The Transcriptional Activator rfiA Is Quorum-Sensing Regulated by Cotranscription with the luxI Homolog pcoI and Is Essential for Plant Virulence in Pseudomonas corrugata

The gram-negative phytopathogen Pseudomonas corrugata has an acyl-homoserine lactone (AHL) quorum-sensing (QS) system called PcoI/PcoR that is involved in virulence on tomato. This work identifies, downstream of pcoI, a gene designated rfiA, which we demonstrate is directly linked to QS by cotranscription with pcoI. The deduced RfiA protein contains a DNA-binding domain characteristic of the LuxR family but lacks the autoinducer-binding terminus characteristic of the QS LuxR-family proteins. We also identified, downstream of rfiA, an operon designated pcoABC, encoding for the three components of a tripartite resistance nodulation-cell-division (RND) transporter system. The expression of pcoABC is regulated by RfiA. We found that lipodepsipeptide (LDP) production is cell density dependent and mutants of pcoI, pcoR, and rfiA are unable to inhibit the growth of the LDP-sensitive microorganisms Rhodotorula pilimanae and Bacillus megaterium. P. corrugata rfiA mutants were significantly reduced in their ability to cause necrosis development in tomato pith. In addition, it was established that PcoR in the absence of AHL also played a role in virulence on tomato. A model for the role of PcoI, PcoR, and RfiA in tomato pith necrosis is presented.

Biological and Molecular Detection of Toxic Lipodepsipeptide-Producing Pseudomonas syringae Strains and PCR Identification in Plants

ABSTRACT Toxin-based identification procedures are useful for differentiating Pseudomonas syringae pathovars. A biological test on peptone-glucose-NaCl agar in which the yeastRhodotorula pilimanae was used proved to be more reliable for detecting lipodepsipeptide-producing strains of P. syringae than the more usual test on potato dextrose agar in which Geotrichum candidum is used. A PCR test performed with primers designed to amplify a 1,040-bp fragment in the coding sequence of the syrD gene, which was assumed to be involved in syringomycin and syringopeptin secretion, efficiently detected the gene in pathovars that produce the lipodepsipeptides. Comparable results were obtained in both tests performed with strains of the syringomycin-producing organisms P. syringae pv. syringae,P. syringae pv. atrofaciens, and P. syringaepv. aptata, but the PCR test failed with a syringotoxin-producingPseudomonas fuscovaginae strain. The specificity of the test was verified by obtaining negative PCR test results for related pathovars or species that do not produce the toxic lipodepsipeptides.P. syringae pv. syringae was detected repeatedly in liquid medium inoculated with diseased vegetative tissue and assayed by the PCR test. Our procedure was also adapted to detect P. syringae pv. morsprunorum with a cfl gene-based PCR test.