Production of Cytokinins by Erwinia herbicolapv. gypsophilae and Isolation of a Locus ConferringCytokinin Biosynthesis. Amnon Lichter. Department of Botany, Tel-Aviv University, Tel-Aviv 69978, Israel. Shulamit Manulis(2), Oded Sagee(3),Yedidya Gafni(4), John Gray(5), Richard Meilan(5), Roy O. Morris(6), and Isaac Barash(1). (1) Department of Botany, Tel-Aviv University, Tel-Aviv 69978, Israel (2) department of Plant Pathology (3) department of Horticulture (4) Department of Plant Genetics, ARO, The Volcani Center, Bet Dagan 50250, Israel (5)Department of Biochemistry, University of Missouri, Columbia 65211, U.S.A. MPMI 8:114-121. Accepted 24 October 1994. Copyright The American Phytopathological Society.

Analysis of cytokinins produced in culture by the gall-forming bacterium Erwinia herbicola pv. gypsophilae indicated that pathogenic but not nonpathogenic strains secreted a significant amount of cytokinin. Chemical identification of cytokinins was performed by immunoaffinity chromatography followed by high-performance liquid chromatography and gas chromatographic mass spec-trometry. The cytokinins were quantified by either radio-immunoassay or enzyme-linked immunosorbent assay using anti-cytokinin monoclonal antibodies. Zcalin, zcatin riboside, iso-pentenyladenine, and two immunoreactive zeatin-type compounds were the predominant cytokinins identified in the supernatant. A locus conferring cylokinin production was cloned from a cosmid library derived from plasmids of a pathogenic strain (Eh824-1). Expression was achieved following mobilization of the cosmid clones into a nonpathogenic strain (Eh3-1) or a deletion mutant (Eh3-106), but not in Escherichia coli. Eh3-106 contained a deletion in the pathogenicity-associated plas-mid and lacked cytokinin production. A 3.9-kb DNA fragment from a cosmid clone was subcloned into a wide-host-range plasmid and found to restore production of all the previously detected cytokinins following its mobilization into nonpathogenic E. herbicola strains. This fragment overlapped the 3' end of the indole-3-acetic acid bio-synthetic genes previously cloned from this plasmid, suggesting that indole-3-acetic acid and the locus specifying cytokinin biosynthesis are clustered on the pathogenicity-associated plasmid.