C. Genetello

The functional organization of the nopaline A. tumefaciens plasmid pTiC58

Abstract We have employed the P type plasmid RP4 and the transposons Tn1 and Tn7 to isolate insertion and deletion mutations in the nopaline Ti-plasmid pTiC58. Mutations that inactivate all known Ti phenotypes have been located on the physical map. Most importantly, we have positioned several regions involved in the determination of oncogenicity. They correspond to regions of homology between octopine and nopaline plasmids. One of these regions is part of the T-DNA, the Ti-plasmid DNA present in transformed plant cells. There are also segments of the T-DNA that are not essential for oncogenicity. One of these determines the biosynthesis of nopaline in tumors. The latter regions might allow insertion of foreign DNA that can then be introduced into plant cells.

Interactions and DNA transfer between Agrobacterium tumefaciens, the Ti-plasmid and the plant host.

Agrobacterium tumefaciens is a gram-negative bacterium with the unique capacity to induce neoplasmic transformations in dicotyledonous plants. Recently, both the mechanism and the biological significance of this transformation have been elucidated. Agrobacterium tumefaciens strains contain a large extrachromosomal DNA plasmid (the Ti-plasmid). This Ti-plasmid is responsible for the oncogenic properties of Agrobacterium strains. A particular segment of the Ti-plasmid, containing information determining the tumorous growth pattern and the synthesis of so-called ‘opines’, e. g. octopine (N-α-(D-l-carboxyethyl)-L-arginine) and nopaline (N-α-(l, 3-dicarboxypropyl)-L-arginine), is transferred and stably maintained and expressed in the transformed plant cells. This phenomenon can be understood as a ‘genetic colonization’ of the plant cells by bacterial plasmid DNA so that the transformed plant cells will produce and secrete into the medium amino acid derivatives (the opines) that Ti-plasmid carrying agrobacteria can selectively use as carbon and nitrogen sources.

Transfer of Ti plasmids between Agrobacterium strains by mobilisation with the conjugative plasmid RP4

SummaryThe P type conjugative plasmid RP4 has been shown to be able to promote the transfer of the Agrobacterium Ti-plasmid. The results provide additional evidence that agrocin 84 sensitivity, exclusion of phage AP1, ability to catabolize the guanidine derivatives octopine and nopaline and tumor inducing ability, are Ti-plasmid determined properties. Furthermore, the results strongly support the notion that at least part of the Ti-plasmid is transferred from the bacterium to the target plant cells, since it was demonstrated that Ti-plasmid linked genes specify the synthesis of octopine or nopaline by crown-gall tumor cells.

Spontaneous formation of cointegrates of the oncogenic Ti-plasmid and the wide-host-range P-plasmid RP4.

A pTiB6S3::RP4 cointegrate and a pTiC58::RP4 cointegrate plasmid were isolated. They are formed by the covalent linkage of the respective pTiB6S3 and pTiC58 plasmids of Agrobacterium tumefaciens and of the RP4 factor. They carry the genetic markers of both parental molecules, and these markers are transferred as a unit under the same circumstances as the RP4 factor alone. The pTiB6S3::RP4 plasmid retains its oncogenic capacity and does not dissociate with a detectable frequency in Agrobacterium strains. The pTiC58::RP4 cointegrate readily dissociates apparently with exact separation of the genetic markers of pTiC58 and of RP4. The cointegrate pTiC58::RP4 state is non-oncogenic whereas after dissociation the oncogenicity of pTiC58 is restored.

In vivo transfer of the Ti-plasmid of Agrobacterium tumefaciens to Escherichia coli

SummaryThe Ti-plasmids are naturally self-transmissible from their normal host Agrobacterium to E. coli. They are however unable to stably establish themselves as a replicon in E. coli. It is nevertheless possible to study the Ti-plasmids in E. coli with the help of Ti::RP4 cointegrate plasmids that transfer and maintain themselves very efficiently in E. coli. An E. coli harbouring such a Ti::RP4 plasmid is unable to catabolize octopine and unable to induce crowngall tumours on plants.

Agrobacterium Tumefaciens: What Segment of the Plasmid is Responsible for the Induction of Crown Gall Tumors ?

Several causes of plant tumors have been described (1). Particularly intriguing is the case of the crown gall tumors. The agent inducing this disease is a Gram-negative bacterium, Agrobacterium tumefaciens belonging to the family of the Rhizobiaceae. Until now it is the only documented case where an oncogenic agent is of bacterial origin.