Fine mapping of the Rj4 locus,a gene controlling nodulation specificity in soybean

Leguminous plants have the ability to make their own nitrogen fertilizer by forming a root nodule symbiosis with nitrogen-fixing soil bacteria, collectively called rhizobia. This biological process plays a critical role in sustainable agriculture because it reduces the need for external nitrogen input. One remarkable property of legume–rhizobial symbiosis is its high level of specificity, which occurs at both inter- and intra-species levels and takes place at multiple phases of the interaction, ranging from initial bacterial infection and nodulation to late nodule development associated with nitrogen fixation. Knowledge of the molecular mechanisms controlling symbiotic specificity will facilitate the development of new crop varieties with improved agronomic potential for nitrogen-fixing symbiosis. In this report, we describe fine mapping of the Rj4 locus, a gene controlling nodulation specificity in soybean (Glycine max). The Rj4 allele prevents the host plant from nodulation with many strains of Bradyrhizobium elkanii, which are frequently present in soils of the southeastern USA. Since B. elkanii strains are poor symbiotic partners of soybean, cultivars containing an Rj4 allele are considered favorable. We have delimited the Rj4 locus within a 57-kb genomic region on soybean chromosome 1. The data reported here will facilitate positional cloning of the Rj4 gene and the development of genetic markers for marker-assisted selection in soybean.     

Silicon promotes nodule formation and nodule function in symbiotic cowpea (Vigna unguiculata)

Applying silicon in the form of metasilicic acid (H₄SiO₃) or silicic acid (H₄SiO₃) to Bradyrhizobium -infected, hydroponically grown cowpea seedlings resulted in a significant ( P 0.05) increase in the number of nodules, nodule dry matter, and nitrogen fixed on a per plant basis. Total dry matter of plants increased with silicon supply, and the differences were significant ( P 0.05) at the higher silicon concentrations. Cowpea plants cultured in sand were also assessed for their response to silicic acid. Nodule number and nodule mass increased with silicon supply to sand cultured plants, though nitrogen fixation was unaltered. Although silicon is not essential for growth of cowpea, it is important for nodule formation and nodule functioning in hydroponically grown plants. Consequently, data collected and conclusions drawn from earlier glasshouse experiments, which have excluded silicon from nutrient solutions, may be flawed. Future studies on nodulation and nitrogen fixation using legumes in liquid culture must therefore include silicon as a nutrient element.     

Responses of Rj(1) and rj(1) Soybean Isolines to Inoculation with Bradyrhizobium japonicum

We evaluated the symbiotic phenotypes of nodulation-restrictive and normal soybean isolines by inoculating Clark (genotypically Rj₁Rj₁) and mutant Clark-rj₁ (genotypically rj₁rj₁) seedlings in plastic growth pouches. Nodules first appeared on Clark seedlings inoculated with Bradyrhizobium japonicum USDA 94 after 6 days. The mean number of nodules per plant was 13.9 ± 0.8 after 24 days. In contrast, Clark-rj₁ seedlings first nodulated at 12 days, and the mean number of nodules per plant was only 1.7 ± 0.3 at 24 days. Segments from infectible zones of primary roots, i.e. near the position occupied by the root tip at the time of inoculation, were sectioned serially. Clark roots contained cortical cell divisions and a few infection threads in question mark-shaped root hairs by 2 days after inoculation. Typical nodules developed soon thereafter. Analogous serially sectioned segments from Clark-rj₁ roots lacked these responses. This prompted us to section nodules and adjacent tissues from other parts of Clark and Clark-rj₁ roots. Clark roots contained cortical cell divisions, many associated with infected root hairs. Cortical cell divisions occasionally were present in Clark-rj₁, and a few infection threads were visible in surface cells. The presence of infection threads within Clark-rj₁ nodules was confirmed by transmission electron microscopy. Thus, although B. japonicum USDA 94 fails to elicit the wild-type spectrum of responses in the infectible zones of primary roots, it can infect Clark-rj₁ via infection threads.     

Effect of phosphorus and zinc nutrition on soybean seed phytic Acid and zinc

The relationships between nutrient P and Zn levels and the phytic acid, P, and Zn concentrations in soybean (Glycine max L. Merr. cv `Williams 79') seed were studied. Phytic acid increased linearly from 4.2 to 19.2 milligrams per gram as nutrient P treatment was varied from 2.0 to 50 milligrams per liter and Zn was held constant at 0.05 milligrams per liter. Leaf P concentration during seed development was found to be closely related to the concentrations of seed P and phytic acid. Leaf and seed Zn concentrations both responded positively to increasing nutrient Zn treatment. The effects of P treatment on plant and seed P and phytic acid were largely independent of the effects of Zn treatment on leaf and seed Zn. Phytic acid to Zn molar ratios ranging from 3.6 to 33.8 were observed.

The effects of nutrient P treatments on the concentrations of phytic acid, seed P, and leaf P were also studied in the P-sensitive (gene np) cultivars `Harosoy' and `Clark' and their respective P-tolerant (gene Np) near-isogenic lines L66-704 and L63-1677. In general, the positive relationships observed among nutrient P, leaf P, seed P, and phytic acid concentrations were similar to those observed in the studies with Williams 79. When fertilized with low or moderate nutrient P (2.5 and 25.0 milligrams P per liter, respectively) no significant differences in any parameter were observed between Harosoy or Clark and their respective P-tolerant isolines. When fertilized with high nutrient P (100 milligrams P per liter), Harosoy seed had a significantly higher concentration of phytic acid (30 milligrams per gram) than did seed of its P-tolerant near-isogenic line L66-704 (24.2 milligrams per gram phytic acid), whereas no significant difference was observed between Clark and its P-tolerant near-isogenic line L63-1677 (22.8 and 21.6 milligrams per gram, respectively). Variation in the phytic acid concentrations in the mature seed of the cultivars and isolines more closely paralleled leaf P concentrations observed during seed development (49 days after flowering), than those observed at the onset of seed development (14 days after flowering). Electrophoresis and ion-exchange chromatography revealed that partially phosphorylated intermediates do not appear when phytic acid accumulation is greatly reduced by limiting the nutrient P or when accumulation is greatly accelerated by excess P.

     

Rj4, a Gene Controlling Nodulation Specificity in Soybeans,Encodes a Thaumatin-Like Protein But Not the One Previously Reported

Rj4 is a dominant gene in soybeans (Glycine max) that restricts nodulation by many strains of Bradyrhizobium elkanii. The soybean-B. elkanii symbiosis has a low nitrogen-fixation efficiency, but B. elkanii strains are highly competitive for nodulation; thus, cultivars harboring an Rj4 allele are considered favorable. Cloning the Rj4 gene is the first step in understanding the molecular basis of Rj4-mediated nodulation restriction and facilitates the development of molecular tools for genetic improvement of nitrogen fixation in soybeans. We finely mapped the Rj4 locus within a small genomic region on soybean chromosome 1, and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We demonstrated that Rj4 encodes a thaumatin-like protein, for which a corresponding allele is not present in the surveyed rj4 genotypes, including the reference genome Williams 82. Our conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (previously annotated as Glyma01g37060). Instead, we provide convincing evidence that Rj4 is Glyma.01g165800-D, a duplicated and unique version of Glyma.01g165800, that has evolved the ability to control symbiotic specificity.Legumes are capable of forming a root nodule symbiosis with nitrogen-fixing soil bacteria called rhizobia. Remarkably, this symbiosis shows a high level of specificity (Broughton et al., 2000; Perret et al., 2000; Wang et al., 2012). The specificity occurs at both between- and within-species levels, such that each legume species or genotype can establish an efficient symbiosis with only a specific group of rhizobial species or strains. Genetic control of symbiosis specificity is complex, involving an exchange of multiple molecular signals between the symbiotic partners. Understanding the molecular mechanisms underlying symbiosis specificity would allow for development of tools for genetic improvement of biological nitrogen fixation in legumes.In most but not all legumes, bacterial infection and nodule organogenesis is mediated by specific perception of bacterially derived lipo-chitooligosaccharides (called Nod factors) by the cognate plant receptors (Lerouge et al., 1990; Geurts et al., 1997; Limpens et al., 2003; Radutoiu et al., 2003, 2007). The Nod factors carry various species-specific chemical decorations, and this structural variation is widely thought to play an important role in defining the recognition specificity at the species level (Lerouge et al., 1990; Perret et al., 2000; Radutoiu et al., 2007). In addition to Nod factors, rhizobial surface polysaccharides, such as exopolysaccharides, lipopolysaccharides, capsular polysaccharides, and cyclic glucans, are also important for development of infected root nodules and for modulating host specificity (D’Haeze and Holsters, 2004; Jones et al., 2008; Deakin and Broughton, 2009). Recently, an exopolysaccharide receptor has been identified in Lotus japonicus that controls rhizobial infection and distinguishes between compatible and incompatible exopolysaccharides (Kawaharada et al., 2015).Despite their unique attributes, the legume-rhizobial interactions share many common features with pathogenic plant-bacterial interactions (D’Haeze and Holsters, 2004; Deakin and Broughton, 2009). As such, plant immunity triggered by microbe-associated molecular patterns or bacterial effector proteins also plays a key role in regulation of strain-specific nodulation (D’Haeze and Holsters, 2004; Deakin and Broughton, 2009; Yang et al., 2010; Wang et al., 2012). It has been demonstrated in soybeans (Glycine max) that plants use classical NBS-LRR resistance genes to restrict nodulation with certain rhizobial strains (Yang et al., 2010). In this case, the host range of rhizobial symbionts is determined by the presence of type III effectors in the bacteria and the corresponding resistance genes in the plant.In this report, we describe positional cloning of the soybean Rj4 gene. The Rj4 gene was first identified in 1972 (Vest and Caldwell, 1972) and subject to extensive study in the 1980s and 1990s (e.g. Devine and O’Neill, 1986; Devine et al., 1990; Sadowsky and Cregan, 1992). Soybean genotypes carrying an Rj4 allele restrict nodulation by many strains of Bradyrhizobium japonicum and Bradyrhizobium elkanii (Sadowsky and Cregan, 1992). B. elkanii is a poor symbiotic partner of soybeans because of its low nitrogen-fixation efficiency. In addition, many of the strains also produce rhizobitoxine, a compound that induces chlorosis in the host plant. Thus, cultivars with the Rj4 genotype are favorable in soils where the B. elkanii population is dominant because Rj4 stops those cultivars from forming invaded nodules with it. The Rj4 allele is frequently present in Gly soja, the wild progenitor of soybean, but less frequent in the modern cultivars from North America (Devine and Breithaupt, 1981). We mapped the Rj4 locus within a small genomic region on soybean chromosome 1 (Tang et al., 2014), and validated one of the candidate genes as Rj4 using both complementation tests and CRISPR/Cas9-based gene knockout experiments. We showed that Rj4 encodes a thaumatin-like protein that does not have a corresponding allele in the analyzed rj4 genetic backgrounds. This conclusion disagrees with the previous report that Rj4 is the Glyma.01G165800 gene (Hayashi et al., 2014). Instead, we provide convincing evidence that Rj4 is a duplicate copy of Glyma.01G165800.     

Effect of plant nutrient supply on nodule effectiveness and rhizobium strain competition for nodulation ofLotus pedunculatus

Summary The effect of nutrient supply on nodule formation and competition between Rhizobium strains for nodulation ofLotus pedunculatus was studied. Limiting plant growth by decreasing the supply of nutrients in an otherwise nitrogen-free medium, increased the size but decreased the number and the nitrogenase activity of nodules formed by a fast-growing strain of Lotus Rhizobium (NZP2037). In contrast decreasing nutrient supply caused only a small decline in the size, number and nitrogenase activity of nodules formed by a slow-growing strain (CC814s). Providing small quantities of NH₄NO₃ (50 to 250 g N) to plants grown with a normal supply of other nutrients stimulated nodule development by both Rhizobium strains and increased the nitrogenase activity of the NZP2037 nodules. Differences in the level of effectiveness (nitrogen-fixing ability) of nodules formed by different Rhizobium strains on plants grown with a normal supply of nutrients were less apparent when the plants were grown with decreased nutrient supply or when the plants were supplied with low levels of inorganic N.Inter-strain competition for nodulation ofL. pedunculatus between the highly effective slow-growing strain CC814s and 7 other fast- and slow-growing strains, showed CC814s to form 42 to 100% of the nodules in all associations. The greater nodulating competitiveness of strain CC814s prevailed despite changes in the nutrient supply to the host plant. A tendency was observed for partially effective Lotus Rhizobium strains to become more competitive in nodule formation when plant growth was supplemented with low levels of inorganic nitrogen.     

Effect of Rj genotype and cultivation temperature on the community structure of soybean-nodulating bradyrhizobia

The nodulation tendency and community structure of indigenous bradyrhizobia on Rj genotype soybean cultivars at cultivation temperatures of 33/28°C, 28/23°C, and 23/18°C for 16/8 h (day/night degrees, hours) were investigated using 780 bradyrhizobial DNA samples from an Andosol with 13 soybean cultivars of four Rj genotypes (non-Rj, Rj(2)Rj(3), Rj(4), and Rj(2)Rj(3)Rj(4)). A dendrogram was constructed based on restriction fragment length polymorphism of the PCR products (PCR-RFLP) of the 16S-23S rRNA gene internal transcribed spacer region. Eleven Bradyrhizobium U.S. Department of Agriculture strains were used as a reference. The dendrogram indicated seven clusters based on similarities among the reference strains. The occupancy rate of the Bj123 cluster decreased with increasing cultivation temperature, whereas the occupancy rates of the Bj110 cluster, Be76 cluster, and Be94 cluster increased with increasing cultivation temperature. In particular, the Rj(2)Rj(3)Rj(4) genotype soybeans were infected with a number of Bj110 clusters, regardless of the increasing cultivation temperature, compared to other Rj genotype soybean cultivars. The ratio of beta diversity to gamma diversity (H'(β)/H'(γ)), which represents differences in the bradyrhizobial communities by pairwise comparison among cultivation temperature sets within the same soybean cultivar, indicated that the bradyrhizobial communities tended to be different among cultivation temperatures. Multidimensional scaling analysis indicated that the infection of the Bj110 cluster and the Bj123 cluster by host soybean genotype and the cultivation temperature affected the bradyrhizobial communities. These results suggested that the Rj genotypes and cultivation temperatures affected the nodulation tendency and community structures of soybean-nodulating bradyrhizobia.     

Synthesis and accumulation of nitrite in soybean nodules supplied with nitrate

Nodulated soybean plants (Glycine max [L.] Merr) were grown in sand culture without combined N or with a continuous supply of nitrate in nutrient solution. Moderate nitrate concentration (30 milligrams N per liter) had little effect on nodule weight/plant while high nitrate concentration (100 milligrams N per liter) depressed nodule weight/plant by 70 to 80% with harvests 30 to 60 days after planting and initiation of nitrate treatments.     

Effects of substrate nitrate concentration on symbiotic nodule formation in actinorhizal plants

The response ofAlnus glutinosa, Casuarina cunninghamiana, Elaeagnus angustifolia andMyrica cerifera to a range of substrate nitrogen levels in solution, in relation to plant growth, infection, nodulation and root fine structure was studied. Nine concentrations of potassium nitrate ranging from 0.05 to 3.0 mM, were tested on each of the species. Plants were inoculated withFrankia pure cultures after a two week exposure to one of the nine levels of added nitrate. After six more weeks with constant exposure to nitrate, plants were harvested and assayed. With the exception of Myrica, regression analyses of whole plant dry weights as a function of added nitrate were highly significant. There was a tendency for nodulated plants grown at intermediate levels of added nitrate to exhibit higher relative growth rates, probably due to the additive effect of substrate nitrogen and fixation of atmospheric nitrogen. The mean numbers of nodules per plant were, with the exception of Alnus, significantly higher at intermediate levels of added nitrate, as were mean nodule dry weights. A highly significant inverse relationship between nodule weight as a percentage of whole plant weight was found in Elaeagnus and Myrica. The observed response of Elaeagnus to added nitrate compared to other actinorhizal plants appears to demonstrate that root hair infected plants are much more sensitive to the inhibitory effects of added nitrate than plants infected by intercellular penetration. A sharp reduction in the presence of root hairs at high concentrations of nitrate was observed. This indicates that the inhibition of nodulation in some actinorhizal plant species results from nitrate induced root hair suppression.     

Effects of nitrogen dioxide and nitrate nutrition on nodulation, nitrogenase activity, growth, and nitrogen content of bean

The influence of nutrient nitrate level (0-20 millimolar) on the effects of NO₂ (0-0.5 parts per million) on nodulation and in vivo acetylene reduction activity of the roots and on growth and nitrate and Kjeldahl N concentration in shoots was studied in bean (Phaseolus vulgaris L. cv Kinghorn Wax) plants. Exposing 8-day old seedlings for 6 hours each day, for 15 days, to 0.02 to 0.5 parts per million NO₂ decreased total nodule weight at 0 and 1 millimolar nitrate, and nitrogenase (acetylene reduction) activity at all concentrations of nitrate. The pollutant had little effect on root fresh or dry weights. Shoot growth was inhibited by NO₂. The NO₂ exposure increased nitrate concentration in roots only at 20 millimolar nutrient nitrate. Exposure to NO₂ markedly increased Kjeldahl N concentration in roots but generally decreased that in shoots. The experiments demonstrated that nutrient N level and NO₂ concentration act jointly in affecting nodulation and N fixing capability, plant growth and composition, and root/shoot relationships of bean plants.     

Selection for increased nodule number in common bean (Phaseolus vulgaris L.)

Common bean (Phaseolus vulgaris L.) plants were grown for 21–28 days in plastic container-modified Leonard jar assemblies and placed in a controlled-environment room. The nodules on each plant were removed, counted; selected plants were repotted, grown and intercrossed to produce progenies for the next cycle of recurrent selection. Among the ten parent lines, Puebla 152 and WBR 22–34 produced the most nodules and Rio Tibagi and Negro Argel the fewest, when averaged over five experiments. An analysis of number of nodules on F₁ plants resulting from crosses made in a partial diallel design among the ten parents revealed highly significants variation for general combining ability (GCA) but not for specific combining ability (SCA). After three cycles of recurrent selection for nodule number per plant, the mean nodule number was 211% of the mean for the 10 parents control. Total nodule weight on selected plants also increased, but individual nodule weight decreased. Nineteen C₁ and 18 C₂ lines resulting from the individual plants selected for greater nodule number, along with the ten parents and two non-nodulating soybean lines included as non-fixing check plants were grown in a single experiment in a low-N field. C₂ lines on average accumulated significantly more N per plant than either the parents or C₁ lines, providing evidence for increased N₂ fixation measured by the N difference method. These data show that more nodules, possibly resulting from greater susceptibility to nodulation, are an important, heritable component of symbiosis and that selection for increased nodule number resulted in lines capable of fixing more atmospheric N₂.     

Nitrate levels affect the development of the black locust-Rhizobium symbiosis

Summary Experiments with black locust (Robinia pseudoacacia L.) seedlings grown under strictly controlled laboratory conditions indicated that the availability of nitrate has a marked impact on nitrogen fixation. When nitrate concentrations were very low, both nodulation and seedling growth were impaired, whereas nitrate concentrations high enough to promote plant growth strongly inhibited symbiotic nitrogen fixation. When nitrate was added to the growth medium after infection, nodulation and nitrogen fixation of the seedlings decreased. This effect was even more marked when nitrate was applied before infection with rhizobia. Higher nitrogen concentrations also reduced nodule number and nodule mass when applied simultaneously with the infecting bacteria. The contribution of symbiotic nitrogen fixation to black locust shoot mass by far exceeded its effects on shoot length and root mass. When nitrate availability was very low, specific nitrogen fixation (i. e. nitrogenase activity per nodule wet weight) was improved with increasing nitrogen supply, but rapidly decreased with higher nitrogen concentrations.     

Positive role of nodulation on the establishment ofRhizobium japonicum in subsequent crops of soybean

The influence of soybean nodulation on the establishment ofRhizobium japonicum inRhizobium-free soil was examined. Seeds of nodulating (Rj ₁) and nonnodulating (rj ₁) isolines of soybeans and four other crop species (cowpeas, mungbeans, corn, and alfalfa) were grown in field plots that were inoculated with a genetically marked strain ofRhizobium (strain I-110 ARS) and the following year nodulating soybeans were grown in these plots and were inoculated with a different genetically marked subline of the same strain (strain I-110 FN). The proportion of nodules containing strain I-110 ARS relative to strain I-110 FN was determined and interpreted as reflecting the relative numbers of the two genetically marked sublines in the soil. The results clearly demonstrate that nodulation with the specific host plant (soybeans) has a significant positive role in the establishment ofRhizobium inRhizobium-free soil and suggests that alfalfa plants diminish the establishment of soybean rhizobia in soil.     

The Rj4 allele in soybean represses nodulation by chlorosis-inducing bradyrhizobia classified as DNA homology group II by antibiotic resistance profiles

Summary To determine the relationship between nodulation restriction by the Rj4 allele of soybean, rhizobitoxine-induced chlorosis, and taxonomic grouping of bradyrhizobia, 119 bradyrhizobial isolates were tested in Leonard jar culture for nodulation response and chlorosis induction. In addition to strain USDA 61, the strain originally reported as defining the Rj4 response, eight other isolates (i.e., USDA 62, 83, 94, 238, 252, 259, 260, and 340) were discovered to elicit the nodulation interdiction of the Rj4 allele. Only 16% of all the bradyrhizobial strains tested induced chlorosis, but seven of the nine strains (78%) interdicted by the Rj4 allele were chlorosis-inducing strains. Furthermore, in tests for antibiotic resistance profile, eight of the nine interdicted strains (89%) were classed in DNA homology group II. This evidence suggests that the Rj4 allele has a positive value to the host plant in shielding it from nodulation by certain chlorosis-inducing bradyrhizobia of a DNA homology group with impaired efficiency of nitrogen fixation with soybean.     

Near isogenic lines of soybeans as tools to identify nodulation specificmutants of Bradyrhizobium elkanii

The dominant allele Rj4 in soybean interdicts or restricts the nodulation of plants by certain strains of bacteria, most of which are classified as Bradyrhizobium elkanii, while the recessive allele permits normal nodulation with the same strains. The near isogenic lines BARC-2 (Rj4) and BARC-3 (rj4) are calculated to be 99.95% identical in their nuclear DNA, but differ specifically in the allele present at the Rj4 locus. These lines were used to identify spontaneous mutants of the Rj4-restricted Bradyrhizobium elkanii strain USDA 61 Nal^r that had the ability to effectively nodulate plants of the Rj4 genotype. Of the eight rare nodules found on roots of soybean plants of the Rj4 genotype inoculated with the genetically marked strain USDA 61 Nal^r, four were identified as containing mutants with the ability to overcome the effects of the Rj4 allele.     

Effect of nodulation on the nitrate assimilation in vegetative soybean plants

Summary Nitrate assimilation in the first trifoliate leaf of vegetative soybean plants (Glycine max L. Merr, cv Hodgson) was studied in relation to nodulation. Nodulated and non-nodulated plants were grown in a nitrate medium (4 mM). As a control nodulated plants were grown in a nutrient medium without combined nitrogen. This study included measurements of the acetylene reduction activity of the whole plant and of thein vitro nitrate reductase, glutamine synthetase and glutamate dehydrogenase activities in the first leaf and of the nitrate concentration. Nitrate accumulation and nitrate reductase activity were depressed in nodulated plants; root growth was decreased in the presence of nitrate. The relationships between nitrate assimilation and nodulation are discussed.