THE GENES INVOLVED IN CYTOKININ BIOSYNTHESIS IN ERWINIA-HERBICOLA PV GYPSOPHILAE - CHARACTERIZATION AND ROLE IN GALL FORMATION

A locus conferring cytokinin production was previously isolated from t he gall-forming bacterium Erwinia herbicola pv. gypsophilae. This locu s resided in a cluster with the genes specifying indole-3-acetic acid production on the pathogenicity-associated plasmid pPATH (A. Lichter, S. Manulis, O. Sagee, Y. Gafni, J. Gray, R. Meilen, R. O. Morris, and I. Barash, Mol. Plant Microbe Interact., 8:114-121, 1995). Sequence an alysis of this locus indicated the presence of a cytokinin biosynthesi s gene (etz) homologous to other described cytokinin biosynthesis gene s. A unique open reading frame (pre-etz) encoding 169 amino acids prec eded etz and together with etz formed a region with a distinctive low G+C content. Northern (RNA) analysis indicated the presence of an etz- specific transcript of 1 kb and a common transcript for pre-etz and et z of 1.4 kb. The level of the 1-kb transcript was high in the late log arithmic phase and very low in the stationary phase. In contrast, the level of the 1.4-kb transcript was lower than that of the 1-kb transcr ipt in the late logarithmic phase and predominant in the stationary ph ase. A marker exchange mutant of etz which did not produce cytokinins exhibited a reduction in gall size on Gypsophila cuttings and almost a bolished disease symptoms in a whole-plant assay. Complementation of t his marker exchange mutant with the intact etz gene on a multicopy pla smid resulted in overproduction of cytokinins and larger plant galls f rom which small shoots emerged. Insertional mutation in pre-eh resulte d in a sharp decrease in both the Level of the etz-specific transcript and cytokinin production. A frameshift mutation in pre-etz caused a s imilar reduction in the cytokinin level. A marker exchange mutation in pre-etz caused a reduction of symptoms but to a lower degree than the etz mutation. In the former mutant, cytokinin production and pathogen icity could not be restored by complementation. Furthermore, attempts to complement the etz marker exchange mutant with a plasmid containing an intact etz gene and a frameshift mutation in the pre-etz gene were unsuccessful. These results suggest that the mutations in pre-etz wer e trans dominant.