Two Host-Inducible Genes of Rhizobium fredii and Characterization of the Inducing Compound

Abstract

Random transcription fusions with Mu dl(Kan lac) generated three mutants in Rhizobium fredii (strain USDA 201) which showed induction of I-galactosidase when grown in root exudate of the host plants Glycine max, Phaseolus vulgaris, and Vigna ungliculata. Two genes were isolated from a library of total plasmid DNA of one of the mutants, 3F1. These genes, present in tandem on a 4.2-kilobase Hindlll fragment, appear in one copy each on the symbiotic plasmid and do not hybridize to the Rhizobium meliloti common nodulation region. Regions homologous to both sequences were detected in EcoRI digests of genomic DNAs from B. japonicum USDA 110, USDA 122, and 61A76, but not in genomic DNA from R. trifolii, Rhizobium leguminosarum, or Rhizobium phaseoli. Mass spectrometry and nuclear magnetic resonance analysis indicated that the inducing compound has properties of 4',7-dihydroxyisoflavone, daidzein. These results suggest that, in addition to common nodulation genes, several other genes appear to be specifically induced by compounds in the root exudate of the host plants.The establishment of the Rhizobium-legume symbiosis requires that complex biochemical, physiological, and molecular changes occur in both partners. The most pronounced changes which occur during the formation of nitrogen-fixing nodules are the production of nodulins (10, 24), and the differentiation of rhizobia into nitrogen-fixing bacteroids (45). However, many subtle changes also occur during the early stages of the symbiotic process and likely involve the induction and repression of a large number of bacterial and plant genes (29; see references 32 and 46).While the early periods of the symbiosis have been shown to be important for nodulation and competition (23), most of our knowledge about plant-bacterium interactions has been limited to postinfection events (3). Recently, evidence has accumulated from several laboratories indicating that some form of bacterium-plant communication is important for the early symbiotic steps (3,4,7,8,13,14,17,18,29,37 (7), cause a phenotypic reversion in slow-to-nodulate B. japonicum HS111 (18), induce symbiosis-associated genes in Rhizobium fredii (29), and increase the competitiveness of some B. japonicum strains (3). Both positive and negative interactions of root-or seed-derived compounds (flavanones, flavanols, flavones, and isoflavones) have been shown with the common nodulation genes of Rhizobium meliloti (28, 30), Rhizobium trifolii (22), Rhizobium leguminosarum (49), and B. japonicum (Kosslak et al., in press). Using Mu dl(Kan lac) transcription fusions, we have identified three R. fredii USDA 201 insertion mutants which have symbiosis-related genes specifically induced by soybean root exudate and extract (29). Here we report on the isolation, cloning, and molecular analysis of two host plant-inducible operons in one of these mutants and the characterization of the inducing substance.MATERIALS AND METHODS Bacteria, plasmids, and growth conditions. R. fredii USDA 201 and the Mu dl(Kan lac) insertion mutants (3F...