Expression of an Engineered Cecropin Gene Cassette in Transgenic Tobacco Plants Confers Disease Resistance to Pseudomonas syringae pv. tabaci.

ABSTRACT 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.

Chromosome mapping in Pseudomonas syringae pv. syringae strain PS224.

A conjugation system for mapping the chromosome of Pseudomonas syringae pv. syringae PS224 has been developed using the IncP-10 plasmid R91-5; pMO22, a Tn501-loaded derivative of R91-5; and pMO75, R91-5 loaded with Tn5. Nine different donor origins were identified with R91-5 and pMO22. By insertion of Tn5 into various sites of the chromosome, an additional six donor origins were available using pMO75 as the donor plasmid. In all, 36 markers were located on three linkage groups. Many donor strains were unstable and the limited availability of stable donor strains has limited the extent to which markers have been located. This instability of donor strains is in marked contrast to the highly stable donor strains found in P. putida using the same plasmids. As in P. aeruginosa and P. putida, auxotrophic markers in P. syringae do not show the clustering of related markers found in enterobacteria.

Generalized Transduction in the Phytopathogen Pseudomonas syringae.

Bacteriophages isolated from culture supernatants of Pseudomonas syringae pv. syringae and from sewage transferred various chromosomal genes to P. syringae PS224. Linkage between arginine and tryptophan loci was demonstrated. The number of transductants recovered per milliliter was not altered appreciably by UV irradiation of selected phage isolates. In addition, the presence of the IncP2 plasmid R38 in a P. syringae PS224 arginine auxotroph did not increase the transduction frequency as it does in Pseudomonas aeruginosa. Increasing the multiplicity of infection of transducing phage Pssy15 from 1 to 10 resulted in up to a 10-fold increase in the number of transductants recovered, although the actual transductional frequency remained about the same. Treatment of transduction mixtures with DNase did not affect transductional frequency.

Isolation and Partial Characterization of Bacteriophages of the Phytopathogen Pseudomonas syringae.

Bacteriophages isolated from culture supernatants of Pseudomonas syringae pv. syringae and from sewage were identified. The DNA from each phage was isolated and digested with the restriction endonuclease EcoRI. Eight isolates were determined to be different, with two phage isolates from sewage having restriction patterns identical to two phages from culture supernatants. The sizes of the phage DNA ranged from 24 to49 kilobases for isolates from sewage and from 39 to 52.5 kilobases for the isolates from culture supernatants. Buoyant densities of phage particles in CsCl varied from 1.498 to 1.507 g/cm for isolates from sewage and from 1.506 to 1.516 g/cm for isolates from culture supernatants. Electron microscopy revealed four morphological types. Based on plaque-forming ability of culture supernatants, 31 out of 47 strains of P. syringae are probably lysogenic.