Rhizobium meliloti has three functional copies of the nodD symbiotic regulatory gene

We have identified two Rhizobium meliloti genes (nodD2 and nodD3) that are highly homologous and closely linked to the regulatory gene nodD (nodD1). R. meliloti strains containing mutations in the three nodD genes in all possible combinations were constructed and their nodulation phenotypes were assayed on Medicago sativa (alfalfa) and Melilotus alba (sweet clover). A triple nodD1-nodD2-nodD3 mutant exhibited a Nod- phenotype on alfalfa and sweet clover, indicating that nodD is an essential nodulation gene in R. meliloti. A nodD2 mutant exhibited no discernable defect in nodulation and nodD3 mutants exhibited a delayed nodulation phenotype of 2-3 days when inoculated onto either host. Alfalfa nodules elicited by a nodD1 mutant appeared 5-6 days after wild-type nodules, and sweet clover nodules elicited by a nodD1 mutant appeared 2-3 days after wild-type nodules. nodD1-nodD2 double mutants formed nodules with the same delay as single nodD1 mutants on both hosts. nodD2-nodD3 double mutants elicited sweet clover nodules at the same rate as single nodD3 mutants, but this same double mutant was slightly more delayed in alfalfa nodule formation than the nodD3 mutant. The nodD1-nodD3 mutant exhibited an extremely delayed nodulation phenotype on alfalfa and elicited no nodules on sweet clover. These experiments indicate that nodD1 and nodD3 have equivalent roles in nodulating sweet clover but that nodD1 plays a more important role than nodD3 in eliciting nodules on alfalfa. The nodD2 gene appears to have some effect on alfalfa nodulation and none on sweet clover. Our results indicate that R. meliloti has three functional nodD genes that modulate the nodulation process in a host-specific manner.