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 the gall-forming bacterium Erwinia herbicola pv. gypsophilae. This locus 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 analysis of this locus indicated the presence of a cytokinin biosynthesis gene (etz) homologous to other described cytokinin biosynthesis genes. A unique open reading frame (pre-etz) encoding 169 amino acids preceded 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 etz of 1.4 kb. The level of the 1-kb transcript was high in the late logarithmic 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 transcript in the late logarithmic phase and predominant in the stationary phase. A marker exchange mutant of etz which did not produce cytokinins exhibited a reduction in gall size on Gypsophila cuttings and almost abolished disease symptoms in a whole-plant assay. Complementation of this marker exchange mutant with the intact etz gene on a multicopy plasmid resulted in overproduction of cytokinins and larger plant galls from which small shoots emerged. Insertional mutation in pre-etz resulted in a sharp decrease in both the level of the etz-specific transcript and cytokinin production. A frameshift mutation in pre-etz caused a similar 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 pathogenicity 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 were trans dominant.