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 Ca²⁺ levels is observed, suggesting that Ca²⁺ represents an important environmental clue, as reported for bacteria infecting mammalians. We demonstrate that Ca²⁺ entry in Pseudomonas savastanoi pv. savastanoi (Psav) strain DAPP-PG 722 is mediated by a Na⁺/Ca²⁺ exchanger critical for virulence. Using the fluorescent Ca²⁺ probe Fura 2-AM, we demonstrate that Ca²⁺ enters Psav cells foremost when they experience low levels of energy, a situation mimicking the apoplastic fluid. In fact, Ca²⁺ entry was suppressed in the presence of high concentrations of glucose, fructose, sucrose or adenosine triphosphate (ATP). Since Ca²⁺ entry was inhibited by nifedipine and LiCl, we conclude that the channel for Ca²⁺ entry is a Na⁺/Ca²⁺ exchanger. In silico analysis of the Psav DAPP-PG 722 genome revealed the presence of a single gene coding for a Na⁺/Ca²⁺ 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 Ca²⁺ 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 Ca²⁺ entry and pathogenicity in olive plants, but failed to restore the HR in tobacco leaves. In conclusion, Ca²⁺ entry acts as a ‘host signal’ that allows and promotes Psav pathogenicity on olive plants.