Summary 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.

Calcium signaling is an emerging mechanism by which bacteria respond to environmental cues. To measure the intracellular free-calcium concentration in bacterial cells, [Ca 2+ ]i, a simple spectrofluorometric method based on the chemical probe Fura 2-acetoxy methyl ester (Fura 2-AM) is here presented using Pseudomonad bacterial cells. This is an alternative and quantitative method that can be completed in a short period of time with low costs, and it does not require the induction of heterologously expressed protein-based probes like Aequorin. Furthermore, it is possible to verify the properties of membrane channels involved in Ca 2+ entry from the extracellular matrix. This method is in particular valuable for measuring [Ca 2+ ]i in the range of 0.1-39.8 µM in small cells like those of prokaryotes.