. Watson, B., Currier, T. C., Gordon, M. P. & Nester, E. W. (1975) infection. The occasional survival of agrobacteria in antibiotic- J. Bacteriol. 123, 255-264. treated plant tissue during transformation experiments (R. A. 18. Garfinkel, D. J., Simpson, R. B., Ream, W., White, F. F. & Ludwig, personal communication) should perhaps be reeval- Gordon, M. P. (1981) Cell 27, 143-153. uated in the light of our findings. In mammalian systems, 19. Beijersbergen, A., Dulk-Ras, A. D., Schilperoort, R. A. & Hooy- invasive bacteria are protected from a variety of antibiotics, kaas, P. J. J. (1992) Science 256, 1324-1327. whereas noninvasive strains are killed (43). 20. Thomashow, M. F., Karlinsey, J. E., Marks, J. R. & Hurlbert, TheAgrobacterium-plant interaction consists of a large and R. E. (1987) J. Bacteriol. 169, 3209-3216. yet partially known series of events in which many bacterial 21. Stachel, S. E. & Nester, E. W. (1986) EMBO J. 5, 1445-1454. genes need to be activated. The microinjection technique 22. Jefferson, R. A. (1987) Plant Mol. Biol. Rep. 5, 387-405. applied here may be useful for studying the function of certain 23. Schultze, M., Hohn, T. & Jiricny, J. (1990) EMBOJ. 9, 1177-1185. bacterial new that are for 24. Shen, W. H., Escudero, J., Schlappi, M., Ramos, C., Hohn, B. & genes (or discovering ones) required Koukolikova-Nicola, Z. (1993) Proc. Natl. Acad. Sci. USA 90, pathogenesis. For instance, virulence functions could be iden- 1488-1492. tified that are required in the early stage of bacterium-plant 25. Koukolikova-Nicola, Z., Raineri, D., Stephens, K., Ramos, C., cell recognition, which are essential for mobilization of T- Tinland, B., Nester, E. W. & Hohn, B. (1993) J. Bacteriol. 175, DNA within the plant cell and which are involved in integra- 723-731. tion of T-DNA. 26. Benfey, P. N. & Chua, N.-H. (1989) Science 244, 174-181. Although the results presented here are based on T-DNA 27. McBride, K E. & Summerfelt, K R. (1990) Plant Mol. Biol. 14, transient expression, our approach may have potential for 269-276. plant transformation in cases inwhich the host-cell recognition 28. Puchta, H. & Hohn, B. (1991) Nucleic Acids Res. 19, 5034-5040. is not efficient under conditions of classical bacterial infection. 29. Sambrook, J., Fritsch, E. F. & Maniatis, T. (1989) Molecular Cloning: A Laboratory Manual (Cold Spring Harbor Lab. Press, We thank the members of our groups, especially Zdena Kouko- Plainview, NY), 2nd Ed. lf'kovi-Nicola for useful discussion and for encouragement, and Ingo 30. Cangelosi, G. A., Best, E. A., Martinetti, G. & Nester, E. W. Potrykus for his support. Holger Puchta and Bruno Tinland provided (1991) Methods Enzymol. 204, 384-397. plasmids pBG5 and pCG5. We are indebted to Paul Hooykaas, Eugene 31. Lichtenstein, C. P. & Draper, J. (1985) in DNA Cloning: A Nester, and Michael Thomashow for their gifts of LBA1100 and Practical Approach, ed. Glover, D. M. (IRL, Washington, DC), LBA1143, A348, and A6.1h bacterial strains, respectively. Fumiaki Vol. 2, p. 78. Katagiri and Nam-Hai Chua are acknowledged for their gifts of 32. Stachel, S. E., An, G., Flores, C. & Nester, E. W. (1985) EMBO TGA-la and pX-GUS-90. We also thank Thomas Boller, Mary-Dell J. 4, 891-898. Chilton, Patrick King, Jacques Tempe, and Bruno Tinland for useful 33. Schnorf, M., Neuhaus-Url, G., Galli, A., Iida, S., Potrykus, I. & suggestions and critical comments on this manuscript. The technical Neuhaus, G. (1991) Transg. Res. 1, 23-30. expertise of Cynthia Ramos and Alessandro Galli is also very much 34. 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