A Na+/Ca2+ exchanger of the olive pathogen Pseudomonas savastanoi pv. savastanoi is critical for its virulence

In a number of compatible plant-bacterium interactions, a rise in apoplastic Ca 2+ levels is observed, suggesting that Ca 2+ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca 2+ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na + /Ca 2+ exchanger critical for virulence. Using the fluorescent Ca 2+ probe Fura 2-AM, we demonstrate that Ca 2+ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca 2+ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca 2+ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca 2+ entry is a Na + /Ca 2+ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na + /Ca 2+ exchanger (cneA), which is a widely conserved and ancestral gene within the P. syringae complex based on gene phylogeny. Mutation of cneA compromised not only Ca 2+ entry, but also compromised the Hypersensitive response (HR) in tobacco leaves and blocked the ability to induce knots in olive stems. The expression of both pathogenicity (hrpL, hrpA and iaaM) and virulence (ptz) genes was reduced in this Psav-cneA mutant. Complementation of the Psav-cneA mutation restored both Ca 2+ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca 2+ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.