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

Abstract

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).

A luminescent reporter evidences active expression of Ralstonia solanacearum type III secretion system genes throughout plant infection

“…The eps and hrpB promoters were amplified from the R. solanacearum GMI1000 genome clone BCC024ZH30 and plasmid pSG315 , using primer pairs PhB-B/PhB-K and Pep-B/Pep-K (Table S1), which introduced BamHI (59) and KpnI (39) restriction sites to clone in pRCGent (Monteiro et al, 2012). This gave rise to pRCGent-Pep and pRCGent-PhB.…”

“…R. solanacearum strains containing integration elements borne by pRC vectors were constructed by natural transformation and chromosomal integration events selected as described previously (Monteiro et al, 2012). To this end, pRCGent-PhB-GFP and pRCGent-Pep-GFP were linearized using HindIII, while SfiI was used to linearize pRCGent-PhB-lux and pRCGent-Pep-lux.…”

“…We evaluated the expression profiles of these key promoters, suspected to be activated at different moments during the infection process Yoshimochi et al, 2009a) using GFP as a non-invasive reporter. For this, we took advantage of the recently-described pRC suicide plasmids, in which promoter : : reporter fusions are cloned between homology regions that enable directed integration in a permissive site of the R. solanacearum chromosome (Monteiro et al, 2012). Integration of the genetic element guarantees genetic stability and stoichiometry conservation (i.e.…”

“…To this end, we chose the luxCDABE operon, a non-invasive reporter that produces light without addition of any substrate (Craney et al, 2007). The promoters under study were cloned in pRCGent-lux, a gentamicin-selection variant of the pRC vectors that contains a synthetic luxCDABE gene cluster optimized for expression in high-GC bacteria (Craney et al, 2007;Monteiro et al, 2012). This gave rise to pRCGent-Pep-lux and pRCGent-PhB-lux, which were linearized and transformed in R. solanacearum GMI1000 to generate strains with either the Peps : : lux or the PhrpB : : lux fusion inserted in the chromosome (Fig.…”

“…NotI was used instead of EcoRI for cloning in pRCGentPep. General molecular biology techniques are detailed in Ausubel et al (1994) and were carried out as described in Monteiro et al (2012). All plasmids used in this work are summarized in Table 1.…”