On understanding the arsenal of the EU quarantine plant pathogen Curtobacterium flaccumfaciens pv. flaccumfaciens: role of a hypothetical serine protease

Phytopathogenic bacteria have evolved a sophisticated arsenal to successfully colonize their host plants and establish the infection. Recently, the involvement in the pathogenicity processes of several phytopathogenic bacteria of some serine proteases has been demonstrated. The plant pathogenic bacterium Curtobacterium flaccumfaciens pv. flaccumfaciens (Cff), which is the causal agent of bean bacterial wilt and soybean tan spot, harbors a hypothetical pathogenicity island (PAI), encoding for three serine proteases highly homologous to some pathogenicity factors found in Clavibacter michiganensis. The Cff PAI is absent in saprophytic and soilborne Curtobacterium spp. Moreover, Cff strains are unequivocally identified by several PCR-based tests targeting one of these proteases, named S1 serine protease (AN: QIH95653.1). Hence, the hypothesis is that S1 serine protease may contribute to Cff virulence as well as to its transition from a saprophytic to a pathogenic lifestyle. Here for the first time, a protocol was developed for the mutagenesis of the gram-positive quarantine bacterium for Europe Cff. The knock-out mutants of serine protease S1, obtained by marker exchange mutagenesis, showed a role of this protein in Cff virulence on artificially infected bean plants. Furthermore, a significant reduction (50%) of proteolytic activity was demonstrated on mutants by an in vitro test, performed by using skim milk medium. Accordingly, S1 serine protease has been expressed by using the vector pMAL-p5X and Escherichia coli Rosetta strains as recipients. However, purified Cff S1 showed no HR in host and nonhost plants, raising new questions about its actual mechanism as an effector of the Cff arsenal.