CHASE-independent cytokinin perception triggers 3′,5′-cAMP signaling in <i>Sinorhizobium meliloti</i>

The <i>Medicago sativa-Sinorhizobium meliloti</i> symbiotic plant-microbe interaction, which results in the formation of nitrogen-fixing root nodules, is subject to sophisticated genetic and metabolic regulation by both partners. <i>S. meliloti</i> is capable of inhibiting secondary plant infections via an adenosine 3',5'-cyclic adenosine monophosphate (cAMP)-dependent regulatory pathway that depends on CHASE2 domain adenylate/guanylate cyclases (AC/GCs). This pathway likely responds to a plant signal of protein nature. Plant cytokinins (CKs) are adenine derivative phytohormones that control many aspects of plant development, including the symbiotic nodule formation. Classical CK receptors in plants and bacteria contain a CHASE domain. In our study, we present a novel, CK-dependent cAMP signaling pathway, specifically mediated by the AC/GC CyaB, which lacks any known receptor domains. The plant CKs N⁶(Δ²isopentenyl)-adenine (iP), trans-zeatin, kinetin, and 6-benzylaminopurine all promoted CyaB-dependent increase in cAMP levels detected through a genetic reporter construct. Among these four CKs, iP exerted the strongest effect. Metabolic profiling confirmed the CyaB-dependent accumulation of cAMP in <i>S. meliloti</i> cells, cultured in the presence of iP. The first enzyme in the terpenoid biosynthetic pathway, 1-deoxyxylulose-5-phosphate synthase Dxs, was identified as a CyaB interaction partner and is proposed to mediate the CK perception. CyaB homologs from closely related members of the <i>Rhizobiaceae</i> were able to interact with Dxs and to mediate cAMP signaling in response to iP.IMPORTANCESymbiotic interactions between nitrogen-fixing bacteria and leguminous plants are important for agriculture, ecological sustainability, and human nutrition. Maintaining an optimal number of symbiotic infections per plant is crucial for efficient symbiosis. Previous studies have shown that <i>S. meliloti</i> 3',5'-cyclic adenosine monophosphate (cAMP) signaling mediates the inhibition of secondary symbiotic infections of <i>Medicago</i> plants. We discovered a molecular mechanism that allows the symbiotic bacterium <i>Sinorhizobium meliloti</i> to respond to the <i>Medicago</i> plant adenosine derivative phytohormones named cytokinins (CKs) via cAMP signaling. This mechanism is mediated by the adenylate/guanylate cyclase CyaB. CyaB lacks any sensory domains and may perceive the CKs via its interaction partner deoxyxylulose-5-phosphate synthase Dxs.