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Infected cells and bacteroids. (А-D) Vicia sativa L., (E-H) Galega orientalis Lam, and (I-L) Cicer arietinum L. (A,E,I,) Symbiosome arrangement in infected cells of the nitrogen-fixation zone. (B-L) Bacteroid morphology based on (B,F,J) scanning electron microscopy, (C,G,K) transmission electron microscopy, and (D,H,L) laser scanning confocal microscopy. (A,E,I,D,H,L) Merged images of differential interference contrast and red channel (DNA staining with propidium iodide (nuclei and bacteria)). bа, bacteroid; cw, cell wall; n, nucleus; ic, infected cell; uic, uninfected cell; arrowheads indicate bacteroids. Bars: (A,E,I) 10 μm, (B,F,J) 2 μm, (C,G,K) 1 μm, and (D,H,L) 5 μm.
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The tubulin cytoskeleton plays an important role in establishing legume–rhizobial symbiosis at all stages of its development. Previously, tubulin cytoskeleton organization was studied in detail in the indeterminate nodules of two legume species, Pisum sativum and Medicago truncatula. General as well as species-specific patterns were revealed. To fu...
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... skeletonized images were analyzed using the AnalyzeSkeleton plugin [36]. The result tables were then processed using a custom R script and six features were calculated for each cell: (1) total number of branches, which is the number of all detected microtubules ( Figure S1A); (2) total length of branches, which is the total length of all detected microtubules ( Figure S1A); (3) mean straightness index of detected microtubules, which is the Euclidian distance between the starting and ending point of each branch divided by its full length ( Figure S1A); (4) total number of junctions ( Figure S1A); (5) degree of branching, which is the number of skeletons (sets of branches connected) with more than one branch divided by the total number of skeletons in the image ( Figure S1A,B); and (6) mean number of junctions per skeleton, which is the average number of branching points across all skeletons in the cell ( Figure S1A,B). Statistically significant differences in these features between different species were determined using Tukey's range test. ...
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... skeletonized images were analyzed using the AnalyzeSkeleton plugin [36]. The result tables were then processed using a custom R script and six features were calculated for each cell: (1) total number of branches, which is the number of all detected microtubules ( Figure S1A); (2) total length of branches, which is the total length of all detected microtubules ( Figure S1A); (3) mean straightness index of detected microtubules, which is the Euclidian distance between the starting and ending point of each branch divided by its full length ( Figure S1A); (4) total number of junctions ( Figure S1A); (5) degree of branching, which is the number of skeletons (sets of branches connected) with more than one branch divided by the total number of skeletons in the image ( Figure S1A,B); and (6) mean number of junctions per skeleton, which is the average number of branching points across all skeletons in the cell ( Figure S1A,B). Statistically significant differences in these features between different species were determined using Tukey's range test. ...
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... skeletonized images were analyzed using the AnalyzeSkeleton plugin [36]. The result tables were then processed using a custom R script and six features were calculated for each cell: (1) total number of branches, which is the number of all detected microtubules ( Figure S1A); (2) total length of branches, which is the total length of all detected microtubules ( Figure S1A); (3) mean straightness index of detected microtubules, which is the Euclidian distance between the starting and ending point of each branch divided by its full length ( Figure S1A); (4) total number of junctions ( Figure S1A); (5) degree of branching, which is the number of skeletons (sets of branches connected) with more than one branch divided by the total number of skeletons in the image ( Figure S1A,B); and (6) mean number of junctions per skeleton, which is the average number of branching points across all skeletons in the cell ( Figure S1A,B). Statistically significant differences in these features between different species were determined using Tukey's range test. ...
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... skeletonized images were analyzed using the AnalyzeSkeleton plugin [36]. The result tables were then processed using a custom R script and six features were calculated for each cell: (1) total number of branches, which is the number of all detected microtubules ( Figure S1A); (2) total length of branches, which is the total length of all detected microtubules ( Figure S1A); (3) mean straightness index of detected microtubules, which is the Euclidian distance between the starting and ending point of each branch divided by its full length ( Figure S1A); (4) total number of junctions ( Figure S1A); (5) degree of branching, which is the number of skeletons (sets of branches connected) with more than one branch divided by the total number of skeletons in the image ( Figure S1A,B); and (6) mean number of junctions per skeleton, which is the average number of branching points across all skeletons in the cell ( Figure S1A,B). Statistically significant differences in these features between different species were determined using Tukey's range test. ...
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... skeletonized images were analyzed using the AnalyzeSkeleton plugin [36]. The result tables were then processed using a custom R script and six features were calculated for each cell: (1) total number of branches, which is the number of all detected microtubules ( Figure S1A); (2) total length of branches, which is the total length of all detected microtubules ( Figure S1A); (3) mean straightness index of detected microtubules, which is the Euclidian distance between the starting and ending point of each branch divided by its full length ( Figure S1A); (4) total number of junctions ( Figure S1A); (5) degree of branching, which is the number of skeletons (sets of branches connected) with more than one branch divided by the total number of skeletons in the image ( Figure S1A,B); and (6) mean number of junctions per skeleton, which is the average number of branching points across all skeletons in the cell ( Figure S1A,B). Statistically significant differences in these features between different species were determined using Tukey's range test. ...
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... skeletonized images were analyzed using the AnalyzeSkeleton plugin [36]. The result tables were then processed using a custom R script and six features were calculated for each cell: (1) total number of branches, which is the number of all detected microtubules ( Figure S1A); (2) total length of branches, which is the total length of all detected microtubules ( Figure S1A); (3) mean straightness index of detected microtubules, which is the Euclidian distance between the starting and ending point of each branch divided by its full length ( Figure S1A); (4) total number of junctions ( Figure S1A); (5) degree of branching, which is the number of skeletons (sets of branches connected) with more than one branch divided by the total number of skeletons in the image ( Figure S1A,B); and (6) mean number of junctions per skeleton, which is the average number of branching points across all skeletons in the cell ( Figure S1A,B). Statistically significant differences in these features between different species were determined using Tukey's range test. ...
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... of V. sativa, G. orientalis, and C. arietinum differed regarding their distribution in infected cells ( Figure 1A,E,I). In infected V. sativa ( Figure 1A) and C. arietinum ( Figure 1I) cells, symbiosomes were randomly oriented, whereas in infected G. orientalis cells, they were mainly oriented perpendicular to the cell surface and parallel to each other ( Figure 1E). ...
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... of V. sativa, G. orientalis, and C. arietinum differed regarding their distribution in infected cells ( Figure 1A,E,I). In infected V. sativa ( Figure 1A) and C. arietinum ( Figure 1I) cells, symbiosomes were randomly oriented, whereas in infected G. orientalis cells, they were mainly oriented perpendicular to the cell surface and parallel to each other ( Figure 1E). Analysis of the ultrastructural organization of the nodules and confocal images of isolated bacteroids demonstrated that bacteroids of G. orientalis and V. sativa belong to the E morphotype ( Figure 1B-D,F-H), whereas bacteroids of C. arietinum belong to the S morphotype ( Figure 1J-L). ...
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... of V. sativa, G. orientalis, and C. arietinum differed regarding their distribution in infected cells ( Figure 1A,E,I). In infected V. sativa ( Figure 1A) and C. arietinum ( Figure 1I) cells, symbiosomes were randomly oriented, whereas in infected G. orientalis cells, they were mainly oriented perpendicular to the cell surface and parallel to each other ( Figure 1E). Analysis of the ultrastructural organization of the nodules and confocal images of isolated bacteroids demonstrated that bacteroids of G. orientalis and V. sativa belong to the E morphotype ( Figure 1B-D,F-H), whereas bacteroids of C. arietinum belong to the S morphotype ( Figure 1J-L). ...
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... of V. sativa, G. orientalis, and C. arietinum differed regarding their distribution in infected cells ( Figure 1A,E,I). In infected V. sativa ( Figure 1A) and C. arietinum ( Figure 1I) cells, symbiosomes were randomly oriented, whereas in infected G. orientalis cells, they were mainly oriented perpendicular to the cell surface and parallel to each other ( Figure 1E). Analysis of the ultrastructural organization of the nodules and confocal images of isolated bacteroids demonstrated that bacteroids of G. orientalis and V. sativa belong to the E morphotype ( Figure 1B-D,F-H), whereas bacteroids of C. arietinum belong to the S morphotype ( Figure 1J-L). ...
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... infected V. sativa ( Figure 1A) and C. arietinum ( Figure 1I) cells, symbiosomes were randomly oriented, whereas in infected G. orientalis cells, they were mainly oriented perpendicular to the cell surface and parallel to each other ( Figure 1E). Analysis of the ultrastructural organization of the nodules and confocal images of isolated bacteroids demonstrated that bacteroids of G. orientalis and V. sativa belong to the E morphotype ( Figure 1B-D,F-H), whereas bacteroids of C. arietinum belong to the S morphotype ( Figure 1J-L). However, the G. orientalis bacteroids are barely branched (ENB morphotype) ( Figure 1F-H), whereas the V. sativa bacteroids are characterized by intensive branching (EB morphotype) ( Figure 1B-D). ...
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... infected V. sativa ( Figure 1A) and C. arietinum ( Figure 1I) cells, symbiosomes were randomly oriented, whereas in infected G. orientalis cells, they were mainly oriented perpendicular to the cell surface and parallel to each other ( Figure 1E). Analysis of the ultrastructural organization of the nodules and confocal images of isolated bacteroids demonstrated that bacteroids of G. orientalis and V. sativa belong to the E morphotype ( Figure 1B-D,F-H), whereas bacteroids of C. arietinum belong to the S morphotype ( Figure 1J-L). However, the G. orientalis bacteroids are barely branched (ENB morphotype) ( Figure 1F-H), whereas the V. sativa bacteroids are characterized by intensive branching (EB morphotype) ( Figure 1B-D). ...
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... of the ultrastructural organization of the nodules and confocal images of isolated bacteroids demonstrated that bacteroids of G. orientalis and V. sativa belong to the E morphotype ( Figure 1B-D,F-H), whereas bacteroids of C. arietinum belong to the S morphotype ( Figure 1J-L). However, the G. orientalis bacteroids are barely branched (ENB morphotype) ( Figure 1F-H), whereas the V. sativa bacteroids are characterized by intensive branching (EB morphotype) ( Figure 1B-D). The bacteroids of the studied species vary significantly in size. ...
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... of the ultrastructural organization of the nodules and confocal images of isolated bacteroids demonstrated that bacteroids of G. orientalis and V. sativa belong to the E morphotype ( Figure 1B-D,F-H), whereas bacteroids of C. arietinum belong to the S morphotype ( Figure 1J-L). However, the G. orientalis bacteroids are barely branched (ENB morphotype) ( Figure 1F-H), whereas the V. sativa bacteroids are characterized by intensive branching (EB morphotype) ( Figure 1B-D). The bacteroids of the studied species vary significantly in size. ...
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... Skeletonize3D software, images of the microtubule were skeletonized (Figure 7). The following parameters were explored: (1) total number of microtubules, (2) total length of microtubules, (3) mean straightness index of detected microtubules, (4) total number of junctions, (5) degree of branching, and (6) mean number of junctions per skeleton ( Figure S1). The quantitative analysis of endoplasmic microtubule organization revealed that infected C. arietinum cells had the highest total number of microtubules, length of microtubules, and number of junctions per cell, and they significantly differed in these parameters from V. sativa cells ( Figure 8A,B,D). ...
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... Skeletonize3D software, images of the microtubule were skeletonized ( Figure 7). The following parameters were explored: (1) total number of microtubules, (2) total length of microtubules, (3) mean straightness index of detected microtubules, (4) total number of junctions, (5) degree of branching, and (6) mean number of junctions per skeleton ( Figure S1). The quantitative analysis of endoplasmic microtubule organization revealed that infected C. arietinum cells had the highest total number of microtubules, length of microtubules, and number of junctions per cell, and they significantly differed in these parameters from V. sativa cells ( Figure 8A,B,D). ...
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... analyzed species differ in terms of bacteroid morphotypes, with the tested rhizobia strains forming the ENB morphotype in G. orientalis nodules, the EB morphotype in V. sativa nodules, and the S morphotype in C. arietinum nodules (Figure 1). In general, these morphotypes correspond well to morphotypes previously described for C. arietinum [38], but not for G. orientalis [30]. ...
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... studies of microtubule patterns in a variety of species that form indeterminate nodules and determinate nodules will contribute to a deeper understanding of the role of the tubulin cytoskeleton in the development of symbiotic nodules. Figure S1: Graphical representation of terms used in the quantitative analysis of the microtubule cytoskeleton; Figure S2: Organization of the cortical microtubules in the infected cells of the nitrogen fixation zone; Figure S3: Organization of microtubules in colonized cells. ...
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... According to many studies, physical activity, overweight, and obesity are the most important and measurable factors in the incidence of cancer, so many studies on breast cancer and risk factors have been conducted [10][11][12]. Few studies have been done at the ecological level, given the geographical distribution [13]. In this study, based on ecological information and mathematical models, changes in breast cancer incidence were considered. ...
... The microtubule cytoskeleton pattern also undergoes changes during symbiosis development [13] starting from reorganization of microtubules on initial stages of root hair responses to rhizobia [74]. In nodule cells, bacteroid positioning correlates with characteristic microtubule rearrangements [75], wherein the pattern in root nodules was found to be host-plant specific [75]. The nodulation-specific kinesin-like calmodulin-binding protein (nKCBP), which crosslinks microtubules with the actin cytoskeleton, controls central vacuole morphogenesis in symbiotic cells in M. truncatula [76]. ...
... The microtubule cytoskeleton pattern also undergoes changes during symbiosis development [13] starting from reorganization of microtubules on initial stages of root hair responses to rhizobia [74]. In nodule cells, bacteroid positioning correlates with characteristic microtubule rearrangements [75], wherein the pattern in root nodules was found to be host-plant specific [75]. The nodulation-specific kinesin-like calmodulin-binding protein (nKCBP), which crosslinks microtubules with the actin cytoskeleton, controls central vacuole morphogenesis in symbiotic cells in M. truncatula [76]. ...
Symbiosis between leguminous plants and soil bacteria rhizobia is a refined type of plant–microbial interaction that has a great importance to the global balance of nitrogen. The reduction of atmospheric nitrogen takes place in infected cells of a root nodule that serves as a temporary shelter for thousands of living bacteria, which, per se, is an unusual state of a eukaryotic cell. One of the most striking features of an infected cell is the drastic changes in the endomembrane system that occur after the entrance of bacteria to the host cell symplast. Mechanisms for maintaining intracellular bacterial colony represent an important part of symbiosis that have still not been sufficiently clarified. This review focuses on the changes that occur in an endomembrane system of infected cells and on the putative mechanisms of infected cell adaptation to its unusual lifestyle.
... Generally, elongated and branched bacteroids are characteristic of indeterminate nodules, and rodshaped ones of determinate nodules (Oono et al., 2010;Montiel et al., 2017). However, in C. arietinum and Glycyrrhiza uralensis indeterminate nodules, bacteroids are spherical and swollen, respectively (Montiel et al., 2016;Kitaeva et al., 2021;Tsyganova et al., 2021). In the current study in L. japonicus nodules, bacteroids were similar to free-living bacteria by shape, but their size showed a twofold increase (Figures 1D,H,P,T, 2). ...
... Nevertheless, in order to confirm the universality of the identified patterns of cortical microtubules for infected cells in determinate and indeterminate nodules, it is necessary to analyze the organization of the tubulin cytoskeleton in a greater number of legume species. To date, of the six legume species forming indeterminate nodules, for which the analysis of the organization of the tubulin cytoskeleton was carried out, five belong to the same Vicioid clade Kitaeva et al., 2016Kitaeva et al., , 2021Tsyganova et al., 2021. ***Differences observed between microtubular patterns in determinate and indeterminate nodules are shown in bold. ...
Plant cell differentiation is based on rearrangements of the tubulin cytoskeleton; this is also true for symbiotic nodules. Nevertheless, although for indeterminate nodules (with a long-lasting meristem) the organization of microtubules during nodule development has been studied for various species, for determinate ones (with limited meristem activity) such studies are rare. Here, we investigated bacteroid morphology and dynamics of the tubulin cytoskeleton in determinate nodules of four legume species: Glycine max, Glycine soja, Phaseolus vulgaris, and Lotus japonicus. The most pronounced differentiation of bacteroids was observed in G. soja nodules. In meristematic cells in incipient nodules of all analyzed species, the organization of both cortical and endoplasmic microtubules was similar to that described for meristematic cells of indeterminate nodules. In young infected cells in developing nodules of all four species, cortical microtubules formed irregular patterns (microtubules were criss-crossed) and endoplasmic ones were associated with infection threads and infection droplets. Surprisingly, in uninfected cells the patterns of cortical microtubules differed in nodules of G. max and G. soja on the one hand, and P. vulgaris and L. japonicus on the other. The first two species exhibited irregular patterns, while the remaining two exhibited regular ones (microtubules were oriented transversely to the longitudinal axis of cell) that are typical for uninfected cells of indeterminate nodules. In contrast to indeterminate nodules, in mature determinate nodules of all four studied species, cortical microtubules formed a regular pattern in infected cells. Thus, our analysis revealed common patterns of tubulin cytoskeleton in the determinate nodules of four legume species, and species-specific differences were associated with the organization of cortical microtubules in uninfected cells. When compared with indeterminate nodules, the most pronounced differences were associated with the organization of cortical microtubules in nitrogen-fixing infected cells. The revealed differences indicated a possible transition during evolution of infected cells from anisotropic growth in determinate nodules to isodiametric growth in indeterminate nodules. It can be assumed that this transition provided an evolutionary advantage to those legume species with indeterminate nodules, enabling them to host symbiosomes in their infected cells more efficiently.
... However, the morphology of bacteroids in the symbiosomes varies considerably and several morphotypes have been distinguished. In some legumes, such as Medicago sativa and Galega orientalis, bacteroids are elongated and not branched (ENB morphotype) [29,30]; in others, such as Pisum sativum and Vicia sativa, they are elongated and branched (EB morphotype) or even pleiomorphic [30,31]; finally, swollen and spherical (S morphotype) bacteroids are typical for Cicer arietinum [30,32]. For determinate nodules, undifferentiated bacteroids (U morphotype) are typical [33]. ...
... However, the morphology of bacteroids in the symbiosomes varies considerably and several morphotypes have been distinguished. In some legumes, such as Medicago sativa and Galega orientalis, bacteroids are elongated and not branched (ENB morphotype) [29,30]; in others, such as Pisum sativum and Vicia sativa, they are elongated and branched (EB morphotype) or even pleiomorphic [30,31]; finally, swollen and spherical (S morphotype) bacteroids are typical for Cicer arietinum [30,32]. For determinate nodules, undifferentiated bacteroids (U morphotype) are typical [33]. ...
... However, the morphology of bacteroids in the symbiosomes varies considerably and several morphotypes have been distinguished. In some legumes, such as Medicago sativa and Galega orientalis, bacteroids are elongated and not branched (ENB morphotype) [29,30]; in others, such as Pisum sativum and Vicia sativa, they are elongated and branched (EB morphotype) or even pleiomorphic [30,31]; finally, swollen and spherical (S morphotype) bacteroids are typical for Cicer arietinum [30,32]. For determinate nodules, undifferentiated bacteroids (U morphotype) are typical [33]. ...
Chinese liquorice (Glycyrrhiza uralensis Fisch. ex DC.) is widely used in the food industry and as a medicine. Like other legumes, G. uralensis forms symbiotic nodules. However, the structural organization of G. uralensis nodules is poorly understood. In this study, we analyzed the histological and ultrastructural organization and dynamics of the tubulin cytoskeleton in various cells from different histological zones of indeterminate nodules formed by two strains of Mesorhizobium sp. The unusual walls of infection threads and formation of multiple symbiosomes with several swollen bacteroids were observed. A large amount of poly-β-hydroxybutyrate accumulated in the bacteroids, while the vacuoles of meristematic and uninfected cells contained drop-shaped osmiophilic inclusions. Immunolocalization of the tubulin cytoskeleton and quantitative analysis of cytoskeletal elements revealed patterns of cortical microtubules in meristematic, infected and uninfected cells, and of endoplasmic microtubules associated with infection structures, typical of indeterminate nodules. The intermediate pattern of endoplasmic microtubules in infected cells was correlated with disordered arrangement of symbiosomes. Thus, analysis of the structural organization of G. uralensis nodules revealed some ancestral features more characteristic of determinate nodules, demonstrating the evolutionary closeness of G. uralensis nodulation to more ancient members of the legume family.
... Differentiation of infected cells primarily leads to an increase in their size. Unlike root cells, which grow anisotropically, infected cells in indeterminate nodules demonstrate isodiametric growth [11,12]. This type of growth produces an increase in the volume of an infected cell up to 80-fold greater than meristematic cells, and allows accommodation of tens of thousands of bacteroids [13]. ...
... These data may indicate a connection between cell differentiation and changes in the organization and functioning of the cytoskeleton in plant cells. Indeed, crucial changes in tubulin and actin cytoskeleton rearrangements during nodule development in different legume species have been described [11,12,[88][89][90]. The observed "switch" when the same functional groups were enriched with both down-and upregulated genes in the same comparisons was reported for several GO categories and KEGG pathways. ...
Garden pea (Pisum sativum L.) is a globally important legume crop. Like other legumes, it forms beneficial symbiotic interactions with the soil bacteria rhizobia, gaining the ability to fix atmospheric nitrogen. In pea nodules, the meristem is long-lasting and results in the formation of several histological zones that implicate a notable differentiation of infected host cells. However, the fine transcriptional changes that accompany differentiation are still unknown. In this study, using laser microdissection followed by RNA-seq analysis, we performed transcriptomic profiling in the early infection zone, late infection zone, and nitrogen fixation zone of 11-day-old nodules of pea wild-type line SGE. As a result, a list of functional groups of differentially expressed genes (DEGs) in different nodule histological zones and a list of genes with the most prominent expression changes during nodule development were obtained. Their analyses demonstrated that the highest amount of DEGs was associated with the nitrogen fixation zone. Among well-known genes controlling nodule development, we revealed genes that can be novel players throughout nodule formation. The characterized genes in pea were compared with those previously described in other legumes and their possible functions in nodule development are discussed.
The legume–rhizobium symbiosis represents the most important system for terrestrial biological nitrogen fixation on land. Efficient nitrogen fixation during this symbiosis depends on successful rhizobial infection and complete endosymbiosis, which are achieved by complex cellular events including cell-wall remodeling, cytoskeletal reorganizations, and extensive membrane expansion and trafficking. In this review, we explore the dynamic remodeling of the plant-specific cell wall-membrane system-cytoskeleton (WMC) continuum during symbiotic nitrogen fixation. We focus on key processes linked to efficient nitrogen fixation, including rhizobial uptake, infection thread formation and elongation, rhizobial droplet release, cytoplasmic bridge formation, and rhizobial endosymbiosis. Additionally, we discuss the advanced techniques for investigating the cellular basis of root-nodule symbiosis and provide insights into the unsolved mysteries of robust symbiotic nitrogen fixation.
