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A Chromosomal Insertion Toolbox for Promoter Probing, Mutant Complementation, and Pathogenicity Studies in Ralstonia solanacearum

April 2012 , Volume 25 , Number  4
Pages  557 - 568

Freddy Monteiro, Montserrat Solé, Irene van Dijk, and Marc Valls

Deptartament de Genètica, Facultat de Biologia, Universitat de Barcelona, Av. Diagonal 645 annex, 08028 Barcelona, Catalonia, Spain

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Accepted 16 November 2011.

We describe here the construction of a delivery system for stable and directed insertion of gene constructs in a permissive chromosomal site of the bacterial wilt pathogen Ralstonia solanacearum. The system consists of a collection of suicide vectors—the Ralstonia chromosome (pRC) series—that carry an integration element flanked by transcription terminators and two sequences of homology to the chromosome of strain GMI1000, where the integration element is inserted through a double recombination event. Unique restriction enzyme sites and a GATEWAY cassette enable cloning of any promoter::gene combination in the integration element. Variants endowed with different selectable antibiotic resistance genes and promoter::gene combinations are described. We show that the system can be readily used in GMI1000 and adapted to other R. solanacearum strains using an accessory plasmid. We prove that the pRC system can be employed to complement a deletion mutation with a single copy of the native gene, and to measure transcription of selected promoters in monocopy both in vitro and in planta. Finally, the system has been used to purify and study secretion type III effectors. These novel genetic tools will be particularly useful for the construction of recombinant bacteria that maintain inserted genes or reporter fusions in competitive situations (i.e., during plant infection).

This article is in the public domain and not copyrightable. It may be freely reprinted with customary crediting of the source. The American Phytopathological Society, 2012.