Legume–rhizobium symbiosis requires a complex dialogue based on the exchange of diffusible signals between the partners. Compatible rhizobia express key nodulation (nod) genes in response to plant signals – flavonoids – before infection. Host plants sense counterpart rhizobial signalling molecules – Nod factors – through transient changes in intracellular free‐calcium. Here we investigate the potential involvement of Ca²⁺ in the symbiotic signalling pathway activated by flavonoids in Rhizobium leguminosarum bv. viciae.

By using aequorin‐expressing rhizobial strains, we monitored intracellular Ca²⁺ dynamics and the Ca²⁺ dependence of nod gene transcriptional activation.

Flavonoid inducers triggered, in R. leguminosarum, transient increases in the concentration of intracellular Ca²⁺ that were essential for the induction of nod genes. Signalling molecules not specifically related to rhizobia, such as strigolactones, were not perceived by rhizobia through Ca²⁺ variations. A Rhizobium strain cured of the symbiotic plasmid responded to inducers with an unchanged Ca²⁺ signature, showing that the transcriptional regulator NodD is not directly involved in this stage of flavonoid perception and plays its role downstream of the Ca²⁺ signalling event.

These findings demonstrate a key role played by Ca²⁺ in sensing and transducing plant‐specific flavonoid signals in rhizobia and open up a new perspective in the flavonoid–NodD paradigm of nod gene regulation.