Identification of plant-induced genes of the bacterial pathogen Xanthomonas campestris pathovar campestris using a promoter-probe plasmid

Abstract

A promoter-probe plasmid suitable for use in Xanthomonas campestris pathovar campestris (causal agent of crucifer black rot) was constructed by ligating a broad host range IncQ replicon into the promoter-probe plasmid pKK232-8, which contains a promoterless chloramphenicol acetyltransferase gene. Xanthomonas chromosomal DNA fragments were 'shotgun' cloned into a restriction site in front of this gene, and the resulting library was transferred en masse into Xanthomonas. Individual transconjugants possessing DNA insertions with promoter activity in plants were identified by virtue of their ability to infect chloramphenicol-treated turnip seedlings. Of 19 transconjugants identified in this way five were chloramphenicol resistant both in turnip seedlings and on agar plates. However the remaining 14 were only chloramphenicol resistant in planta, and thus apparently contained plant-inducible promoter fragments. Resistance to chloramphenicol was correlated with increased chloramphenicol acetyltransferase activity for the transconjugants assayed. The promoter fragments were used to isolate genomic clones from a library, and the role of the genes contained in these clones in pathogenicity is being investigated.