Proc. Natl. Acad. Sci. USA Vol. 83, pp. 4428-4432, June 1986 Genetics Rapid assay of foreign gene expression in leaf discs transformed by Agrobacterium tumefaciens: Role of T-DNA borders in the transfer process (nopaline/plants/gene transfer)

R. B. HORSCH AND H. J. KLEE Monsanto Company, 700 Chesterfield Village Parkway, St. Louis, MO 63198 Communicated by Howard A. Schneiderman, February 24, 1986

ABSTRACT We have developed a sensitive leaf disc trans- These data suggest that either transfer or integration is being formation procedure for studying early and/or transient T- directed in a polar fashion from the right border sequence. It DNA expression during Agrobacterium tumefaciens-mediated should be noted that none of these experiments can distin- transformation of plant cells. Using this system, we have guish between transfer and integration, since formation of a examined the function of T-DNA border sequences on the early tumor requires both processes to occur. expression of T-DNA genes and on the stable integration of We have developed a system to measure early expression those genes in infected cells. Deletion of the right border from of T-DNA genes in a plant cell and subsequent stable the T-DNA appears to permit transfer ofT-DNA genes from the integration of those genes. Plant cells are cocultured with A. tumor-inducing (Ti) plasmid but greatly reduces the frequency tumefaciens carrying a T-DNA with the genes for nopaline of their stable integration. A binary vector has been construct- synthase (NOS) and a selectable marker encoding kanamycin ed to permit examination of T-DNA border function in trans to resistance (NOS-NPTII-NOS) (24). Early expression is mea- the Ti plasmid. In this situation, a single T-DNA border is sured by accumulation ofnopaline, while stable integration is necessary for early expression of T-DNA genes and is sufficient measured by the ability of transformed plant cells to form for stable integration in any orientation. calli on kanamycin-containing medium. Since plants lack enzymes capable of catabolizing nopaline, it accumulates in Agrobacterium tumefaciens is the causative agent of crown cells expressing the nos gene, thus providing a rapid and gall disease, a neoplastic transformation of most dicotyle- sensitive assay system. Results of these experiments dem- donous plants (for recent reviews, see refs. 1-3). Transfor- onstrate that a single copy of the 25-nucleotide border mation is the result of transfer and integration of a portion of sequence is necessary and sufficient to direct transfer to plant the tumor-inducing (Ti) plasmid, the T-DNA, into plant cells, and subsequent stable integration of the T-DNA. nuclear DNA. The T-DNA encodes several gene products that direct the synthesis of phytohormones, which in turn METHODS stimulate plant cell division (4-7). DNA sequence analyses have shown that the T-DNA is bordered by 25-nucleotide Bacterial Media and Strains. Unless otherwise noted, all direct repeats (8). The T-DNA appears to be a discrete piece Escherichia coli and A. tumefaciens strains were grown on of DNA defined by the border sequence: the junction LB medium (25). Some A. tumefaciens strains were selected between plant and bacterial DNA has been observed to occur by growth on AB minimal medium (26). All DNA manipu- within or adjacent to the border sequences in a number of lations were done in E. coli MM294 or 901 (chromosomal independent tumor lines (9-11). Genetic data indicate that TnJO insertion). A. tumefaciens strain LBA4404 (17) and none of the T-DNA gene products are necessary for transfer GV3111-SE (24) have been described. and integration (12, 13). Indeed, any DNA placed between Construction of Binary Vectors. The various binary vectors the T-DNA border sequences can be directed to integrate into were derived from the integrating vectors (24) pMON200 or plant DNA. pMON8015 (Fig. 1). The first step in construction of these A second region of the Ti plasmid, the virulence or vir vectors involved deleting a 700-base-pair (bp) (Nde I) frag- region is also required for T-DNA transfer (14, 15). This ment from pMON200 and pMON8015, creating pMON503 region is not normally found in transformed plant tissue, but and pMON504, respectively. The large Sma I/HindIII frag- it must be present in the bacterium to direct transfer of ment from each of these plasmids was then ligated to the T-DNA. Workers in several laboratories have demonstrated 3.8-kilobase (kb) Sma I/HindIII fragment from pTJS75, an that the T-DNA need not be physically linked to the virulence RK2 derivative containing oriV, oriT, and trfA (27). The region but must only be maintained within the same cell in resulting pMON503 derivative, pMON505, is a binary vector order to be transferred (16, 17). This has led to the develop- containing only the right border sequence. The pMON504 ment of a number of binary transformation vectors based on derivative, pMON506, is a borderless binary vector. The the T-DNA borders and a selectable marker (18-20). vectors containing a right and left border were derived from The role of the 25-nucleotide direct repeats in T-DNA pMON505 by cloning a 2.8-kb HindIII fragment containing transfer and integration is particularly intriguing. Work from the left border from the octopine Ti plasmid, pTiA6, into the several laboratories (21-23) has suggested that there is unique HindIII site. Two such plasmids were isolated; something unique about the right T-DNA border. Whereas pMON507a, in which the left border is correctly oriented deletions of the left border have little or no effect on the with respect to the right border, and pMON507b, in which the ability to incite tumors, deletions of the right border either left border orientation is reversed. Vectors containing only a eliminate or severely attenuate the ability to incite tumors. left border were constructed by insertion of the same 2.8-kb HindIII fragment into the unique HindIII site of pMON506. The publication costs of this article were defrayed in part by page charge Both possible orientations of the border with respect to the payment. This article must therefore be hereby marked "advertisement" in accordance with 18 U.S.C. §1734 solely to indicate this fact. Abbreviations: Ti, tumor inducing; bp, base pair(s); kb, kilobase(s).

4428 Downloaded by guest on September 29, 2021 Genetics: Horsch and Klee Proc. Natl. Acad. Sci. USA 83 (1986) 4429

Right Border Nopaline p/BR322 Synthase pBR322 SynthaseNopaline Spc/Str5 Spc StrR pMON 200 pMON 8015 Smal-\ / pTiA6 Nos-nptll pTiA6 Nos-nptll LIN

Hind~lll HindilII

Right Border I

pB32 Nopaline Sma p Synthase SpcIStr\ pMON 508W

pTJS75 Nos-npIIV'

_ XX ~~~~~Hindil L Border HindIlil Hindlil HindIlIl

FIG. 1. Construction binary vectors for testing ofborder functions. Details ofthe constructions are provided in Methods. Plasmid pMON200 was used to derive a binary vector containing the pTiT37 right border (pMON505). Plasmid pMON505 was then used to construct vectors containing the pTiT37 right border and the pTiA6 left border either in the same orientation (pMON507a) or in the opposite orientation (pMON507a). Plasmid pMON8015 was used to derive a borderless binary vector, pMON506. A pTiA6 left border was then added to this plasmid in each orientation (pMON508 and pMON508W). Broad host range (pTJS75) and pBR322 plasmid replication origins are indicated. 4, Border sequence; Spc/StrR, gene conferring bacterial spectinomycin/streptomycin resistance; Nos-nptlI, chimeric gene conferring plant kanamycin resistance.

chimeric NOS-NPTII-NOS gene were obtained; pMON508 random in the discs. Discs were placed upside down on and pMON508W. medium containing MS salts (Gibco), B5 vitamins, 3% Bacterial Conjugations. All of the binary vectors were sucrose, benzyladenine (1.0 ,ug/ml), naphthalene acetic acid mobilized into A. tumefaciens strain LBA4404 or GV3111-SE (0.1 4g/ml), 0.8% agar, adjusted to pH 5.7. After 2 days at using the triparental conjugation system of Ditta et al. (28). 260C, the discs that had expanded to 7 or 8 mm in diameter The helper plasmid was pRK2013. Selection of transconju- were selected for inoculation with A. tumefaciens. gants was done on AB plates containing spectinomycin at 100 Expanded discs were randomly distributed into equal ,ug/ml. All transconjugants were checked by remobilizing the groups and each group of discs was randomly assigned to a plasmids back into E. coli strain 901 (tetracycline resistant) particular treatment. An overnight culture ofA. tumefaciens and verifying their structures with plasmid mini-preps (29). grown in Luria broth was poured over the discs in a screw cap Leaf Disc Transformation. Diploid Petunia hybrida tube and gently shaken for a few seconds to ensure contact (Mitchell) plants were grown from seed in flats of Promix ofall leafdisc edges with the bacterial culture. The discs were (Premere Brands, New Rochelle, NY) medium under fluo- then quickly blotted dry and placed upside down on nurse rescent lights with a 16-hr day and were fertilized weekly with culture plates prepared as described (30). Peters soluble 10: 10: 10 plus microelements. Six- to 10-week- After 3 days ofcoculture with the Agrobacterium, the discs old plants gave the best quality leaves: medium green, were transferred to the same medium containing carbenicillin moderately thick without being leathery, and with a semi- at 500 ,ug/ml to kill the bacteria and 0.5 mM arginine to opaque lower epidermis. Unblemished leaves were sterilized enhance production of opines, or kanamycin at 300 ,g/ml to in a solution of 10% bleach with 0.1% Tween 20 for 20 min, select for stable transformants. and then rinsed three times with sterile distilled water. Analysis of Transformants. Opines were assayed after 2 Leaf discs were punched with a 6-mm paper punch. The days on the arginine-containing medium by freezing discs in center vein was avoided but branch veins were included at liquid nitrogen, thawing and macerating with a plastic rod, Downloaded by guest on September 29, 2021 4430 Genetics: Horsch and Klee Proc. Natl. Acad. Sci. USA 83 (1986)

and centrifuging briefly to obtain a semi-clear supernatant. gave background readings identical with the negative con- Four microliters of supernatant was spotted for electropho- trols, showing a comigrating spot of blue color that could be retic separation and detection as described (31). visually distinguished from the yellow-green color of Kanamycin resistance was scored 2 to 6 weeks later by nopaline. A red filter could be used to reduce the intensity of observing vigorous growth of confluent or semi-confluent the blue spot relative to the nopaline. No kanamycin-resistant masses of callus from the edge of a disc or single discrete callus ever grew from more than 60 pMON506 infected leaf colonies (depending on the frequency of stable transforma- discs. As a further confirmation, a pair ofvectors was derived tion). from pMON506 that differed only by the presence or absence of a 25-base synthetic oligonucleotide that matched the RESULTS pTiB6S3 TL-DNA right border. The pMON519 plasmid, which lacked the border sequence, was completely negative Binary Vectors. A number of transformation vectors have in the nopaline assay while pMON520, which contained the been constructed for the purpose of analyzing the role(s) of 25-base sequence, gave strong expression of nopaline al- the T-DNA border sequences in transfer and integration of though at a noticeably reduced level compared to pMON505 T-DNA into plants. These vectors are all capable of auton- (data not shown). omous replication in A. tumefaciens that carry a Ti plasmid Cointegrate Vectors. The split end vector system described with no T-DNA and are thus referred to as binary or trans earlier (24) uses an intermediate vector that integrates into a vectors. They were constructed by deleting the region of modified Ti plasmid containing a left border sequence and a T-DNA homology (LIH) normally used to enable cointegrate small piece of the original T-DNA (LIH). The intermediate formation with a resident Ti plasmid (24) and substituting vector, pMON200 (24), contains the right border and the broad host range plasmid origins of replication and transfer. DNA to be transferred to plant cells. When the vector The plasmid origin was isolated from an RK2 derivative, integrates through the region of homology, a complete pTJS75 (27). The resulting transformation vectors all contain T-DNA is reconstructed. We have described a borderless a pBR322 replication origin, genes encoding bacterial derivative of pMON200, pMON8015, and its attenuated spectinomycin resistance (24), plant kanamycin resistance behavior in cocultivation experiments (24). (24), and nopaline synthase. Plasmids containing either a In leaf disc transformation experiments, pMON8015 also right or left border alone, no border, or two borders were gave attenuated transformation: much less kanamycin-resist- constructed. Since there is evidence indicating that orienta- ant callus and severely reduced accumulation of nopaline tion of the border sequence may be important (21-23, 32), compared to pMON200 (Fig. 2, lanes H and I). When infected plasmids containing the left border in the correct as well as discs were cultured for 7 days nonselectively (no kanamycin, the reversed orientation with respect to the right border were but with carbenicillin to kill the bacteria) before transfer to constructed. medium containing kanamycin, callus was able to form at the Nopaline was detectable in leaf discs within 36 hr of edges of the discs. After transfer to medium containing infection by A. tumefaciens containing pMON200 when kanamycin, most of the callus stopped growth rapidly but cultured on arginine and reached a maximum after 5 days. sectors appeared on approximately one-half to two-thirds of Fig. 2 shows the nopaline produced in leafdiscs infected by the discs that were able to continue rapid growth (stable the various binary or cointegrate-type vectors. All border transformants). Discs that lacked any growing sectors could orientations and combinations allowed efficient transfer and be found to contain nopaline, indicating transient expression integration of the vector into leaf disc cells. There was little of nopaline synthase. The inhibited callus did not turn brown difference in nopaline production between tissue transformed at a uniform rate, and the green (but nongrowing) regions with the single and double border binary vectors (pMON505, were found to contain more nopaline than the brown regions. -507a, -507b, -508, -508W) and the integrating vector We interpret the above observations to indicate that (pMON200), although tissue transformed with the single pMON8015 is capable of being transferred to plant cells but border binary vectors sometimes produced slightly lower that it does not efficiently and/or stably integrate into the levels of nopaline. plant genome. Whether we are measuring transient expres- Fig. 2 shows the effect of omitting a border sequence from sion from nonintegrated copies of the T-DNA or unstably the binary vector (pMON506). Without the border sequence, integrated copies is not known. Also, the efficiency of initial no transfer or integration could be detected. The opine assay T-DNA transfer might be attenuated in this situation as well.

A B c D E F G _H l

-W -,q A. ;i

Nopaline - J& i __ r l

_-I _

_ 1 ;s* _l FIG. 2. Opine assay showing accumulated nopaline from leaf discs transformed with different vector configurations. Leaf discs were transformed as described in the text and assayed 5 days after infection. Lanes A, pMON505; B, pMON506; C, pMON507a; D, 0.5-,ug nopaline standard; E, pMON507b; F, pMON508; G, pMON508W; H, pMON200; I, pMON8015.

Downloaded by guest on September 29, 2021 _I_ | Genetics: Horsch and Klee Proc. Natl. Acad. Sci. USA 83 (1986) 4431

We have clearly demonstrated that a right border sequence is directing transfer in the opposite polarity at a greatly reduced not essential for DNA transfer from the Ti plasmid and frequency. integration of the T-DNA into the plant genome. Similar The observation that a binary vector containing any single observations were made by Joos et al. (21). border will not only be transferred to the nucleus but lead to stable kanamycin-resistant transformants suggests that there DISCUSSION is an inherent difference between binary and cointegrate vectors. The most obvious difference between pMON508 and Several binary transformation vectors based on the A. pTiB6S35E::pMON8015 (both containing only a left border) tumefaciens Ti plasmid have been constructed. The proto- is the size of the two plasmids, 11 kb vs. 250 kb. A single type vector, pMON505, can be easily mobilized into A. border may be sufficient to permit Ti plasmid transfer, but tumefaciens by using a triparental mating system (27). It transfer of a particularly large plasmid may be relatively contains a chimeric NOS-NPTII-NOS gene (24) for selection inefficient. Thus, the primary role of a circular T-DNA of transformed plant cells, as well as a selectable bacterial intermediate that was recently identified by Koukolikova- marker, ColE1 and RK2 replication origins and several Nicola et al. (35) may be to reduce the size of the Ti plasmid unique restriction endonuclease cleavage sites (EcoRI, to a more manageable size than 250 kb. Transfer of Ti EcoRV, Xba I, Bgl II, Cla I, Xho I, HindIII). The nopaline sequences is probably not limited to the T-DNA, but under synthase gene also provides a convenient marker for follow- normal circumstances the processed T-DNA intermediate is ing the T-DNA through plant regeneration and meiosis. The the predominant species. Supporting this idea, we have small size of the vector allows for efficient cloning of large observed low frequency stable transformation with Ti plas- DNA fragments. Transformation efficiency using the binary mids containing a chimeric NOS-NPTII-NOS gene in the vir vectors described here with the petunia leaf disc system (30) region or in the octopine catabolism region, but not in the is the same as with the cointegrate vectors. However, the chromosome (data not shown). Similar results were reported binary vectors we have examined seem to transform certain by Joos et al. (36). plant species such as tomato less efficiently than do integrat- The leaf disc transformation system is simple, rapid, and ing vectors (33). reproducible (when practiced with care), thus providing a We have used both binary and integrating vectors contain- means to quantitatively compare differences in transforma- ing various combinations of left and right border sequences tion efficiency between various vectors. Where precise to elucidate the roles of the border sequences in transfer and quantitation is important, more analytical measurements integration ofT-DNA into plants. The experiments presented could be applied to assess the opine content of leaf discs or, have demonstrated that a single 25-nucleotide border se- alternatively, to assay the enzyme activity directly. This quence is both necessary and sufficient for transfer to the procedure should also be useful to measure the effects of plant nucleus. This is consistent with the data of Wang et al. various modifications of gene sequences to determine the (23) that the 25-bp sequence is sufficient to restore regions that affect transcriptional, translational, and post- tumorigenicity to a strain with a Ti plasmid lacking the right translational aspects of gene expression. border sequence. However, we have consistently observed that transfer of plasmids containing only the 25 bp is not as We thank Steve Rogers and Robb Fraley for many helpful high as with the larger Ti-derived border fragments. This suggestions in the course of this work and in the preparation of the suggests that additional sequences surrounding the T-DNA manuscript. We thank Barbara Schiermeyer and Vicki Grant for play some role in the transfer/integration process. Peralta typing the manuscript. and Ream (32) have observed a similar phenomenon. Ifother sequences outside of the 25-bp repeat are important, they 1. Bevan, M. W. & Chilton, M.-D. (1982) Annu. Rev. Genet. 16, might be present within the left border fragment used in these 357-384. experiments, since we observed no difference in transforma- 2. Depicker, A., Van Montagu, M. & Schell, J. (1983) in Genetic tion frequency between vectors containing only the left Engineering of Plants: An Agricultural Perspective, eds. border (pMON508, 508W) and the right border (pMON505). Kosuge, T., Meredith, C. P. & Hollaender, A. (Plenum, New At this point, we cannot determine the fate of a T-DNA York), pp. 143-176. lacking a border sequence, but it apparently does not reach 3. Nester, E. W., Gordon, M. P., Amasino, R. M. & Yanofsky, the nucleus in a be transcribed at detectable M. F. (1984) Annu. Rev. Plant Physiol. 35, 387-413. form that can 4. Schrcder, G., Waffenschmidt, S., Weiler, E. W. & Schroder, levels. It should be noted that the leaf disc system provides J. (1984) Eur. J. Biochem. 138, 387-391. an extremely sensitive assay, as we have been able to obtain 5. Thomashow, L. S., Reeves, S. & Thomashow, M. F. (1984) rare transformants with certain vir mutants of A. tumefaci- Proc. Natl. Acad. Sci. USA 81, 5071-5075. ens, which are completely avirulent in a typical gall induction 6. Akiyoshi, D. E., Klee, H., Amasino, R. M., Nester, E. W. & assay (34). Gordon, M. P. (1984) Proc. Natl. Acad. Sci. USA 81, The results obtained with the cointegrate vector 5994-5998. pMON8015 are particularly intriguing. Previous results 7. Barry, G. F., Rogers, S. G., Fraley, R. T. & Brand, L. (1984) (21-23) indicated that deletion of the right border results in Proc. Natl. Acad. Sci. USA 81, 4776-4780. avirulence or severe attenuation. The dramatically reduced 8. Barker, R. F., Idler, K. B., Thompson, D. V. & Kemp, J. D. number of stable kanamycin-resistant transformants with (1984) Plant Mol. Biol. 2, 335-350. strain this 9. Simpson, R. B., O'Hara, P. J., Kwok, W., Montoya, A. L., pMON8015 (24) compared to a wild-type confirms Lichtenstein, C., Gordon, M. P. & Nester, E. W. (1982) Cell observation. The data suggest that either the left border or 29, 1005-1014. other border-like sequences in pMON8015 can act as an 10. Zambryski, P., Depicker, A., Kruger, K. & Goodman, H. origin of transfer, allowing the nopaline synthase gene to (1982) J. Mol. Appl. Genet. 1, 361-370. reach the nucleus and be transcribed. The reduced level of 11. Yadav, N. S., Vanderleyden, J., Bennett, D. R., Barnes, nopaline produced by pMON8015 compared to complete W. M. & Chilton, M. D. (1982) Proc. Natl. Acad. Sci. USA 79, T-DNA construction could be due to the necessity for polar 6322-6326. transfer of the entire Ti plasmid starting from the left border 12. Garfinkel, D. J., Simpson, R. B., Ream, L. W., White, F. F., before the nopaline synthase gene can enter the plant cell. Gordon, M. P. & Nester, E. W. (1981) Cell 27, 143-153. This could explain why the right border sequence is more 13. Leemans, J., Deblaere, R., Willmitzer, L., De Greve, H., critical to T-DNA transfer than the left border sequence Hernalsteens, J. P., Van Montagu, M. & Schell, J. (1982) (21-23). Alternatively, the left border may be capable of EMBO J. 1, 147-152. Downloaded by guest on September 29, 2021 4432 Genetics: Horsch and Klee Proc. Natl. Acad. Sci. USA 83 (1986)

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