Regulation of the soybean-Rhizobium nodule symbiosis by shoot and root factors

The availability of soybean mutants with altered symbiotic properties allowed an investigation of the shoot or root control of the relevant phenotype. By means of grafts between these mutants and wild-type plants (cultivar Bragg and Williams), we demonstrated that supernodulation as well as hypernodulation (nitrate tolerance in nodulation and lack of autoregulation) is shoot controlled in two mutants (nts382 and nts1116) belonging most likely to two separate complementation groups. The supernodulation phenotype was expressed on roots of the parent cultivar Bragg as well as the roots of cultivar Williams. Likewise it was shown that non-nodulation (resistance to Bradyrhizobium) is root controlled in mutant nod49. The shoot control of nodule initiation is epistatically suppressed by the non-nodulation, root-expressed mutation. These findings suggest that different plant organs can influence the expression of the nodulation phenotype.     

Regulation of nodulation in Alnus incana-Frankia symbiosis

We have studied regulation of nodulation in Alnus incana (L.) Moench using double inoculations in plastic pouches and a slide technique to observe root hair deformation. Initially, the distribution of nodules between main and lateral roots appeared quite constant, independent of the concentration of inoculum (1 to 250 μg of crushed nodules plant⁻¹). Susceptibility to infection after the second inoculation was restricted to lateral roots after the initial infections developed. When pre-existing nodules were excised before the second inoculation, subsequent nodules appeared to arise where infections had arrested at stages earlier than actual nodule emergence. We observed that root hairs formed postinoculation were very crowded and short with a pronounced deformation. No nodules were found later on this region of the root, suggesting a loss of susceptibility in this region. Split-root experiments with delays between inoculation of the first and second side of the root system showed irreversible, systemic inhibition of nodulation on the second side starting between 3 and 6 days after the inoculation of the first side. Only when compatible, infective strains were used in the first inoculation, was nodule formation inhibited after the second inoculation. We conclude that autoregulation of nodulation operates in Alnus incana and on a time scale similar to what is found in some legumes.     

Some USDA studies on the soybean-Rhizobium symbiosis

Summary The ecology, strain evaluation, genetics of host strain interactions and physiology of nitrogen fixation ofRhizobium japonicum in association with the soybean,Glycine max, were studied. Results of inoculation experiments with selected strains ofRhizobium japonicum indicated that indigenous strains occupied most of the nodules of soybeans grown in highRhizobium japonicum populated soils. Nodule sampling indicated that inoculation did not result in quicker nodulation or a higher incidence of root nodules (primary or secondary) than uninoculated checks. Rhizosphere studies indicated that colonization by introduced strains did occur but did not compete successfully with field strains for nodule sites. Recovery of specific serological types from nodules was influenced by planting intervals. The distribution of the serotypes varied with the time of planting and the age of the plant. Temperature studies indicated that the distribution of serotypes recovered from the nodules was influenced by temperature. Field studies showed the selectivity of soybean genotypes on strains ofRhizobium japonicum. Some strains were more common in the nodules of some varieties than in others. Closely related varieties had similar populations in their nodules. Three genes which control nodule response in soybeans are reported. Nitrogen fixation profiles were determined for some variety-strain interactions. Combinations previously classified as inefficient showed some nitrogenase activity as measured by the acetylene reduction technique. Research Microbiologist; Research Agronomist; Research Plant Physiologist, Soybean Investigations, Crops Research Division, Beltsville, Md. (USDA, ARS); and Plant Pathologist currently located at Michigan State University, East Lansing, Michigan.     

Review: Infection and nodulation signalling in Rhizobium-Phaseolus vulgaris symbiosis

During the Rhizobium–legume symbiosis, a mutual exchange of signalling molecules occurs. Distinct oligo- and polysaccharides are involved in nodule formation and rhizobial invasion. The common bean is a promiscuous host plant that can be nodulated by a wide range of rhizobia. Reviewing the literature on nodulation suggests that the Nod factor oligosaccharide backbone of bean-nodulating rhizobia does not require a specific attached group, except for the acyl chain at the non-reducing end. However, in Rhizobium strains that elicit nitrogen-fixing nodules on Phaseolus vulgaris and that produce methylated Nod factors, NodS mediated decorations are indispensable for invasion and/or subsequent nitrogen-fixation. Finally, we present a model that links the pathways for methylation and sulphation in nodule signalling and invasion processes.     

Host recognition in the Rhizobium-soybean symbiosis : evidence for the involvement of lectin in nodulation

Rhizobium japonicum mutant strain HS111 was previously shown to be defective in the rate of initiation of infection leading to subsequent nodule formation (1984 Plant Physiol 74: 84-89). Mutant strain HS111's defect in nodulation can be phenotypically reversed to wild type levels by pretreatment with root exudates from all soybean varieties that have been tested. The data indicate that lectin-Rhizobium interaction is necessary for the phenotypic reversal of the nodulation characteristics of mutant strain HS111. Pretreatment of strain HS111 with soybean seed lectin mimics the effect of root exudate pretreatment. In addition, the presence of 30 millimolar d-galactose, a hapten of soybean seed lectin, in the root exudate or soybean seed lectin pretreatment solution prevents enhancement of nodulation of strain HS111. Pretreatment of mutant strain HS111 in soybean root exudate which has had galactose-specific lectin(s) removed by affinity chromatography (affinity eluate) results in no enhancement of nodulation by strain HS111. Lectin(s) subsequently removed from the affinity column possesses 100% of the stimulatory activity originally found in the root exudate. Pretreatment of strain HS111 in root exudate from a soybean seed line (T102) known to lack seed lectin due to an insertion in the structural gene results in the reversal of the defective nodulation phenotype. This latter result indicates that the lectin found in soybean root exudate is genetically distinct from the seed lectin. It is apparently this root lectin that is involved in nodulation.     

Structural determination of bacterial nodulation factors involved in the Rhizobium meliloti-alfalfa symbiosis

Extracellular signals produced by Rhizobium meliloti are able to induce root hair deformations and nodule organogenesis on alfalfa. The production of these signals is controlled by bacterial nod genes. To enable their isolation in significant amounts, an overproducting strain was constructed. These Nod factors were first extracted by butanol from the culture medium and further purified by reverse-phase high performance liquid chromatography, ion-exchange, and Sephadex LH-20 chromatographies. The structure of the major signal, called NodRm-1, was determined by mass spectrometry, nuclear magnetic resonance, 35S labeling, chemical analysis, and enzymatic degradation, and was shown to be a sulfated and acylated tetramer of glucosamine namely, beta-D-GlcpN(2,9-hexadecadie-noyl) - (1----4) - beta - D - Glc p Nac - (1----4) - beta - D - Glc p NAc - (1----4) - D - GlcpNAc-6-SO3H. Another Nod factor (called Ac-NodRm-1) was co-purified and identified as NodRm-1 acetylated on the C-6 of the nonreducing end sugar. NodRm-1 elicits root hair deformation specifically on alfalfa at a concentration less than 10(-10) M but has no effect on vetch (a heterologous host plant).     

Soil P-status and cultivar maturity effects on pea-Rhizobium symbiosis

In a green-house experiment, five cultivars of Pisum sativum L. grown on soils from 10 different locations in Tunisia, showed significant differences in nodulation, shoot dry matter (shDM) yield and shoot nitrogen content (shNC). The effect of soil on biological nitrogen fixation, as evidenced by the number and weight of nodules, was mainly attributable to the available phosphorus content. Cate-Nelson ANOVA analysis established a critical value of soil test phosphorus (STP) of 20 mg P kg^–1 soil for nodule weight and number for the majority of cultivars. Within cultivars, nodulation varied with maturation period and was correlated with shoot NC. Thus, the overall interaction of soil-P content and cultivar-maturation period were correlated positively with nodulation and to symbiotic effectiveness of strains of Rhizobium leguminosarum bv. viceae indigenous to these soils. Based on an antibiotic susceptibility test and main variable factor analysis of the data obtained, 70 isolates of Rhizobia that nodulate pea, obtained from soils from agricultural sites throughout Tunisia, were identified as belonging to 18 distinct strains. These classes were identified on the basis of symbiotic efficiency parameters (shoot DM yield and shoot NC) as: ineffective (33 isolates), moderately effective (27 isolates), and efficient strains (10 isolates). This study shows that the Mateur site, an agricultural area for millennia in the northern region of Tunisia, harbors rhizobial strains that are highly efficient in fixing N₂ with peas. These results also indicate the importance of strain-cultivar interrelationships and specificity.     

Identification and cloning of the bacterial nodulation specificity gene in the Sinorhizobium meliloti--Medicago laciniata symbiosis

Medicago laciniata (cut-leaf medic) is an annual medic that is highly nodulation specific, nodulating only with a restricted range of Sinorhizobium meliloti. e.g., strain 102L4, but not with most strains that nodulate Medicago sativa (alfalfa), e.g., strains RCR2011 and Rm41. Our aim was to identify and clone the S. meliloti 102L4 gene implicated in the specific nodulation of M. laciniata and to characterize the adjacent nodulation (nod) region. An 11-kb EcoRI DNA fragment from S. meliloti 102L4 was shown to complement strain RCR2011 for nodulation of M. laciniata. Nucleotide sequencing revealed that this fragment contained nodABCIJ genes whose overall arrangement was similar to those found in strains RCR2011 and Rm41, which do not nodulate M. laciniata. Data for Tn5 mutagenesis of the nodABCIJ region of strain 102L4 suggested that the nodC gene was involved in the specific nodulation of M. laciniata. Tn5 insertions in the nodIJ genes gave mutants with nodulation delay phenotypes on both M. laciniata and M. sativa. Only subclones of the 11-kb DNA fragment containing a functional nodC gene from strain 102L4 were able to complement strain RCR2011 for nodulation of M. laciniata. The practical implications of these findings are discussed in the context of the development of a specific M. sativa - S. meliloti combination that excludes competition for nodulation by bacterial competitors resident in soil.     

Regulation of nodulation inRhizobium leguminosarum

World Journal of Microbiology and Biotechnology -     

Root nodulation: a paradigm for how plant-microbe symbiosis influences host developmental pathways

Legume plants have an exceptional capacity for association with microorganisms, ranging from largely nonspecific to very specific interactions. Legume-rhizobial symbiosis results in major developmental and metabolic changes for both the microorganism and host, while providing the plant with fixed nitrogen. A complex signal exchange leads to the selective rhizobial colonization of plant cells within nodules, new organs that develop on the roots of host plants. Although the nodulation mechanism is highly specific, it involves the same subset of plant phytohormones, namely auxin, cytokinin, and ethylene, which are required for root development. In addition, nodulation triggered by the rhizobia affects the development of the host root system, indicating that the microorganism can alter host developmental pathways. Nodulation by rhizobia is a prime example of how microorganisms and plants have coevolved and exemplifies how microbial colonization may affect plant developmental pathways.     

Comparison of twoPisum sativum nodulation mutants with their parental cultivar

In comparison with the parental cv. Finale the ‘RisfixC’ supernodulator exhibited higher, continuously increasing nodule number and fresh mass accumulation, but substantially lower individual nodule fresh mass, leghemoglobin concentration, and specific acetylene reduction activity of nodule tissue. There were no substantial differences between Finale and ‘RisfixC’ in total acetylene reduction, nodule leghemoglobin accumulation per nodulated root, total and specific CO₂ evolution from nodulated roots and gross CO₂ respiratory costs of acetylene reduction. The ‘RisfixC’ also exhibited a substantially lower plant dry mass production (by 30%), but nitrogen concentration in shoots and carotenoid concentration in leaf tissue were significantly higher by 33 and 14%, and the chlorophylla+b content insignficantly higher than in the parental cultivar. In contrast, the nodulation mutant ‘Risnod29’, exhibited a somewhat higher nodule fresh mass accumulation (by 21%) and individual nodule fresh mass (by 23%), total and specific acetylene reduction (by 49 and 19%) and a somewhat more rapid plant dry mass accumulation compared with the cv. Finale.     

Regulation of the plant defence response in arbuscular mycorrhizal symbiosis

The response of plants to arbuscular mycorrhizal fungi involves a temporal and spatial activation of different defence mechanisms. The activation and regulation of these defences have been proposed to play a role in the maintenance of the mutualistic status of the association, however, how these defences affect the functioning and development of arbuscular mycorrhiza remains unclear. A number of regulatory mechanisms of plant defence response have been described during the establishment of the arbuscular mycorrhizal symbiosis, including elicitor degradation, modulation of second messenger concentration, nutritional and hormonal plant defence regulation, and activation of regulatory symbiotic gene expression. The functional characterization of these regulatory mechanisms on arbuscular mycorrhiza, including cross-talk between them, will be the aim and objective of future work on this topic.     

Nodulation gene regulation and quorum sensing control density-dependent suppression and restriction of nodulation in the Bradyrhizobium japonicum-soybean symbiosis

The nodulation of Glycine max cv. Lambert and the nodulation-restricting plant introduction (PI) genotype PI 417566 by wild-type Bradyrhizobium japonicum USDA110 is regulated in a population-density-dependent manner. Nodulation on both plant genotypes was suppressed (inhibited) when plants received a high-density inoculum (10(9) cells/ml) of strain USDA110 grown in complex medium, and more nodules were produced on plants receiving a low-cell-density inoculum (10(5) cells/ml). Since cell-free supernatants from strain USDA110 grown to high cell density in complex medium decreased the expression of an nodY-lacZ fusion, this phenomenon was attributed to bradyoxetin-induced repression of nod gene expression. Inoculation of either the permissive soybean genotype (cv. Lambert) or PI 417566 with 10(9) cells/ml of the nodD2, nolA, nodW, and nwsB mutants of USDA110 enhanced nodulation (up to 24%) relative to that seen with inoculations done with 10(5) cells/ml of the mutants or the wild-type strain, indicating that these genes are involved in population-density-dependent nodulation of soybeans. In contrast, the number of nodules produced by an nodD1 mutant on either soybean genotype was less than those seen with the wild-type strain inoculated at a low inoculum density. The nodD2 mutant outcompeted B. japonicum strain USDA123 for nodulation of G. max cv. Lambert at a high or low inoculum density, and the results of root-tip-marking and time-to-nodulate studies indicated that the nolA and nodD2 mutants nodulated this soybean genotype faster than wild-type USDA110. Taken together, the results from these studies indicate that the nodD2 mutant of B. japonicum may be useful to enhance soybean nodulation at high inoculum densities and that NodD2 is a key repressor influencing host-controlled restriction of nodulation, density-dependent suppression of nodulation, perception of bradyoxetin, and competitiveness in the soybean-B. japonicum symbiosis.     

Lectins and the soybean-Rhizobium symbiosis: I. Immunological investigations of soybean lines,the seeds of which have been reported to lack the 120 000 dalton soybean lectin

Seeds of six soybean lines (Glycine max (L.) Merr. cv. Columbia, D68-127, Norredo, Sooty, T-102, Wilson 5) have been reported to lack the 120 000 dalton soybean lectin. Immunofiffusion and radioimmunoassay using anti-soybean lectin immunoglobulin failed to detect the lectin in seeds of five lines, but D68-127 seeds contained as much soybean lectin as the control line, Harosoy 63. The D68-127 seed lactin could be purified by affinity chromatography on Sepharose-N-caproylgalactosamine, and was indistinguishable from the conventional soybean lectin by the following criteria: electrophoretic migration in acidic and alkaline buffers, subunit molecular weight and composition, analytical isoelectric focusing, gel filtration chromatography.Phosphate buffered saline extracts of roots, hypocotyls, stems, and leaves of 3–66-day-old Norredo and Harosoy 63 plants lacked soybean lectin, as determined by hemagglutination and radioimmunoassay (detection limit: 1.4 μg soybean lectin/g dry weight tissue). Cotyledons of Harosoy 63 (but not Norredo) contained large quantities of the lectin, which diminished as the plants aged. 5-day-old roots and hypocotyls of 20 soybean lines did not contain soybean lectin. Roots of Columbia, Norredo, Sooty, T-102, Wilson 5, and Harosoy 63 (control) were modulated by a variety of strains of Rhizobium japonicum and Rhizobium sp.