Agrobacterium tumefaciens mutants affected in attachment to plant cells

An analysis of Agrobacterium tumefaciens mutants with Tn5 insertions in chromosomal DNA showed that the chromosome of A. tumefaciens codes for a specific ability of this bacterium to attach to plant cells. This ability is associated with tumorigenesis by A. tumefaciens, the ability of avirulent A. tumefaciens to inhibit tumorigenesis, and the ability to adsorb certain phages. A second class of chromosomal mutations affects tumorigenesis without altering the ability to attach to plant cells. The attachment of A. tumefaciens to plant cells was assayed by mixing radiolabeled bacteria with suspensions of tobacco tissue culture cells or freshly isolated Zinnia leaf mesophyll cells. Under the conditions of this assay, an avirulent Ti plasmid-cured strain attached to the same extent as the same strain containing pTiB6806. Six of eight avirulent mutants with Tn5 insertions in chromosomal DNA showed defective attachment, whereas two retained wild-type attachment ability. In contrast to the strains showing wild-type attachment, the attachment-defective mutants failed to inhibit tumorigenesis when inoculated onto Jerusalem artichoke slices before inoculation of a virulent strain and also showed a loss of sensitivity to two Agrobacterium phages. The loss of phage sensitivity appeared to be due to a loss of ability to adsorb the phages. Staining with Calcofluor indicated that the mutants retained the ability to synthesize cellulose fibrils, which have been implicated in the attachment process. Southern filter hybridizations demonstrated that each mutant contained a single Tn5 insertion, and genetic linkage between the Tn5 insertion in one mutant and the attachment phenotype has also been demonstrated.

Starch-enhanced synthesis and release of amylolytic enzymes from normal and crown gall tumor tobacco tissue culture cells

The response of tobacco crown gall tumor cells growing in tissue culture to a substitution of starch for sucrose as the carbon source in the medium was compared with the response of normal tobacco tissue culture cells. In both cases amylolytic activity was secreted into the medium. The increase in extracellular amylolytic activity was preceded by an increase in intracellular activity. The increase in intracellular amylolytic enzymes was sensitive to cycloheximide and to actinomycin D indicating that enzyme induction at the level of mRNA synthesis was required. No significant difference between the responses of normal and tumor cells was observed. Thus crown gall tumor cells were as capable as normal cells of sensing and responding to an alteration in the carbon source in the external medium.