A chimeric gene fusion cassette, consisting of a secretory sequence from barley alpha-amylase joined to a modified cecropin (MB39) coding sequence and placed under control of the promoter and terminator from the potato proteinase inhibitor II (PiII) gene, was introduced into tobacco by Agrobacterium-mediated transformation. Transgenic and control plants reacted differently when inoculated with tobacco wildfire pathogen Pseudomonas syringae pv. tabaci at various cell concentrations. With control plants (transformed with a PiII-GUS [beta-D-glucuronidase] gene fusion), necrosis was clearly visible in leaf tissue infiltrated with bacterial inoculum levels of 10(2), 10(3), 10(4), 10(5), and 10(6) CFU/ml. With MB39-transgenic plants, however, necrosis was observed only in the areas infiltrated with the two highest levels (10(5) and 10(6) CFU/ml). No necrosis was evident in areas infiltrated with bacterial concentrations of 10(4) CFU/ml or less. Bacterial multiplication in leaves of MB39-transgenic plants was suppressed more than 10-fold compared to control plants, and absence of disease symptom development was associated with this growth suppression. We conclude that the pathogen-induced promoter and the secretory sequence were competent elements for transforming a cecropin gene into an effective disease-control gene for plants.

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protein suggest that it may be a membrane-bound protein, whereas the predicted iaaL gene product possesses considerable hydrophilic character, consistent with the demonstration of (indole-3-acetyl)-L-lysine synthetase activity in cell-free aqueous extracts. No nucleotide or protein homologies were found between iaaL and any sequences contained within the GenBank or National Biomedical Research Foundation data bases (April 13, 1989).Infection and colonization of olive and oleander plants by the plant pathogenic bacterium Pseudomonas syringae pv. savastanoi (P. savastano) is manifested by the appearance of hyperplasias termed "galls" (1-3). The production of indole-3-acetic acid (IAA) and cytokinins by the bacterium is necessary for the elicitation of this response in the plant, and therefore, these bacterially produced compounds are virulence factors in the disease interaction (4,5). Further metabolism of IAA has been observed in P. savastanoi oleander isolates, and the amino acid conjugate, N6-(indole-3-acetyl)-L-lysine (IAA-lysine), and an acetylated derivative, N"-acetyl-N6-(indole-3-acetyl)-L-lysine (N-acetyl-IAA-lysine) are found in culture filtrates of P. savastanoi (6-8).The genes for synthesis of phytohormones reside on the 52-kilobase (kb) plasmid, pIAA1, in oleander strain EW2009 (9, 10). A 4.25-kb EcoRI fragment was found to confer on Escherichia coli the ability to synthesize IAA-lysine from IAA, and transposon mutations in the IAA-lysine synthetase locus in P. savastanoi were obtained by marker exchange (11,12). The mutants showed an increase in IAA accumulation and were attenuated in virulence and in their ability to grow within the plant (12). Therefore, the IAA-lysine synthetase gene represents an additional bacterial locus that influences microbial-plant interactions.The locus for IAA-lysine synthesis has not been fully characterized, and the number of genes involved is not known. We report here the determination of the nucleotide sequence** of the IAA-lysine synthetase gene iaaL and demonstrate that iaaL is sufficient for IAA-lysine production in E. coli.MATERIALS AND METHODS Bacterial Strains, Media, and Plasmids. E. coli HB101 (pLG87) (11), HB101 (pLG87X), E. coli K38 (pMON686), K38 (pMON686ARV), K38 (pMON676), and pLG/SK were used for plasmid DNA isolations, DNA sequencing, and enzyme assays. Restriction enzyme sites for these plasmids derived from P. savastanoi pIAA1 and their orientation relative to one another are shown in Fig. 1. Bacteria were maintained on LB agar plates incubated at 370C, and cells used for plasmid isolations were grown in LB liquid medium (broth). Plasmids were maintained on medium supplemented with ampicillin (50 ,ug/ml).Recombinant DNA Techniques. Restriction enzyme digestions, ligations, plasmid isolation, and purification were performed as described by Maniatis et al. (13).Nucleotide Sequence Determination. Unidirectional exonuclease III deletions (14) were performed to produce a set of nested clones derived from pLG87, cloned in both orientations within pUC8. Exc...

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