Nodule Primordia Research Articles

The Small GTPase ROP6 Interacts with NFR5 and Is Involved in Nodule Formation inLotus japonicus

Nod Factor Receptor5 (NFR5) is an atypical receptor-like kinase, having no activation loop in the protein kinase domain. It forms a heterodimer with NFR1 and is required for the early plant responses to Rhizobium infection. A Rho-like small GTPase from Lotus japonicus was identified as an NFR5-interacting protein. The amino acid sequence of this Rho-like GTPase is closest to the Arabidopsis (Arabidopsis thaliana) ROP6 and Medicago truncatula ROP6 and was designated as LjROP6. The interaction between Rop6 and NFR5 occurred both in vitro and in planta. No interaction between Rop6 and NFR1 was observed. Green fluorescent protein-tagged ROP6 was localized at the plasma membrane and cytoplasm. The interaction between ROP6 and NFR5 appeared to take place at the plasma membrane. The expression of the ROP6 gene could be detected in vascular tissues of Lotus roots. After inoculation with Mesorhizobium loti, elevated levels of ROP6 expression were found in the root hairs, root tips, vascular bundles of roots, nodule primordia, and young nodules. In transgenic hairy roots expressing ROP6 RNA interference constructs, Rhizobium entry into the root hairs did not appear to be affected, but infection thread growth through the root cortex were severely inhibited, resulting in the development of fewer nodules per plant. These data demonstrate a role of ROP6 as a positive regulator of infection thread formation and nodulation in L. japonicus.

The diversity of actinorhizal symbiosis

Filamentous aerobic soil actinobacteria of the genus Frankia can induce the formation of nitrogen-fixing nodules on the roots of a diverse group of plants from eight dicotyledonous families, collectively called actinorhizal plants. Within nodules, Frankia can fix nitrogen while being hosted inside plant cells. Like in legume/rhizobia symbioses, bacteria can enter the plant root either intracellularly through an infection thread formed in a curled root hair, or intercellularly without root hair involvement, and the entry mechanism is determined by the host plant species. Nodule primordium formation is induced in the root pericycle as for lateral root primordia. Mature actinorhizal nodules are coralloid structures consisting of multiple lobes, each of which represents a modified lateral root without a root cap, a superficial periderm and with infected cells in the expanded cortex. In this review, an overview of nodule induction mechanisms and nodule structure is presented including comparisons with the corresponding mechanisms in legume symbioses.

A MAP Kinase Kinase Interacts with SymRK and Regulates Nodule Organogenesis in Lotus japonicus

The symbiosis receptor kinase, SymRK, is required for root nodule development. A SymRK-interacting protein (SIP2) was found to form protein complex with SymRK in vitro and in planta. The interaction between SymRK and SIP2 is conserved in legumes. The SIP2 gene was expressed in all Lotus japonicus tissues examined. SIP2 represents a typical plant mitogen-activated protein kinase kinase (MAPKK) and exhibited autophosphorylation and transphosphorylation activities. Recombinant SIP2 protein could phosphorylate casein and the Arabidopsis thaliana MAP kinase MPK6. SymRK and SIP2 could not use one another as a substrate for phosphorylation. Instead, SymRK acted as an inhibitor of SIP2 kinase when MPK6 was used as a substrate, suggesting that SymRK may serve as a negative regulator of the SIP2 signaling pathway. Knockdown expression of SIP2 via RNA interference (RNAi) resulted in drastic reduction of nodules formed in transgenic hairy roots. A significant portion of SIP2 RNAi hairy roots failed to form a nodule. In these roots, the expression levels of SIP2 and three marker genes for infection thread and nodule primordium formation were downregulated drastically, while the expression of two other MAPKK genes were not altered. These observations demonstrate an essential role of SIP2 in the early symbiosis signaling and nodule organogenesis.

Nodule numbers are governed by interaction between CLE peptides and cytokinin signaling

CLE peptides are involved in the balance between cell division and differentiation throughout plant development, including nodulation. Previously, two CLE genes of Medicago truncatula, MtCLE12 and MtCLE13, had been identified whose expression correlated with nodule primordium formation and meristem establishment. Gain-of-function analysis indicated that both MtCLE12 and MtCLE13 interact with the SUPER NUMERIC NODULES (SUNN)-dependent auto-regulation of nodulation to control nodule numbers. Here we demonstrate that cytokinin, which is essential for nodule organ formation, regulates MtCLE13 expression. In addition, simultaneous knockdown of MtCLE12 and MtCLE13 resulted in an increase in nodule number, implying that both genes play a role in controlling nodule number. Additionally, a weak link may exist with the ethylene-dependent mechanism that locally controls nodule number.

Modeling a cortical auxin maximum for nodulation: different signatures of potential strategies.

Lateral organ formation from plant roots typically requires the de novo creation of a meristem, initiated at the location of a localized auxin maximum. Legume roots can form both root nodules and lateral roots. From the basic principles of auxin transport and metabolism only a few mechanisms can be inferred for increasing the local auxin concentration: increased influx, decreased efflux, and (increased) local production. Using computer simulations we investigate the different spatio-temporal patterns resulting from each of these mechanisms in the context of a root model of a generalized legume. We apply all mechanisms to the same group of preselected cells, dubbed the controlled area. We find that each mechanism leaves its own characteristic signature. Local production by itself can not create a strong auxin maximum. An increase of influx, as is observed in lateral root formation, can result in an auxin maximum that is spatially more confined than the controlled area. A decrease of efflux on the other hand leads to a broad maximum, which is more similar to what is observed for nodule primordia. With our prime interest in nodulation, we further investigate the dynamics following a decrease of efflux. We find that with a homogeneous change in the whole cortex, the first auxin accumulation is observed in the inner cortex. The steady state lateral location of this efflux reduced auxin maximum can be shifted by slight changes in the ratio of central to peripheral efflux carriers. We discuss the implications of this finding in the context of determinate and indeterminate nodules, which originate from different cortical positions. The patterns we have found are robust under disruption of the (artificial) tissue layout. The same patterns are therefore likely to occur in many other contexts.

Reproducible hairy root transformation and spot-inoculation methods to study root symbioses of pea.

Pea has lagged behind other model legumes in the molecular study of nodulation and mycorrhizae-formation because of the difficulty to transform its roots and its poor growth on agar plates. Here we describe for pea 1) a transformation technique which permits the complementation of two known non-nodulating pea mutants, 2) a rhizobial inoculation method which allows the study of early cellular events giving rise to nodule primordia, and 3) a targeted fungal inoculation method which allows us to study short segments of mycorrhizal roots assured to be infected. These tools are certain to advance our knowledge of pea root symbioses.

Cytokinin Induction of Root Nodule Primordia in Lotus japonicus Is Regulated by a Mechanism Operating in the Root Cortex

Cytokinin plays a central role in the formation of nitrogen-fixing root nodules following inoculation with rhizobia. We show that exogenous cytokinin induces formation of discrete and easily visible nodule primordia in Lotus japonicus roots. The expression of nodulin genes was up-regulated upon cytokinin treatment, suggesting that the genuine nodulation program was indeed activated. This offers a simple approach for dissecting the underlying mechanism. Cytokinin-induced nodule primordia formation was unperturbed in several loss-of-function mutants impaired in epidermal responses to either rhizobial infection, Nod factor application, or both. However, absence of primordia in nsp1, nsp2, and nin mutants showed the requirement for these transcriptional regulators in the cytokinin-mediated activation of the root cortex. Distinguishing the epidermal and cortical responses further, we found that external cytokinin application induced expression of the Nin::GUS reporter gene within the root cortex but not in the root epidermis. Using L. japonicus lhk1-1 and har1 mutants, we demonstrate that discrete activation of root cortical cells by cytokinin depends on the LHK1 cytokinin receptor and is subjected to HAR1-mediated autoregulation.

A Phylogenetic Strategy Based on a Legume-Specific Whole Genome Duplication Yields Symbiotic Cytokinin Type-A Response Regulators

Legumes host their Rhizobium spp. symbiont in novel root organs called nodules. Nodules originate from differentiated root cortical cells that dedifferentiate and subsequently form nodule primordia, a process controlled by cytokinin. A whole-genome duplication has occurred at the root of the legume Papilionoideae subfamily. We hypothesize that gene pairs originating from this duplication event and are conserved in distinct Papilionoideae lineages have evolved symbiotic functions. A phylogenetic strategy was applied to search for such gene pairs to identify novel regulators of nodulation, using the cytokinin phosphorelay pathway as a test case. In this way, two paralogous type-A cytokinin response regulators were identified that are involved in root nodule symbiosis. Response Regulator9 (MtRR9) and MtRR11 in medicago (Medicago truncatula) and an ortholog in lotus (Lotus japonicus) are rapidly induced upon Rhizobium spp. Nod factor signaling. Constitutive expression of MtRR9 results in arrested primordia that have emerged from cortical, endodermal, and pericycle cells. In legumes, lateral root primordia are not exclusively formed from pericycle cells but also require the involvement of the root cortical cell layer. Therefore, the MtRR9-induced foci of cell divisions show a strong resemblance to lateral root primordia, suggesting an ancestral function of MtRR9 in this process. Together, these findings provide a proof of principle for the applied phylogenetic strategy to identify genes with a symbiotic function in legumes.

Soybean Nodule-Enhanced CLE Peptides in Roots Act as Signals in GmNARK-Mediated Nodulation Suppression

The number of nodules formed in the roots of leguminous plants is systemically controlled by autoregulation of nodulation (AON). This study characterized two of the CLAVATA3/endosperm-surrounding region (CLE) genes involved in AON signal transduction. The GmRIC1 and GmRIC2 genes initiated expression solely in the roots at approximately 3 days after inoculation (DAI) with Nod factor-producing rhizobia, corresponding to the time point of AON, and the expression was up-regulated by cytokinins. Levels of GmRIC1 and GmRIC2 gene expression were much higher in the supernodulation mutant, SS2-2, than in wild-type (WT) soybeans during nodule development, even after initiation of nitrogen fixation. At 3 DAI, GmRIC2 was induced in the cells of the pericycle and the outer cortex, which undergo cell division to form nodule primordia and spreads from the central region to the whole nodule as it develops. Overexpression of GmRIC1 and GmRIC2 strongly suppressed the nodulation of WT roots as well as transgenic hairy roots in a GmNARK-dependent manner. This systemic suppression of nodulation was caused by the secretion of two CLE proteins into the extracellular space. Double grafting between WT and SS2-2 soybeans showed that signal Q is larger in SS2-2 than in WT roots during nodulation. The results of this study suggest that GmRIC1 and GmRIC2 are good candidates for root-derived signal Q in AON signal transduction.

Lotus japonicus symRK‐14 uncouples the cortical and epidermal symbiotic program

SYMRK is a leucine-rich-repeat (LRR)-receptor kinase that mediates intracellular symbioses of legumes with rhizobia and arbuscular mycorrhizal fungi. It participates in signalling events that lead to epidermal calcium spiking, an early cellular response that is typically considered as central for intracellular accommodation and nodule organogenesis. Here, we describe the Lotus japonicus symRK-14 mutation that alters a conserved GDPC amino-acid sequence in the SYMRK extracellular domain. Normal infection of the epidermis by fungal or bacterial symbionts was aborted in symRK-14. Likewise, epidermal responses of symRK-14 to bacterial signalling, including calcium spiking, NIN gene expression and infection thread formation, were significantly reduced. In contrast, no major negative effects on the formation of nodule primordia and cortical infection were detected. Cumulatively, our data show that the symRK-14 mutation uncouples the epidermal and cortical symbiotic program, while indicating that the SYMRK extracellular domain participates in transduction of non-equivalent signalling events. The GDPC sequence was found to be highly conserved in LRR-receptor kinases in legumes and non-legumes, including the evolutionarily distant bryophytes. Conservation of the GDPC sequence in nearly one-fourth of LRR-receptor-like kinases in the genome of Arabidopsis thaliana suggests, however, that this sequence might also play an important non-symbiotic function in this plant.

MtbHLH1, a bHLH transcription factor involved in Medicago truncatula nodule vascular patterning and nodule to plant metabolic exchanges.

This study aimed at defining the role of a basic helix–loop–helix (bHLH) transcription factor gene from Medicago truncatula, MtbHLH1, whose expression is upregulated during the development of root nodules produced upon infection by rhizobia bacteria.We used MtbHLH1 promoter::GUS fusions and quantitative reverse-transcription polymerase chain reaction analyses to finely characterize the MtbHLH1 expression pattern. We altered MtbHLH1 function by expressing a dominantly repressed construct (CRES-T approach) and looked for possible MtbHLH1 target genes by transcriptomics.We found that MtbHLH1 is expressed in nodule primordia cells derived from pericycle divisions, in nodule vascular bundles (VBs) and in uninfected cells of the nitrogen (N) fixation zone. MtbHLH1 is also expressed in root tips, lateral root primordia cells and root VBs, and induced upon auxin treatment. Altering MtbHLH1 function led to an unusual phenotype, with a modified patterning of nodule VB development and a reduced growth of aerial parts of the plant, even though the nodules were able to fix atmospheric N. Several putative MtbHLH1 regulated genes were identified, including an asparagine synthase and a LOB (lateral organ boundary) transcription factor.Our results suggest that the MtbHLH1 gene is involved in the control of nodule vasculature patterning and nutrient exchanges between nodules and roots.

Rhizobial Nod factors are required for cortical cell division in the nodule morphogenetic programme of the Aeschynomeneae legume Arachis

Nod factors are among the best-studied molecules implicated in the signal exchange that leads to legume-rhizobia symbiosis. The role of these molecules in symbiosis development has been primarily studied in legumes invaded through infection threads. In these plants, Nod factors generate several responses required for nodulation, including the induction of cortical cell division to form the nodule primordium. Arachis hypogaea L. (peanut) exhibits a specific mode of rhizobial infection and nodule morphogenetic programme in which infection threads are never formed. The role of Nod factors in this particular mechanism is unknown. In this work, a peanut symbiont mutant strain unable to produce Nod factors was obtained and characterised. The strain Bradyrhizobium (Arachis) sp. SEMIA 6144 V2 is altered in the nodC gene, which encodes an N-acetylglucosaminyl transferase involved in the first step of the Nod factor biosynthetic pathway. Further research revealed that, although its ability to colonise peanut roots was unaffected, it is not capable of inducing the division of cortical cells. The results obtained indicate that rhizobial Nod factors are essential for the induction of cortical cell division that leads to nodule primordium formation.

Search for nodulation-related CLE genes in the genome of Glycine max

CLE peptides are potentially involved in nodule organ development and in the autoregulation of nodulation (AON), a systemic process that restricts nodule number. A genome-wide survey of CLE peptide genes in the soybean glycine max genome resulted in the identification of 39 GmCLE genes, the majority of which have not yet been annotated. qRT-PCR analysis indicated two different nodulation-related CLE expression patterns, one linked with nodule primordium development and a new one linked with nodule maturation. Moreover, two GmCLE gene pairs, encoding group-III CLE peptides that were previously shown to be involved in AON, had a transient expression pattern during nodule development, were induced by the essential nodulation hormone cytokinin, and one pair was also slightly induced by the addition of nitrate. Hence, our data support the hypothesis that group-III CLE peptides produced in the nodules are involved in primordium homeostasis and intertwined in activating AON, but not in sustaining it.

Role of lupeol synthase in Lotus japonicus nodule formation

• Triterpenes are plant secondary metabolites, derived from the cyclization of 2,3-oxidosqualene by oxidosqualene cyclases (OSCs). Here, we investigated the role of lupeol synthase, encoded by OSC3, and its product, lupeol, in developing roots and nodules of the model legume Lotus japonicus. • The expression patterns of OSC3 in different developmental stages of uninfected roots and in roots infected with Mesorhizobium loti were determined. The tissue specificity of OSC3 expression was analysed by in situ hybridization. Functional analysis, in which transgenic L. japonicus roots silenced for OSC3 were generated, was performed. The absence of lupeol in the silenced plant lines was determined by GC-MS. • The expression of ENOD40, a marker gene for nodule primordia initiation, was increased significantly in the OSC3-silenced plant lines, suggesting that lupeol influences nodule formation. Silenced plants also showed a more rapid nodulation phenotype, consistent with this. Exogenous application of lupeol to M. loti-infected wild-type plants provided further evidence for a negative regulatory effect of lupeol on the expression of ENOD40. • The synthesis of lupeol in L. japonicus roots and nodules can be solely attributed to OSC3. Taken together, our data suggest a role for lupeol biosynthesis in nodule formation through the regulation of ENOD40 gene expression.

Misexpression of miR482, miR1512, and miR1515 Increases Soybean Nodulation

MicroRNAs (miRNAs) are important regulators of plant growth and development. Previously, we identified a group of conserved and novel miRNA families from soybean (Glycine max) roots. Many of these miRNAs are specifically induced during soybean-Bradyrhizobium japonicum interactions. Here, we examined the gene expression levels of six families of novel miRNAs and investigated their functions in nodule development. We used northern-blot analyses to study the tissue specificity and time course of miRNA expression. Transgenic expression of miR482, miR1512, and miR1515 led to significant increases of nodule numbers, while root length, lateral root density, and the number of nodule primordia were not altered in all tested miRNA lines. We also found differential expression of these miRNAs in nonnodulating and supernodulating soybean mutants. The expression levels of 22 predicted target genes regulated by six novel miRNAs were studied by real-time polymerase chain reaction and quantitative real-time polymerase chain reaction. These results suggested that miRNAs play important roles in soybean nodule development.

Sesbania rostrata: a case study of natural variation in legume nodulation

Legumes acquired the ability to engage in a symbiotic interaction with soil-borne bacteria and establish a nitrogen-fixing symbiosis in a novel root organ, the nodule. Most legume crops and the model legumes Medicago truncatula and Lotus japonicus are infected intracellularly in root hairs via infection threads that lead the bacteria towards a nodule primordium in the root cortex. This infection process, however, does not reflect the great diversity of infection strategies that are used by leguminous plants. An alternative, intercellular invasion occurs in the semiaquatic legume Sesbania rostrata. Bacteria colonize epidermal fissures at lateral root bases and trigger cortical cell death for infection pocket formation and subsequent intercellular and intracellular infection thread progression towards the primordium. This infection mode evolved as an adaptation to waterlogged conditions that inhibit intracellular invasion. In this review, we discuss the molecular basis for this adaptation and how insights into this process contribute to general knowledge of the rhizobial infection process.

Nodulation Gene Mutants of Mesorhizobium loti R7A—nodZ and nolL Mutants Have Host-Specific Phenotypes on Lotus spp.

Rhizobial Nod factors induce plant responses and facilitate bacterial infection, leading to the development of nitrogen-fixing root nodules on host legumes. Nodule initiation is highly dependent on Nod-factor structure and, hence, on at least some of the nodulation genes that encode Nod-factor production. Here, we report the effects of mutations in Mesorhizobium loti R7A nodulation genes on nodulation of four Lotus spp. and on Nod-factor structure. Most mutants, including a DeltanodSDeltanolO double mutant that produced Nod factors lacking the carbamoyl and possibly N-methyl groups on the nonreducing terminal residue, were unaffected for nodulation. R7ADeltanodZ and R7ADeltanolL mutants that produced Nod factors without the (acetyl)fucose on the reducing terminal residue had a host-specific phenotype, forming mainly uninfected nodule primordia on Lotus filicaulis and L. corniculatus and effective nodules with a delay on L. japonicus. The mutants also showed significantly reduced infection thread formation and Nin gene induction. In planta complementation experiments further suggested that the acetylfucose was important for balanced signaling in response to Nod factor by the L. japonicus NFR1/NFR5 receptors. Overall the results reveal differences in the sensitivity of plant perception with respect to signaling leading to root hair deformation and nodule primordium development versus infection thread formation and rhizobial entry.

CERBERUS, a novel U‐box protein containing WD‐40 repeats, is required for formation of the infection thread and nodule development in the legume–Rhizobium symbiosis

Endosymbiotic infection of legume plants by Rhizobium bacteria is initiated through infection threads (ITs) which are initiated within and penetrate from root hairs and deliver the endosymbionts into nodule cells. Despite recent progress in understanding the mutual recognition and early symbiotic signaling cascades in host legumes, the molecular mechanisms underlying bacterial infection processes and successive nodule organogenesis are still poorly understood. We isolated a novel symbiotic mutant of Lotus japonicus, cerberus, which shows defects in IT formation and nodule organogenesis. Map-based cloning of the causal gene allowed us to identify the CERBERUS gene, which encodes a novel protein containing a U-box domain and WD-40 repeats. CERBERUS expression was detected in the roots and nodules, and was enhanced after inoculation of Mesorhizobium loti. Strong expression was detected in developing nodule primordia and the infected zone of mature nodules. In cerberus mutants, Rhizobium colonized curled root hair tips, but hardly penetrated into root hair cells. The occasional ITs that were formed inside the root hair cells were mostly arrested within the epidermal cell layer. Nodule organogenesis was aborted prematurely, resulting in the formation of a large number of small bumps which contained no endosymbiotic bacteria. These phenotypic and genetic analyses, together with comparisons with other legume mutants with defects in IT formation, indicate that CERBERUS plays a critical role in the very early steps of IT formation as well as in growth and differentiation of nodules.

LIN, a Novel Type of U-Box/WD40 Protein, Controls Early Infection by Rhizobia in Legumes

The formation of a nitrogen-fixing nodule requires the coordinated development of rhizobial colonization and nodule organogenesis. Based on its mutant phenotype, lumpy infections (lin), LIN functions at an early stage of the rhizobial symbiotic process, required for both infection thread growth in root hair cells and the further development of nodule primordia. We show that spontaneous nodulation activated by the calcium- and calmodulin-dependent protein kinase is independent of LIN; thus, LIN is not necessary for nodule organogenesis. From this, we infer that LIN predominantly functions during rhizobial colonization and that the abortion of this process in lin mutants leads to a suppression of nodule development. Here, we identify the LIN gene in Medicago truncatula and Lotus japonicus, showing that it codes for a predicted E3 ubiquitin ligase containing a highly conserved U-box and WD40 repeat domains. Ubiquitin-mediated protein degradation is a universal mechanism to regulate many biological processes by eliminating rate-limiting enzymes and key components such as transcription factors. We propose that LIN is a regulator of the component(s) of the nodulation factor signal transduction pathway and that its function is required for correct temporal and spatial activity of the target protein(s).

A Small GTPase of the Rab Family Is Required for Root Hair Formation and Preinfection Stages of the Common Bean–RhizobiumSymbiotic Association

Legume plants are able to establish a symbiotic relationship with soil bacteria from the genus Rhizobium, leading to the formation of nitrogen-fixing root nodules. Successful nodulation requires both the formation of infection threads (ITs) in the root epidermis and the activation of cell division in the cortex to form the nodule primordium. This study describes the characterization of RabA2, a common bean (Phaseolus vulgaris) cDNA previously isolated as differentially expressed in root hairs infected with Rhizobium etli, which encodes a protein highly similar to small GTPases of the RabA2 subfamily. This gene is expressed in roots, particularly in root hairs, where the protein was found to be associated with vesicles that move along the cell. The role of this gene during nodulation has been studied in common bean transgenic roots using a reverse genetic approach. Examination of root morphology in RabA2 RNA interference (RNAi) plants revealed that the number and length of the root hairs were severely reduced in these plants. Upon inoculation with R. etli, nodulation was completely impaired and no induction of early nodulation genes (ENODs), such as ERN1, ENOD40, and Hap5, was detected in silenced hairy roots. Moreover, RabA2 RNAi plants failed to induce root hair deformation and to initiate ITs, indicating that morphological changes that precede bacterial infection are compromised in these plants. We propose that RabA2 acts in polar growth of root hairs and is required for reorientation of the root hair growth axis during bacterial infection.