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.