© 2014 BSPP AND JOHN WILEY & SONS LTD. In a recent screen for novel virulence factors involved in the interaction between Pseudomonas savastanoi pv. savastanoi and the olive tree, a mutant was selected that contained a transposon insertion in a putative cyclic diguanylate (c-di-GMP) phosphodiesterase-encoding gene. This gene displayed high similarity to bifA of Pseudomonas aeruginosa and Pseudomonas putida. Here, we examined the role of BifA in free-living and virulence-related phenotypes of two bacterial plant pathogens in the Pseudomonas syringae complex, the tumour-inducing pathogen of woody hosts, P.savastanoi pv. savastanoiNCPPB 3335, and the pathogen of tomato and Arabidopsis, P.syringae pv. tomatoDC3000. We showed that deletion of the bifA gene resulted in decreased swimming motility of both bacteria and inhibited swarming motility of DC3000. In contrast, overexpression of BifA in P.savastanoi pv. savastanoi had a positive impact on swimming motility and negatively affected biofilm formation. Deletion of bifA in NCPPB 3335 and DC3000 resulted in reduced fitness and virulence of the microbes in olive (NCPPB 3335) and tomato (DC3000) plants. In addition, real-time monitoring of olive plants infected with green fluorescent protein (GFP)-tagged P.savastanoi cells displayed an altered spatial distribution of mutant ΔbifA cells inside olive knots compared with the wild-type strain. All free-living phenotypes that were altered in both ΔbifA mutants, as well as the virulence of the NCPPB 3335 ΔbifA mutant in olive plants, were fully rescued by complementation with P.aeruginosaBifA, whose phosphodiesterase activity has been demonstrated. Thus, these results suggest that P.syringae and P.savastanoiBifA are also active phosphodiesterases. This first demonstration of the involvement of a putative phosphodiesterase in the virulence of the P.syringae complex provides confirmation of the role of c-di-GMP signalling in the virulence of this group of plant pathogens.

This research was supported by the Spanish Plan Nacional I+D+i grant AGL2011-30343-CO2-01. IMA was supported by an Formación del Profesorado Universitario (FPU) fellowship (Ministerio de Ciencia e Innovación/Ministerio de Economía y Competitividad, Madrid, Spain).

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