A possible approach for altering alkaloid biosynthesis in plants is the expression of genes encoding key enzymes of a pathway such as lysine decarboxylase (ldc) in transgenic plants. Two strategies were followed here: one focused on expression of the gene in the cytoplasm, the other on subsequent targeting of the protein to the chloroplasts. The ldcgene from Hafnia alvei was therefore (a) placed under the control of the 1' promoter of the bidirectional Tr promoter from Agrobacterium tumefaciens Ti-plasmid, and (b) cloned behind the rbcS promoter from potato fused to the coding region of the rbcS transit peptide. Both ldc constructs, introduced into Nicotiana tabacum with the aid of A. tumefaciens, were integrated into the plant genome and transcribed as shown by Southern and northern hybridization. However, LDC activity was only detectable in plants expressing mRNA under the control of the rbcS promoter directing the LDC fusion protein into chloroplasts with the aid of the transit peptide domain. In plants expressing the processed bacterial enzyme cadaverine levels increased from nearly zero to 0.3-1% of dry mass.

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P. syringae pv. syringae strain R32 causes the bacterial brown spot disease on bush beans. A 31 kD protein was detected which is involved in the pathogenic response. Monospecific antibodies directed against this 31 kD-protein were used to screen a protein expression gene bank made from the wild type strain R32. A 0.8 kb DNA insert of a clone which gave a positive reaction with the monospecific antibodies was used in hybridizations to clone a larger chromosomal fragment. A km-cassette (kmresistance) was integrated into this chromosomal DNA-fragment preventing the expression of the 31 kD-protein. This construct was integrated into the chromosome of the wild type strain R32 via homologous recombination resulting in the 31 kD-protein deficient mutant LMI. Biotests with the host plant (bean) and with tobacco leaves showed no symptoms or hypersensitive reaction (HR) when the mutant LMI was inoculated. However, atypical chlorotic and necrotic lesions compared to the wild type strain R32 were found on tobacco leaves when the mutant LMI was incubated for more than 2 weeks. Complementation of the LMI mutant with a plasmid harbouring the corresponding wild type R32 DNA fragment resulted in an isolate (LMIC) which showed a partial restoration of the HR on tobacco but no brown spot disease symptoms on bush beans. The 31 kD-protein could be detected serologically in LMIC. Zusammenfassung Teilcharakterisierung eines an der pathogenese beteiligten 31 kD Protein-Gens vonPseudomonas syringae pv. syringae Bei P. syringae pv. syringae Isolat R32, dem Erreger der bakteriellen Braunfleckenkrankheit an Buschbohnen, wurde serologisch ein 31 kD-Protein nachgewiesen, das bei der Pathogenese involviert ist. Eine Protein-Expressions-Klonbank vom Wildtyp Isolat R32 wurde mit monospezifischen Antikorpern getestet, die mit dem 31 kD-Protein reagierten. Es wurde ein positiv reagierender Klon gefunden. Dessen 0,8 kb-Insert wurde zur Hybridisierung mit einer chromosomalen DNA-Klonbank vom Wildtyp Isolat R32 verwendet, um einen grofieren Genbereich zu klonieren. In den fur das 31 kD-Protein codierenden Bereich wurde eine Kanamycin-Kassette (Km-Resistenz) integriert. Durch homologe Rekombination dieses Konstrukts in das Chromosom des Wildtyps Isolat R32 wurde die Expression des 31 kD-Proteins verhindert. Bei dieser Mutante (LMI) war es serologisch nicht mehr U. S.