Cloning of genes involved in pathogenicity of Xanthomonas campestris pv. campestris using the broad host range cosmid pLAFR1

Abstract

A genomic library was prepared in Escherichia coli from DNA of wild-type Xanthomonas campestris pv. campestris (aetiological agent of crucifer black rot), partially digested with endonuclease EcoRI, using the mobilisable broad host range cosmid vector pLAFR1. Recombinant plasmids contained inserts ranging in size from 19.1 to 32.3 kb (mean 26.6). Certain of the clones complemented E. coli auxotrophic markers. Using the narrow host range plasmid pRK2013 as a helper the pooled recombinant plasmids were transferred conjugally to X. c. campestris mutants, and clones were identified which restored yellow pigmentation to white mutants, prototrophy to amino acid auxotrophs and pathogenicity towards turnip plants to two non-pathogenic mutants. The lesion in one mutant (8288, complemented by the plasmid pIJ3000) is unknown. However mutant 8237 is defective in production of extracellular protease and polygalacturonate lyase and restoration of pathogenicity by complementation with the plasmid pIJ3020 concomitantly restored both enzyme levels to wild-type values.