The early events in legume nodulation by Rhizobium spp. involve a conserved gene cluster known as the common nod region. A broad-host-range plasmid (pEA2-21) containing a Bradyrhizobium japonicum nodDABC-acZ translational fusion was constructed and used to monitor nod gene expression in response to soybean root extract. Two inducing compounds were isolated and identified. Analysis using ultraviolet absorption spectra, proton nuclear magnetic resonance, and mass spectrometry showed that the two inducers were 4',7-dihydroxyisoflavone (daidzein) and 4',5,7-trihydroxyisoflavone (genistein). Induction was also seen with some, but not all, of the flavonoid compounds that induce nod genes in fast-growing Rhizobium strains that nodulate clover, alfalfa, or peas. When pEA2-21 was introduced into Rhizobium trifohii, it was inducible by flavones but not by daidzein and genistein. In Rhizobium fredii, pEA2-21 was induced by isoflavones and flavones. Thus, the specificity of induction appears to be influenced by the host-strain genome.Members of the bacterial genus Rhizobium form symbiotic associations with leguminous plants that result in the formation of nitrogen-fixing root nodules. In three agronomically important Rhizobium/legume associations, R. trifolii/clover, R. meliloti/alfalfa, and R. leguminosarum/pea, important bacterial nodulation genes (1-4) and plant compounds that induce them (5-7) have been identified. In these associations flavones (5-7) or flavanones (7) have been found to induce the nodABC genes, as well as other nod genes involved in host specificity (1). Isoflavones have been found to inhibit the induction of nodABC in R. leguminosarum (7).The Bradyrhizobium japonicum/soybean symbiosis is of considerable agricultural importance. In contrast to Rhizobium spp., Bradyrhizobium species are slow-growing (8), nitrogen-fixation and nodulation genes are located on the chromosome (9) and not on plasmids (10)(11)(12), and less is known about the genetic requirements for nodulation (13)(14)(15). In particular, the compounds produced by the soybean host that interact with the common nod genes have not been characterized.In this study, a nodABC-lacZ translational fusion was used to monitor nod gene expression in B. japonicum in response to soybean root extract. Two major components from soybeans (Glycine max cv. Williams) were isolated that induced the expression of the nodABC-lacZ fusion when it was present in the soybean-nodulating bacteria B. japonicum and Rhizobium fredii, but not when it was present in R. trifolii. MATERIALS AND METHODSStrains and Plasmids. Standard procedures (16) were used for DNA manipulations. A HindIII fragment containing the nod region of B. japonicum USDA 123 was cloned in both orientations into the single HindIII site of plC19R (17). BamHI-Bgl II fragments containing the nod genes and flanking polylinker sequences were then cloned into the BamHI site of the broad-host-range vector pGD926 (18) resulting in pEA2-21 and pEA4-10 (Fig. 1) (19) agar and germinated for 3 days in the d...

In a split-root system of soybeans (Glycine max L. Merr), inoculation of one half-side suppressed subsequent development of nodules on the opposite side. At zero time, the first side of the split-root system of soybeans received Rhizobium japonicum strain USDA 138 as the primary inoculum. At selected time intervals, the second side was inoculated with the secondary inoculum, a mixture of R. japonicum strain USDA 138 and strain USDA 110. In a short-day season, nodulation by the secondary inoculum was inhibited 100% when inoculation was delayed 10 days. Nodulation on the second side was significantly suppressed when the secondary inoculum was delayed for only 96 hours. In a long-day season, nodule suppression on the second side was highly significant, but not always 100%. Nodule suppression on the second side was not related to the appeaance of nodules or nitrogenase activity on the side of splitroots which were inoculated at zero time. When the experiments were done under different light intensities, nodule suppression was significantly more pronounced in the shaded treatments.The nitrogen-fixing symbiotic association formed between leguminous plants and soil bacteria of the genus Rhizobium has recently become an area of intense scientific research because of the economic and agricultural benefits derived from cropping systems using nodulated legumes. One of the specific goals of recent investigations has been to understand the mechanisms involved in the formation of the legume-Rhizobium association. Although both the host plant and the bacteria contribute to the specificity of the association (6), the mechanisms by which each partner exerts its influence remain poorly understood (1,4,13

The soybean root necrosis (rn) mutation causes a progressive browning of the root soon after germination that is associated with accumulation of phytoalexins and pathogenesis-related proteins and an increased tolerance to root-borne infection by the fungal pathogen, Phytophthora sojae. Grafting and decapitation experiments indicate that the rn phenotype is root-autonomous at the macroscopic level. However, the onset and severity of browning was modulated in intact plants by exposure to light, as was the extent of lateral root formation, suggesting that both lateral roots and the rn phenotype could be directly or indirectly controlled by similar shoot-derived factors. Browning first occurs in differentiated inner cortical cells adjacent to the stele and is preceded by a wave of autofluorescence that emanates from cortical cells opposite the xylem poles and spreads across the cortex. Before any visible changes in autofluorescence or browning, fragmented DNA was detected by TUNEL (Terminal deoxynucleotidyl transferase-mediated dUTP-digoxigenin nick end labeling) in small clusters of inner cortical cells that subsequently could be distinguished cytologically from neighboring cells throughout rn root development. Inner cortical cells overlying lateral root primordia in either Rn or rn plants also were stained by TUNEL. Features commonly observed in animal cell apoptosis were confirmed by electron microscopy but, surprisingly, cells with a necrotic morphology were detected alongside apoptotic cells in the cortex of rn roots when TUNEL-positive cells were first observed. The two morphologies may represent different stages of a common pathway for programmed cell death (pcd) in plant roots, or two separate pathways of pcd could be involved. The phenotype of rn plants suggests that the Rn gene could either negatively regulate cortical cell death or be required for cortical cell survival. The possibility of a mechanistic link between cortical cell death in rn plants and during lateral root emergence is discussed.

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...

The effect of several biotic and abiotic factors on the pattern of competition between two strains of Rhizobium japonicum was examined. In two Minnesota soils, Waseca and Waukegan, strain USDA 123 occupied 69% (Waseca) and 24% (Waukegan) of the root nodules on Glycine max L. Merrill cv. Chippewa. USDA 110 occupied 2% of the root nodules in the Waseca soil and 12% of the nodules in the Waukegan soil. Under a variety of other growth conditions-vermiculite, vermiculite amended with Waseca soil, and two Hawaiian soils devoid of naturalized Rhizobium japonicum strains-USDA 110 was more competitive than USDA 123. The addition of nitrate to or the presence of antibiotic-producing actinomycetes in the rhizosphere of soybeans did not affect the pattern of competition between the two strains. However, preexposure of young seedlings to USDA 110 or USDA 123 before transplantation into soil altered the pattern of competition between the two strains significantly. In the Waseca soil, preexposure of cv. Chippewa to USDA 110 for 72 h increased the percentage of nodules occupied by USDA 110 from 2 to 55%. Similarly, in the Hawaiian soil Waimea, nodule occupancy by USDA 123 increased from 7 to 33% after a 72-h preexposure.