Expression of nodD1 and nodD2 in Sinorhizobium fredii, a Nitrogen-Fixing Symbiont of Soybean and Other Legumes

Abstract

The ability of Sinorhizobium fredii strains USDA191 and USDA257 to form nitrogen-fixing root nodules on legume plants is regulated by nodD1 and nodD2, which sense flavonoid signals from host roots and then activate the expression of inducible nodulation genes. We assessed the interactions between these two loci with nodD1-negative and nodD2-negative mutants and with strains containing extra copies of these genes. Although both nodD1 and nodD2 are expressed constitutively, levels of nodD1 are much higher, as measured by RNA dot blots. Extra plasmid-borne copies of nodD2 reduced transcription of nodD1 to below the level of detection. We employed gel mobility shift assays to demonstrate that cellular proteins from flavonoid-treated cultures of strain USDA191 bind to DNA sequences that lie upstream from nodD1, but not to the corresponding region upstream from nodD2. Extra plasmid-borne copies of nodD2 enhanced protein binding to the nodD1-associated region and rendered the process flavonoid independent. DNase I footprinting analysis and gel retardation experiments with an oligonucleotide DNA probe localized the protein-binding site to a nod box-like sequence that lies just upstream from the nodD1 coding region. Antibodies raised against a NodD2 fusion protein and capable of reacting both with NodD1 and NodD2 blocked formation of the retarded protein-DNA complex.