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Rhizobia, soil bacteria of the Rhizobiales, enter the roots of homologous legumes, where they induce the formation of nitrogen-fixing nodules. Signals emanating from both symbiotic partners control nodule development. Efficient nodulation requires precise, temporal regulation of symbiotic genes. Roots continuously release flavonoids that interact with transcriptional activators of the LysR family. NodD proteins, which are members of this family, act both as sensors of the environment and modulate the expression of genes preceded by conserved promoter sequences called nod-boxes. The symbiotic plasmid of the broad host-range Rhizobium sp. NGR234 caries 19 nod-boxes (NB1 to NB19), all of which were cloned upstream of a lacZ-reporter gene. A flavonoid, daidzein was able to induce 18 of the 19 nod-boxes in a NodD1-dependent manner. Interestingly, induction of four nod-boxes (NB6, NB15, NB16 and NB17) is highly dependent on NodD2 and was delayed in comparison with the others. In turn, NodD2 is involved in the repression of the NB8 nodABCIJnolOnoeI operon. Activation of transcription of nodD2 is also dependent on flavonoids despite the absence of a nod-box like sequence in the upstream promoter region. Mutational analysis showed that syrM 2 (another member of the LysR family), which is controlled by NB19, is also necessary for expression of nodD 2. Thus, NodD1, NodD2 and SyrM2 co-modulate a flavonoid-inducible regulatory cascade that coordinates the expression of symbiotic genes with nodule development.