ELABORATION OF FLAVONOID-INDUCED PROTEINS BY THE NITROGEN-FIXING SOYBEAN SYMBIONT RHIZOBIUM-FREDII IS REGULATED BY BOTH NODD1 AND NODD2, AND IS DEPENDENT ON THE CULTIVAR-SPECIFICITY LOCUS, NOLXWBTUV

Genistein and other flavonoids from host legumes are known to stimulate cells of the nitrogen-fixing soybean symbiont Rhizobium fredii to synthesize Nod factors, which function as signals during nodule initiation. Flavonoids also trigger R. fredii to secrete a set of signal-responsive (SR) proteins into the environment. By insertion mutagenesis, we showed that secretion of SR proteins by this organism has an absolute dependence on the regulatory gene nodD1. We isolated and sequenced nodal and nodD2 of R, fredii USDA257 and constructed strains containing additional, plasmid-borne copies of these genes. Extra copies of nodD1 had no effect on secretion of SR proteins, but extra copies of nodD2 rendered the process constitutive. Extracts from seeds of the soybean cultivars McCall and Peking can substitute for purified flavonoids as inducers of SR proteins. The nolXWBTUV locus is known to control cultivar-specific nodulation of McCall soybean in a negative, flavonoid-dependent manner. Inactivation of any of these genes prevented SR proteins from accumulating in culture fluids. Protein secretion in response to host signals was a characteristic of nine out of ten R, fredii strains tested. Immunological probes failed to detect SR3 or SRS in mature soybean or cowpea nodules, Although the functions of these proteins remain unknown, their potential role in symbiosis is strengthened by the discovery that their accumulation depends on nodD1, nodD2 and nolXWBTUV.