Scott E. Stachel

New cloning vehicles for transformation of higher plants.

We have constructed a set of small vectors based on the tumor‐inducing (Ti) plasmid of Agrobacterium tumefaciens which allow the transfer of exogenous DNA into plant chromosomes. These vectors contain: (i) a chimeric gene containing the transcriptional control signals from the nopaline synthase gene and the coding sequence for neomycin phosphotransferase; (ii) the ColE1 replicon; (iii) the cos site of bacteriophage λ; (iv) the border sequences from the ends of the T‐DNA region of the Ti plasmid; and (v) a wide host range replicon. Due to the small size of these cosmid vectors, DNA fragments up to 35 kbp can be inserted by an in vitro packaging method in Escherichia coli. The ability of these vectors to be stably replicated in both E. coli and A. tumefaciens allows their subsequent transfer to and maintenance in Agrobacterium without intermediate genetic manipulations. We demonstrate that DNA cloned into these vectors in A. tumefaciens can efficiently transform plants when in trans with a wild‐type Ti plasmid which donates the functions necessary for DNA transfer and integration. We also show that only the right border of the T‐DNA is necessary for DNA transformation.

A Tn3 lacZ transposon for the random generation of beta-galactosidase gene fusions: application to the analysis of gene expression in Agrobacterium.

The construction and use of a Tn3‐lac transposon, Tn3‐HoHo1, is described. Tn3‐HoHo1 can serve as a transposon mutagen and provides a new and useful system for the random generation of both transcriptional and translational lacZ gene fusions. In these fusions the production of beta‐galactosidase, the lacZ gene product, is placed under the control of the gene into which Tn3‐HoHo1 has inserted. The expression of the gene can thus be analyzed by monitoring beta‐galactosidase activity. Tn3‐HoHo1 carries a non‐functional transposase gene; consequently, it can transpose only if transposase activity is supplied in trans, and is stable in the absence of this activity. A system for the insertion of Tn3‐HoHo1 into sequences specifically contained within plasmids is described. The applicability of Tn3‐HoHo1 was demonstrated studying three functional regions of the Agrobacterium tumefaciens A6 Ti plasmid. These regions code for octopine catabolism, virulence and plant tumor phenotype. The regulated expression of genes contained within each of these regions was analyzed in Agrobacterium employing Tn3‐HoHo1 generated lac fusions.

The genetic and transcriptional organization of the vir region of the A6 Ti plasmid of Agrobacterium tumefaciens

The genetic transformation of plant cells by Agrobacterium tumefaciens is mediated by the genes of the Ti plasmid vir region. To determine the genetic and transcriptional organization of the vir region of pTiA6, vir plasmid clones were saturated with insertion mutations of a Tn3‐lacZ transposon. This element is both an insertion mutagen and a reporter for the expression of the sequences into which it has inserted. One hundred and twenty‐four vir::Tn3‐lac insertions were analyzed for their mutagenic effect on Agrobacterium virulence, and for their expression of beta‐galactosidase activity, the lacZ gene product, in vegetative bacteria and in bacteria cocultivated with plant cells. These data in conjunction with genetic complementation results show that the pTiA6 vir region contains six distinct vir complementation groups: virA, virB, virC, virD, virE and virG. Mutations in these loci eliminate (virA, virB, virD and virG) or significantly restrict (virC and virE) the ability of Agrobacterium to transform plant cells. Each of the vir loci corresponds to a single vir transcription unit: virA is constitutively expressed and non‐inducible; virB, virC, virD and virE are expressed only upon activation by plant cells; and virG is both constitutively expressed and plant‐inducible. The two largest vir operons, virB and virD, are probably polycistronic. The pTiA6 vir region also contains plant‐inducible loci (pin) which are non‐essential for virulence.

virA and virG control the plant-induced activation of the T-DNA transfer process of A. tumefaciens

The Ti plasmid vir loci of Agrobacterium tumefaciens are transcriptionally activated in response to signal molecules produced by plant cells to initiate the T-DNA transfer process. We show that the pTiA6 vir loci are organized as a single regulon whose induction by plants is controlled by virA and virG. Mutations in virA result in attenuated induction. This locus is constitutively transcribed and noninducible. Mutations in virG eliminate vir induction. This locus is constitutively transcribed, plant-inducible, and self-regulated in a complex fashion, and it produces two distinct and differentially regulated transcripts. virA is proposed to encode a transport protein for the plant signal molecule, and virG a positive regulatory protein that together with the plant molecule activates vir expression.

Activation of Agrobacterium tumefaciens vir gene expression generates multiple single-stranded T-strand molecules from the pTiA6 T-region: requirement for 5' virD gene products

Agrobacterium tumefaciens transfers its Ti‐plasmid T‐DNA to plant cells. This process is initiated by plant‐induced activation of the Ti‐plasmid virulence loci, resulting in the generation of single stranded (ss) cleavages of the Ti‐plasmid T‐DNA border sequences (border nicks) and ss linear unipolar T‐DNA molecules (T‐strands). A single T‐strand is produced from the two‐border T‐region of the pGV3850 nopaline plasmid. In this paper the induced molecular events for the complex T‐region of the pTiA6 octopine plasmid are analyzed. This T‐region carries four T‐DNA borders delimiting three T‐DNA elements (TR, TC and TL). Induction of pTiA6 generates cleavages independently at its border repeats, and six distinct T‐strand species corresponding to TR, TR/TC, TR/TC/TL, TC, TC/TL and TL. These T‐strand molecules are linear and correspond to the bottom strand of the pTiA6 T‐region. Thus, borders can function for both initiation and termination of T‐strand synthesis. We propose that the different pTiA6 T‐strands are independently generated, and that the distribution of border nicks within the parental T‐region determines which T‐strand is produced. To identify genes involved in T‐strand production, pTiA6 virulence (vir) and chromosomal virulence (chv) mutant strains were analyzed. VirA and VirG, the vir regulatory loci are required. Furthermore, the two 5′ cistrons of virD are required for both border nicks and T‐strands, suggesting that these genes encode the border endonuclease, and that T‐strand production is dependent on border nicks. That no mutants are defective for T‐strands alone suggests that functions encoded outside of vir and chv might mediate some of the later reactions of T‐strand synthesis.

Agrobacterium tumefaciens and the susceptible plant cell: a novel adaptation of extracellular recognition and DNA conjugation.

The soil phytopathogen Agrobacterium tumefaciens genetically transforms plant cells through a unique interaction that demonstrates both the diversity and conservation of evolution. A. tumefaciens first recognizes and attaches to a susceptible target plant cell in the complex soil environment. This recognition sets in motion a coordinated seriies of events: a specific set of bacterial genes becomes transcriptionally activated; and a distinct DNA element, the T-DNA, becomes mobilized from the large (approximately 200 kb) Ti-plasmid of the bacterium, is transferred across the bacterial and plant cell walls, and is integrated intact into the plant nuclear genome. Subsequent expression of T-DNA genes in the transformed cell results in a crown gall tumor that synthesizes novel compounds (opines) beneficial to the bacterium. While the A. tumefaciens-plant interaction is the only known example of interkingdom DNA transfer, recent results suggest that the underlying processes are not unique, but instead represent novel adaptations of two common prokaryotic processes: activation of gene expression in response to an external stimulus through a two-component positive regulatory system; and conjugative transfer of DNA from a donor to a recipient cell. Bacterial Recognition of Plant Cells The T-DNA functions solely as a structural element during its transfer, and does not encode products essential for this process (Garfinkel et al., Cell 27: 143-153, 1981; Zambryski et al., EMBO J. 2, 2143-2150, 1983). These transacting functions are specified in the bacterium by its chromosomal virulence loci, chvA and chvB (Douglas et al., J. 13act. 767, 850-860, 1985) and Ti-plasmid virulence loci, nirA, vi/B, virC, t&D, virE, and virG (Stachel and Nester, EMBO J. 5, 1445-1454, 1986). The target plant cell probahly also specifies functions necessary for its transforImation. The chv loci mediate attachment of the bacterium to the /plant cell. This activity might be generally useful to the Ibacterium, since chv expression is constitutive and nonregulated. The vir loci direct the more specialized events ‘of plant cell recognition and subsequent reactions of T-DNA transfer. These loci span an approximately 35 kb region of the Ti-plasmid that is separate from the T-region. In con-

Site-specific nick in the T-DNA border sequence as a result of Agrobacterium vir gene expression

The T-DNA transfer process of Agrobacterium tumefaciens is activated by the induction of the expression of the Ti plasmid virulence (vir) loci by plant signal molecules such as acetosyringone. The vir gene products act in trans to mobilize the T-DNA element from the bacterial Ti plasmid. The T-DNA is bounded by 25—base pair direct repeat sequences, which are the only sequences on the element essential for transfer. Thus, specific reactions must occur at the border sites to generate a transferable T-DNA copy. The T-DNA border sequences were shown in this study to be specifically nicked after vir gene activation. Border nicks were detected on the bottom strand just after the third or fourth base (� one or two nucleotides) of the 25—base pair transferpromoting sequence. Naturally occurring and base-substituted derivatives of the 25—base pair sequences are effective substrates for acetosyringone-induced border cleavage, whereas derivatives carrying only the first 15 or last 19 base pairs of the 25—base pair sequence are not. Site-specific border cleavages occur within 12 hours after acetosyringone induction and probably represent an early step in the T-DNA transfer process.