Formation Of Root Nodules Research Articles

Involvement of programmed cell death in suppression of the number of root nodules formed in soybean induced by Bradyrhizobium infection

ABSTRACTLegumes establish symbiosis with nitrogen-fixing rhizobia through root nodules to acquire nitrogen. Legumes control nodule number through systemic (autoregulation of nodulation) as well as local regulation. Moreover, plants defend themselves against bacteria and other pathogens through the induction of localized (localized acquired resistance) and systemic (SAR, systemic acquired resistance; ISR, induced systemic resistance) responses. Herein, we show that the number of root nodules is suppressed by programmed cell death (PCD), and is simultaneously controlled by SAR and ISR in soybean (Glycine max [L.] Merr.). The wild-type soybean cultivar Williams 82 showed markedly fewer root nodule primordia and PCD symptoms, including accelerated DNA degradation, enhanced generation of reactive oxygen species (visualized by 3,3′-diaminobenzidine staining), and excessive cell death (detected on staining with trypan blue) compared to the hypernodulation mutant NOD1-3. These results suggest that PCD suppresses the formation of root nodules in wild-type soybean. In addition, microarray and gene ontology analyses showed that essential components of hypersensitive response (HR) or disease resistance, such as resistance (R) genes, mitogen-activated protein kinase cascade, SAR, salicylic acid, jasmonic acid, ethylene, etc., were activated in wild-type plants. These analyses corroborate the above findings, demonstrating that the suppression of root nodule formation by PCD is accompanied by HR, and is simultaneously controlled by SAR and ISR in soybean. These findings provide new insight into the control of nodulation to balance nutritional requirements and energy status in legumes.

DELLA1-Mediated Gibberellin Signaling Regulates Cytokinin-Dependent Symbiotic Nodulation.

In legume plants, low-nitrogen soils promote symbiotic interactions with rhizobial bacteria, leading to the formation of nitrogen-fixing root nodules. Among critical signals regulating this developmental process are bacterial Nod Factors (NFs) and several plant hormones, including cytokinins (CKs) and gibberellins (GAs). Here, we show in Medicago truncatula that GA signaling mediated by DELLA1 decreases the amount of bioactive CKs in roots and negatively impacts the Cytokinin Response1 (CRE1)-dependent NF activation of a subset of CK-signaling genes as well as of the CK-regulated Nodulation Signaling Pathway2 and Ethylene Response Factor Required for Nodulation1 early nodulation genes. Consistently, a dominant-active DELLA1 protein can partially rescue the reduced nodulation of the cre1 mutant and triggers the formation of nodule-like structures when expressed in the root cortex or in the root epidermis. This suggests a model where the DELLA1-mediated GA signaling interplays with the CRE1-dependent CK pathway to regulate early nodulation in response to both NF and CK signals critical for this symbiotic interaction.

Effects of nitrogen concentrations on nodulation and nitrogenase activity in dual root systems of soybean plants

ABSTRACTCurrently, the mechanisms underlying the effects of nitrogen on nodulation and nitrogen fixation of leguminous plants remain uncertain. To further investigate the relationship between nitrogen with nodulation and nitrogen fixation of soybean plants, a young seedling grafting technique was applied to generate soybean plants with dual root systems. The two root systems were separately cultured in sand medium, with one irrigated with nutrient solution containing or as nitrogen sources (the N+ side) and another irrigated with nitrogen-free nutrient solution (the N− side). Various nitrogen concentrations, 0, 25, 50, 75, and 100 mg/L, were applied to the N+-side root system to determine the effects of nitrogen concentrations on nodulation and nitrogenase activity of soybean plants. The results showed that in the dual root systems constructed by seedling grafting, the number and weight of root nodules at the N+ side both exhibited a single-peak changing trend of first increasing and then decreasing with nitrogen concentrations. Root nodulation was accelerated by low concentrations of nitrogen (<50 mg/L) and significantly suppressed by high concentrations of nitrogen (>50 mg/L). In contrast, the number and weight of root nodules at the N− side showed a constant increasing trend with the increase in nitrogen concentrations. Taken together, these results indicated that high nitrogen concentration exerts a contact-dependent localized inhibitory effect on the formation (number of nodules) and growth of root nodules (weight of nodules). The nitrogenase activities in the root nodules at both the N+ and the N− sides displayed trends of first increasing and then decreasing with nitrogen concentrations, reaching a single peak when the nitrogen concentration was 50 mg/L. In addition, the promotive or inhibitory effects of nitrogen at different concentrations on nitrogenase activity in the root nodules were synchronous between the N+ and the N− sides, suggesting that the effect of nitrogen is systematically regulated via interactions between the aboveground and underground parts of the plants.

Effect of seed pre-treatment on Kidney Beans (Phaseolus vulgarisss L.) yield

The article presents the results of the impact the application of seed inoculation bean with strains of microorganisms, treatment common bean varieties Slavia with growth and biological adhesive Regoplant EPAA. In experiments rhizobia strains from the collection of the Institute of Microbiology and Virology NAN of Ukraine were used. Most favorable conditions for the formation of grain yield kidney beans were produced in those crops that best meet the needs of plants. It has been revealed that all versions of inoculation of nodules on the roots of bacteria in the phase of budding, flowering plants were pink and large in comparison with the control options, which were observed in the formation of small root nodules that formed when soil ryzobia population was infected. It was found that the yield of common bean seed depends on biologics. These measures affect the biological development of bean plants and seed production of culture, but a reliable grain yield increase was observed in cases where the bean seeds were treated with microbial strains F-16 and biological agents Regoplant + EPAA

Nitrate transporters: an overview in legumes.

The nitrate transporters, belonging to NPF and NRT2 families, play critical roles in nitrate signaling, root growth and nodule development in legumes. Nitrate plays an essential role during plant development as nutrient and also as signal molecule, in both cases working via the activity of nitrate transporters. To date, few studies on NRT2 or NPF nitrate transporters in legumes have been reported, and most of those concern Lotus japonicus and Medicago truncatula. A molecular characterization led to the identification of 4 putative LjNRT2 and 37 putative LjNPF gene sequences in L. japonicus. In M. truncatula, the NRT2 family is composed of 3 putative members. Using the new genome annotation of M. truncatula (Mt4.0), we identified, for this review, 97 putative MtNPF sequences, including 32 new sequences relative to previous studies. Functional characterization has been published for only two MtNPF genes, encoding nitrate transporters of M. truncatula. Both transporters have a role in root system development via abscisic acid signaling: MtNPF6.8 acts as a nitrate sensor during the cell elongation of the primary root, while MtNPF1.7 contributes to the cellular organization of the root tip and nodule formation. An in silico expression study of MtNPF genes confirmed that NPF genes are expressed in nodules, as previously shown for L. japonicus, suggesting a role for the corresponding proteins in nitrate transport, or signal perception in nodules. This review summarizes our knowledge of legume nitrate transporters and discusses new roles for these proteins based on recent discoveries.

Microsymbiont discrimination mediated by a host-secreted peptide in Medicago truncatula

The legume-rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant-bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix+) nodules on one plant genotype but nonfunctional (Fix-) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts' differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background.

The Ethylene Responsive Factor Required for Nodulation 1 (ERN1) Transcription Factor Is Required for Infection-Thread Formation in Lotus japonicus.

Several hundred genes are transcriptionally regulated during infection-thread formation and development of nitrogen-fixing root nodules. We have characterized a set of Lotus japonicus mutants impaired in root-nodule formation and found that the causative gene, Ern1, encodes a protein with a characteristic APETALA2/Ethylene Responsive Factor (AP2/ERF) transcription-factor domain. Phenotypic characterization of four ern1 alleles shows that infection pockets are formed but root-hair infection threads are absent. Formation of root-nodule primordia is delayed and no normal transcellular infection threads are found in the infected nodules. Corroborating the role of ERN1 (ERF Required for Nodulation1) in nodule organogenesis, spontaneous nodulation induced by an autoactive CCaMK and cytokinin-induced nodule primordia were not observed in ern1 mutants. Expression of Ern1 is induced in the susceptible zone by Nod factor treatment or rhizobial inoculation. At the cellular level, the pErn1:GUS reporter is highly expressed in root epidermal cells of the susceptible zone and in the cortical cells that form nodule primordia. The genetic regulation of this cellular expression pattern was further investigated in symbiotic mutants. Nod factor induction of Ern1 in epidermal cells was found to depend on Nfr1, Cyclops, and Nsp2 but was independent of Nin and Nf-ya1. These results suggest that ERN1 functions as a transcriptional regulator involved in the formation of infection threads and development of nodule primordia and may coordinate these two processes.

Microbial inoculation of seeds characteristically shapes the rhizosphere microbiome in desi and kabuli chickpea types

Chickpea is generally cultivated after seed treatment with host-specific Mesorhizobium ciceri, the nitrogen-fixing bacterium forming root nodules. Some species of free-living cyanobacteria are capable of nitrogen fixation. We examined the rhizosphere microbiota changes and the potential for plant growth promotion by applying a free-living, nitrogen-fixing cyanobacterium and the biofilm formulation of cyanobacterium with M. ciceri, relative to M. ciceri applied singly, to two each of desi and kabuli varieties of chickpea. Denaturing gradient gel electrophoresis (DGGE) profiles of archaeal, bacterial and cyanobacterial communities and those of phospholipid fatty acids (PLFAs) were obtained to evaluate the changes of the microbial communities in the chickpea rhizosphere. Plant growth attributes, including the pod yields and the availabilities of soil macronutrients and micronutrients, were monitored. The DGGE profiles showed distinct and characteristic changes due to the microbial inoculation; varietal differences exerted a marked influence on the archaeal and cyanobacterial communities. However, bacterial communities were modulated more by the type of microbial inoculants. Abundance of Gram-negative bacteria (in terms of notional PLFAs) differed between the desi and the kabuli varieties inoculated with M. ciceri alone, and the principal component analysis of PLFA profiles confirmed the characteristic effect of microbial inoculants tested. Microbial inoculation led to increases in the 100-seed weight and differential effects on the concentrations of available nitrogen and phosphorus, and those of iron, zinc and copper, suggesting their increased cycling in the rhizosphere. Microbial inoculation of chickpea brought out the characteristic changes in rhizosphere microbiota. Consequently, the growth promotion of chickpea and nutrient cycling in its rhizosphere distinctively differed. Further studies are needed to analyse the association and dynamic changes in the microbial communities to define the subset of microorganisms selected by chickpea in its rhizosphere and the influence of microbial inoculation.

Sybr Green- and TaqMan-Based Quantitative PCR Approaches Allow Assessment of the Abundance and Relative Distribution of Frankia Clusters in Soils.

The nodule-forming actinobacterial genus Frankia can generally be divided into 4 taxonomic clusters, with clusters 1, 2, and 3 representing nitrogen-fixing strains of different host infection groups and cluster 4 representing atypical, generally non-nitrogen-fixing strains. Recently, quantitative PCR (qPCR)-based quantification methods have been developed for frankiae of clusters 1 and 3; however, similar approaches for clusters 2 and 4 were missing. We amended a database of partial 23S rRNA gene sequences of Frankia strains belonging to clusters 1 and 3 with sequences of frankiae representing clusters 2 and 4. The alignment allowed us to design primers and probes for the specific detection and quantification of these Frankia clusters by either Sybr Green- or TaqMan-based qPCR. Analyses of frankiae in different soils, all obtained from the same region in Illinois, USA, provided similar results, independent of the qPCR method applied, with abundance estimates of 10 × 105 to 15 × 105 cells (g soil)-1 depending on the soil. Diversity was higher in prairie soils (native, restored, and cultivated), with frankiae of all 4 clusters detected and those of cluster 4 dominating, while diversity in soils under Alnus glutinosa, a host plant for cluster 1 frankiae, or Betula nigra, a related nonhost plant, was restricted to cluster 1 and 3 frankiae and generally members of subgroup 1b were dominating. These results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.IMPORTANCE Root nodule formation by the actinobacterium Frankia is host plant specific and largely, but not exclusively, correlates with assignments of strains to specific clusters within the genus. Due to the lack of adequate detection and quantification tools, studies on Frankia have been limited to clusters 1 and 3 and generally excluded clusters 2 and 4. We have developed tools for the detection and quantification of clusters 2 and 4, which can now be used in combination with those developed for clusters 1 and 3 to retrieve information on the ecology of all clusters delineated within the genus Frankia Our initial results indicate that vegetation affects the basic composition of frankiae in soils, with higher diversity in prairie soils compared to much more restricted diversity under some host and nonhost trees.

Complete Genome Sequence of Bradyrhizobium japonicum J5, Isolated from a Soybean Nodule in Hokkaido, Japan.

ABSTRACTSoybean bradyrhizobia form root nodules on soybean plants and symbiotically fix N2. Strain J5 is phylogenetically far from well-known representatives within the Bradyrhizobium japonicum linage. The complete genome showed the largest single chromosomal (10.1 Mb) and symbiosis island (998 kb) among complete genomes of soybean bradyrhizobia.

Ensifer shofinae sp. nov., a novel rhizobial species isolated from root nodules of soybean (Glycine max)

Two bacterial strains isolated from root nodules of soybean were characterized phylogenetically as members of a distinct group in the genus Ensifer based on 16S rRNA gene comparisons. They were also verified as a separated group by the concatenated sequence analyses of recA, atpD and glnII (with similarities ≤93.9% to the type strains for defined species), and by the average nucleotide identities (ANI) between the whole genome sequence of the representative strain CCBAU 251167T and those of the closely related strains in Ensifer glycinis and Ensifer fredii (90.5% and 90.3%, respectively). Phylogeny of symbiotic genes (nodC and nifH) grouped these two strains together with some soybean-nodulating strains of E. fredii, E. glycinis and Ensifer sojae. Nodulation tests indicated that the representative strain CCBAU 251167T could form root nodules with capability of nitrogen fixing on its host plant and Glycine soja, Cajanus cajan, Vigna unguiculata, Phaseolus vulgaris and Astragalus membranaceus, and it formed ineffective nodules on Leucaena leucocephala. Strain CCBAU 251167T contained fatty acids 18:1 ω9c, 18:0 iso and 20:0, differing from other related strains. Utilization of l-threonine and d-serine as carbon source, growth at pH 6.0 and intolerance of 1% (w/v) NaCl distinguished strain CCBAU 251167T from other type strains of the related species. The genome size of CCBAU 251167T was 6.2Mbp, comprising 7,581 predicted genes with DNA G+C content of 59.9mol% and 970 unique genes. Therefore, a novel species, Ensifer shofinae sp. nov., is proposed, with CCBAU 251167T (=ACCC 19939T=LMG 29645T) as type strain.

Characterization of Diversity of Bradyrhizobia on Cowpea in Iraq Reveals Unusual Strain Characteristics

Rhizobium-legume symbiosis is considered as one of the most well established symbiotic nitrogen fixing system for agronomic studies. Association between legumes and rhizobia results in the formation of root nodules where symbiotic nitrogen fixation occurs. The current study aimed to authenticate 110 isolates from 20 sites belonging to 10 governorates in Iraq, tested their capacity of nodulation with cowpea and classified them depending on the phenotype and genotype presented by sequence analysis of 16S rRNA. To fulfill these goals, many approaches have been implemented such as Authentication Tests, Bromothymol Blue Reaction, Colony Size and Morphology, Antibiotic Test, Sequencing of 16S rRNA and Phylogenetic analysis. This study provides an easy way to classify the Bradyrhizobia sp. strains by genotype analysis depending on the phenotypes (i.e. motility and colony size) by sample preservation and high quality DNA isolation from environmental soil samples followed by 16S rRNA sequencing. This molecular technique has demonstrated the usefulness of these methods, easy technologies, and their applications to microbiome analysis and environmental science. Interestingly, a group of Bradyrhizobia identified in the current study was able to secrete acidic products before switching and starting to secrete alkali products after 1, 2 and 3 days. This is an unusual phenotype observed within rhizobia strains.

PENGGUNAAN RHIZOBIUM DAN MIKORIZA UNTUK PERTUMBUHAN Calliandra calothyrsus UNGGUL

Calliandra calothyrsus, a rhizobium associated legume, fixes atmospheric nitrogen by forming root nodules. Rhizobium availability is crucial for Calliandra’s growth especially on new sites. Additive or synergic effects of rhizobium and mycorrhiza are found to improve seedling quality. Genetically improved Calliandras require optimum silvicultural practices including rhizobium and mycorrhiza application and differences among families need to be observed. This followed with combination treatment of 5g rhizobium and different mycorrhiza level at 0, 5 and 10g applied to 5 families in 30 x 30 cm polybag. Assessments were for leaf number, height, diameter at 1, 4 and 8 weeks and number of root nodules at 4 and 8 weeks after application. Results show that rhizobium application has no significant effects although it enhances grow, while mycorrhiza application improve leaf number after 14 weeks. Second experiment for 3.5 month seedlings, indicates interaction on family-mycorrhiza level to seedling height and root nodules. Very positive correlations show that more root nodules improved leaf number (r=0.41), height (r=0.3) and diameter (r=0.45) up to planting time. Quite cheap rhizobium and mycorrhiza application is therefore beneficial to optimize the growth of genetically improved C. calothyrsus, although genotype differences may eXist.

Experimental evolution of rhizobia may lead to either extra- or intracellular symbiotic adaptation depending on the selection regime.

Experimental evolution is a powerful approach to study the process of adaptation to new environments, including the colonization of eukaryotic hosts. Facultative endosymbionts, including pathogens and mutualists, face changing and spatially structured environments during the symbiotic process, which impose diverse selection pressures. Here, we provide evidence that different selection regimes, involving different times spent in the plant environment, can result in either intra- or extracellular symbiotic adaptations. In previous work, we introduced the symbiotic plasmid of Cupriavidus taiwanensis, the rhizobial symbiont of Mimosa pudica, into the phytopathogen Ralstonia solanacearum and selected three variants able to form root nodules on M.pudica, two (CBM212 and CBM349) being able to rudimentarily infect nodule cells and the third one (CBM356) only capable of extracellular infection of nodules. Each nodulating ancestor was further challenged to evolve using serial ex planta-in planta cycles of either 21 (three short-cycle lineages) or 42days (three long-cycle lineages). In this study, we compared the phenotype of the 18 final evolved clones. Evolution through short and long cycles resulted in similar adaptive paths on lineages deriving from the two intracellularly infectious ancestors, CBM212 and CBM349. In contrast, only short cycles allowed a stable acquisition of intracellular infection in lineages deriving from the extracellularly infecting ancestor, CBM356. Long cycles, instead, favoured improvement of extracellular infection. Our work highlights the importance of the selection regime in shaping desired traits during host-mediated selection experiments.

EFFECT OF PHOSPHATE ON NODULE PRIMORDIA OF SOYBEAN (Glycine max Merrill) IN ACID SOILS IN RHIZOTRON EXPERIMENTS

To clarify whether P had a direct or indirect effect on the nodulation process of soybean grown in acid soils from Sitiung, West Sumatra, Indonesia, a series of rhizotron experiments, with special attention given to formation of nodule primordia, was conducted at Laboratory of Microbiology, Wageningen University in 1998-2000. It was shown that Ca and P were essential nutrients for root growth, nodule formation, and growth of soybean in the acid soils (Oxisols). Ca increased root growth, number of nodule primordia, nodules, and growth of the soybean plant. This positive effect of Ca was increased considerably by the application of P. Ca and P have a synergistic effect on biological nitrogen fixation (BNF) of soybean in acid soils. Ca is important for the establishment of nodules, whilst P is essential for the development and function of the formed nodules. P increased number of nodule primordia, thus it also has an important role in the initiation of nodule formation. From this study, it can be concluded that Ca and P are the most limiting nutrients for BNF of soybean in the acid soils of Sitiung, West Sumatra, Indonesia.

EFFECT OF PHOSPHATE ON NODULE PRIMORDIA OF SOYBEAN (Glycine max Merrill) IN ACID SOILS IN RHIZOTRON EXPERIMENTS

To clarify whether P had a direct or indirect effect on the nodulation process of soybean grown in acid soils from Sitiung, West Sumatra, Indonesia, a series of rhizotron experiments, with special attention given to formation of nodule primordia, was conducted at Laboratory of Microbiology, Wageningen University in 1998-2000. It was shown that Ca and P were essential nutrients for root growth, nodule formation, and growth of soybean in the acid soils (Oxisols). Ca increased root growth, number of nodule primordia, nodules, and growth of the soybean plant. This positive effect of Ca was increased considerably by the application of P. Ca and P have a synergistic effect on biological nitrogen fixation (BNF) of soybean in acid soils. Ca is important for the establishment of nodules, whilst P is essential for the development and function of the formed nodules. P increased number of nodule primordia, thus it also has an important role in the initiation of nodule formation. From this study, it can be concluded that Ca and P are the most limiting nutrients for BNF of soybean in the acid soils of Sitiung, West Sumatra, Indonesia.

Starting points in plant-bacteria nitrogen-fixing symbioses: intercellular invasion of the roots.

Agricultural practices contribute to climate change by releasing greenhouse gases such as nitrous oxide that are mainly derived from nitrogen fertilizers. Therefore, understanding biological nitrogen fixation in farming systems is beneficial to agriculture and environmental preservation. In this context, a better grasp of nitrogen-fixing systems and nitrogen-fixing bacteria-plant associations will contribute to the optimization of these biological processes. Legumes and actinorhizal plants can engage in a symbiotic interaction with nitrogen-fixing rhizobia or actinomycetes, resulting in the formation of specialized root nodules. The legume-rhizobia interaction is mediated by a complex molecular signal exchange, where recognition of different bacterial determinants activates the nodulation program in the plant. To invade plants roots, bacteria follow different routes, which are determined by the host plant. Entrance via root hairs is probably the best understood. Alternatively, entry via intercellular invasion has been observed in many legumes. Although there are common features shared by intercellular infection mechanisms, differences are observed in the site of root invasion and bacterial spread on the cortex reaching and infecting a susceptible cell to form a nodule. This review focuses on intercellular bacterial invasion of roots observed in the Fabaceae and considers, within an evolutionary context, the different variants, distribution and molecular determinants involved. Intercellular invasion of actinorhizal plants and Parasponia is also discussed.

A putative molybdate transporter LjMOT1 is required for molybdenum transport in Lotus japonicus.

Molybdenum (Mo) is an essential micronutrient that is required for plant growth and development, and it affects the formation of root nodules and nitrogen fixation in legumes. In this study, Lotus japonicus was grown on MS solid media containing 0 nmol l-1 (-Mo), 103 nmol l-1 (+Mo) and 1030 nmol l-1 (10 × Mo) of Mo. The phenotypes of plants growing on the three different media showed no obvious differences after 15 days, but the plants growing on -Mo for 45 days presented typical symptoms of Mo depletion, such as a short taproot, few lateral roots and yellowing leaves. A Mo transporter gene, LjMOT1, was isolated from L. japonicus. It encoded 468 amino acids, including two conserved motifs, and was predicted to locate to chromosome 3 of the L. japonicus genome. A homology comparison indicated that LjMOT1 had high similarities to other MOT1 proteins and was closely related to GmMOT1. Subcellular localization indicated that LjMOT1 is localized to the plasma membrane. qRT-PCR analyses showed that increasing Mo concentrations regulated the relative expression level of LjMOT1. Moreover, the Mo concentration in shoots was positively correlated to the expression of LjMOT1, but there was no such evident correlation in the roots. In addition, changes in the nitrate reductase activity were coincident with changes in the Mo concentration. These results suggest that LjMOT1 may be involved in the transport of Mo and provide a theoretical basis for further understanding of the mechanism of Mo transport in higher plants.

Enhanced rhizobial symbiotic capacity in an allopolyploid species of Glycine (Leguminosae).

Previous studies have shown that polyploidy can alter biotic interactions, and it has been suggested that these effects may contribute to the increased ability for colonization of new habitats shown by many allopolyploids. Little is known, however, about the effects of allopolyploidy, which combines hybridity and genome doubling, on symbiotic interactions with rhizobial bacteria. We examined interactions of the allopolyploid Glycine dolichocarpa (designated T2) with novel rhizobial partners, such as might occur in a context of colonization, and compared these with the responses of its diploid progenitors, G. tomentella (D3) and G. syndetika (D4). We assessed root hair response, nodule formation, nodule mass, nodule number, and plant biomass. The allopolyploid (T2) showed a greater root hair deformation response when exposed to rhizobia, compared with either diploid. T2 had a greater probability of forming nodules with NGR234 compared with diploid D4, and greater total nodule mass per nodulated plant compared with diploid D3. T2 also had greater plant biomass responses to nitrogen and when exposed to NGR234. The allopolyploid is characterized by transgressive responses to rhizobia for some variables, while also combining certain parental diploid responses such that its capacity for interactions with rhizobia appears to be greater than for either diploid progenitor. This overall enhanced nodulation capacity and the ability to make greater gains from exposure to both rhizobia and additional nitrogen indicate a greater potential of the allopolyploid to benefit from these factors both generally and in a context of colonization.

The systemic nodule number regulation kinase SUNN in Medicago truncatula interacts with MtCLV2 and MtCRN.

Autoregulation of nodulation (AON), a systemic signaling pathway in legumes, limits the number of nodules formed by the legume in its symbiosis with rhizobia. Recent research suggests a model for the systemic regulation in Medicago truncatula in which root signaling peptides are translocated to the shoot where they bind to a shoot receptor complex containing the leucine-rich repeat receptor-like kinase SUNN, triggering signal transduction which terminates nodule formation in roots. Here we show that a tagged SUNN protein capable of rescuing the sunn-4 phenotype is localized to the plasma membrane and is associated with the plasmodesmata. Using bimolecular fluorescence complementation analysis we show that, like its sequence ortholog Arabidopsis CLV1, SUNN interacts with homologous CLV1-interacting proteins MtCLAVATA2 and MtCORYNE. All three proteins were also able to form homomers and MtCRN and MtCLV2 also interact with each other. A crn Tnt1 insertion mutant of M. truncatula displayed a shoot controlled increased nodulation phenotype, similar to the clv2 mutants of pea and Lotus japonicus. Together these data suggest that legume AON signaling could occur through a multi-protein complex and that both MtCRN and MtCLV2 may play roles in AON together with SUNN.