David J. Garfinkel

Genetic Analysis of Crown Gall: Fine Structure Map of the T-DNA by Site-Directed Mutagenesis

Abstract Seventy-five Tn5 and three Tn3 insertions were generated, characterized and mapped in the pTiA6NC plasmid sequences which are stably integrated in crown gall tumors (T-DNA). Four mutants containing Tn5 insertions in a specific region of the T-DNA incited tumors that no longer synthesized octopine. No single insertion resulted in complete loss of oncogenicity. Twenty-five transposon insertions defined three distinct loci affecting tumor morphology. The first group ( tml ), of seven mutants, contained Tn5 insertions within a 1.25 kilobase (kb) region and incited tumors two to three times larger than normal. The second group ( tmr ), of nine mutants, incited tumors with a massive amount of roots proliferating from the tumor callus and contained Tn5 insertions in a 1 kb cluster. The third group ( tms ), of nine mutants, incited tumors with shoots growing from the tumor callus and contained three Tn3 insertions and six Tn5 insertions distributed over a 3.1 kb region. Each of these loci was separated by Tn5 insertions that did not noticeably change tumor formation. We identified two more regions of the T-DNA where transposon insertions did not appear to affect tumor morphology: one contained 28 Tn5 insertions distributed over 3.2 kb; the other contained 17 Tn5 insertions distributed over 7 kb and the octopine synthesis locus. A detailed functional map of the T-DNA of pTiA6NC resulted from the characterization of these insertions. The incorporation of Tn5 sequences into the plant genome was also demonstrated. We discuss these results in relation to the map location of tumor-derived RNA transcribed from the T-DNA, the role of phytohormones in crown gall tumorigenesis and the eventual use of the Ti plasmid as a vehicle for introducing genes of choice into the genomes of higher plants.

A Mutational and Transcriptional Analysis of a Tumor Inducing Plasmid of Agrobacterium tumefaciens

The large tumor inducing (Ti) plasmids (Zaenen et al., 1974) of Agrobacterium tumefaciens are the causitive agents of gall tumors on dicotylendonous plants. The plant cell transformation is brought about by the stable integration of a portion of the bacterial Ti-plasmid into plant nuclear DNA (Chilton et al., 1978a; Thomashow et al., 1980a; Thomashow et al., 1980b; Lemmers et al., 1981; Chilton et al., 1980; Yadav et al., 1980). Transformed plant cells are characterized by the following properties: the ability to grow in azenic culture without an exogenous supply of the plant hormones auxin and cytokinin (Braun, 1958) and the synthesis of unusual amino acids called opines (Petit et al., 1968; Menage and Morel, 1964; Goldman et al., 1968; Goldman et al., 1969; Fermin and Fenwick, 1978). The transferred plasmid DNA (T-DNA) is transcribed, (Drummond et al., 1978; Yang et al., 1979; Gelvin et al., 1981; Ledeboer, 1978; Gurley et al., 1979) influences the levels of plant hormones, and directs the synthesis opines (Bomhoff et al., 1976; Montoya et al., 1977; Kemp et al., 1979; Hack and Kemp, 1980; Guyon et al., 1980) in transformed plant cells. Thus, crown gall tumorigenesis is a model system for the study of the mechanism by which a bacterial plasmid transforms a eukaryotic cell causing a neoplastic disease.