MtNRLK1, a CLAVATA1-like leucine-rich repeat receptor-like kinase upregulated during nodulation in Medicago truncatula.
ABSTRACT: Peptides are signaling molecules regulating various aspects of plant development, including the balance between cell division and differentiation in different meristems. Among those, CLAVATA3/Embryo Surrounding Region-related (CLE-ESR) peptide activity depends on leucine-rich-repeat receptor-like-kinases (LRR-RLK) belonging to the subclass XI. In legume plants, such as the Medicago truncatula model, specific CLE peptides were shown to regulate root symbiotic nodulation depending on the LRR-RLK SUNN (Super Numeric Nodules). Amongst the ten M. truncatula LRR-RLK most closely related to SUNN, only one showed a nodule-induced expression, and was so-called MtNRLK1 (Nodule-induced Receptor-Like Kinase 1). MtNRLK1 expression is associated to root and nodule vasculature as well as to the proximal meristem and rhizobial infection zone in the nodule apex. Except for the root vasculature, the MtNRLK1 symbiotic expression pattern is different than the one of MtSUNN. Functional analyses either based on RNA interference, insertional mutagenesis, and overexpression of MtNRLK1 however failed to identify a significant nodulation phenotype, either regarding the number, size, organization or nitrogen fixation capacity of the symbiotic organs formed.
Project description:In plants, root system architecture is determined by the activity of root apical meristems, which control the root growth rate, and by the formation of lateral roots. In legumes, an additional root lateral organ can develop: the symbiotic nitrogen-fixing nodule. We identified in Medicago truncatula ten allelic mutants showing a compact root architecture phenotype (cra2) independent of any major shoot phenotype, and that consisted of shorter roots, an increased number of lateral roots, and a reduced number of nodules. The CRA2 gene encodes a Leucine-Rich Repeat Receptor-Like Kinase (LRR-RLK) that primarily negatively regulates lateral root formation and positively regulates symbiotic nodulation. Grafting experiments revealed that CRA2 acts through different pathways to regulate these lateral organs originating from the roots, locally controlling the lateral root development and nodule formation systemically from the shoots. The CRA2 LRR-RLK therefore integrates short- and long-distance regulations to control root system architecture under non-symbiotic and symbiotic conditions.
Project description:The number of legume root nodules resulting from a symbiosis with rhizobia is tightly controlled by the plant. Certain members of the CLAVATA3/Embryo Surrounding Region (CLE) peptide family, specifically MtCLE12 and MtCLE13 in Medicago truncatula, act in the systemic autoregulation of nodulation (AON) pathway that negatively regulates the number of nodules. Little is known about the molecular pathways that operate downstream of the AON-related CLE peptides. Here, by means of a transcriptome analysis, we show that roots ectopically expressing MtCLE13 deregulate only a limited number of genes, including three down-regulated genes encoding lysin motif receptor-like kinases (LysM-RLKs), among which are the nodulation factor (NF) receptor NF Perception gene (NFP) and two up-regulated genes, MtTML1 and MtTML2, encoding Too Much Love (TML)-related Kelch-repeat containing F-box proteins. The observed deregulation was specific for the ectopic expression of nodulation-related MtCLE genes and depended on the Super Numeric Nodules (SUNN) AON RLK. Moreover, overexpression and silencing of these two MtTML genes demonstrated that they play a role in the negative regulation of nodule numbers. Hence, the identified MtTML genes are the functional counterpart of the Lotus japonicus TML gene shown to be central in the AON pathway. Additionally, we propose that the down-regulation of a subset of LysM-RLK-encoding genes, among which is NFP, might contribute to the restriction of further nodulation once the first nodules have been formed.
Project description:Legumes can survive in nitrogen-deficient environments by forming root-nodule symbioses with rhizobial bacteria; however, forming nodules consumes energy, and nodule numbers must thus be strictly controlled. Previous studies identified major negative regulators of nodulation in Lotus japonicus, including the small peptides CLAVATA3/ESR (CLE)-RELATED-ROOT SIGNAL1 (CLE-RS1), CLE-RS2, and CLE-RS3, and their putative major receptor HYPERNODULATION AND ABERRANT ROOT FORMATION1 (HAR1). CLE-RS2 is known to be expressed in rhizobia-inoculated roots, and is predicted to be post-translationally arabinosylated, a modification essential for its activity. Moreover, all three CLE-RSs suppress nodulation in a HAR1-dependent manner. Here, we identified PLENTY as a gene responsible for the previously isolated hypernodulation mutant plenty. PLENTY encoded a hydroxyproline O-arabinosyltransferase orthologous to ROOT DETERMINED NODULATION1 in Medicago truncatula. PLENTY was localized to the Golgi, and an in vitro analysis of the recombinant protein demonstrated its arabinosylation activity, indicating that CLE-RS1/2/3 may be substrates for PLENTY. The constitutive expression experiments showed that CLE-RS3 was the major candidate substrate for PLENTY, suggesting the substrate preference of PLENTY for individual CLE-RS peptides. Furthermore, a genetic analysis of the plenty har1 double mutant indicated the existence of another PLENTY-dependent and HAR1-independent pathway negatively regulating nodulation.
Project description:Autoregulatory negative-feedback loops play important roles in fine-balancing tissue and organ development. Such loops are composed of short-range intercellular signaling pathways via cell-cell communications. On the other hand, leguminous plants use a long-distance negative-feedback system involving root-shoot communication to control the number of root nodules, root lateral organs that harbor symbiotic nitrogen-fixing bacteria known as rhizobia. This feedback system, known as autoregulation of nodulation (AON), consists of two long-distance mobile signals: root-derived and shoot-derived signals. Two Lotus japonicus CLAVATA3/endosperm surrounding region (CLE)-related small peptides, CLE root signal1 (CLE-RS1) and CLE-RS2, function as root-derived signals and are perceived by a shoot-acting AON factor, the hypernodulation aberrant root formation1 (HAR1) receptor protein, an ortholog of Arabidopsis CLAVATA1, which is responsible for shoot apical meristem homeostasis. This peptide-receptor interaction is necessary for systemic suppression of nodulation. How the onset of nodulation activates AON and how optimal nodule numbers are maintained remain unknown, however. Here we show that an RWP-RK-containing transcription factor, nodule inception (NIN), which induces nodule-like structures without rhizobial infection when expressed ectopically, directly targets CLE-RS1 and CLE-RS2. Roots constitutively expressing NIN systemically repress activation of endogenous NIN expression in untransformed roots of the same plant in a HAR1-dependent manner, leading to systemic suppression of nodulation and down-regulation of CLE expression. Our findings provide, to our knowledge, the first molecular evidence of a long-distance autoregulatory negative-feedback loop that homeostatically regulates nodule organ formation.
Project description:BACKGROUND: Plants encode a large number of leucine-rich repeat receptor-like kinases. Legumes encode several LRR-RLK linked to the process of root nodule formation, the ligands of which are unknown. To identify ligands for these receptors, we used a combination of profile hidden Markov models and position-specific iterative BLAST, allowing us to detect new members of the CLV3/ESR (CLE) protein family from publicly available sequence databases. RESULTS: We identified 114 new members of the CLE protein family from various plant species, as well as five protein sequences containing multiple CLE domains. We were able to cluster the CLE domain proteins into 13 distinct groups based on their pairwise similarities in the primary CLE motif. In addition, we identified secondary motifs that coincide with our sequence clusters. The groupings based on the CLE motifs correlate with known biological functions of CLE signaling peptides and are analogous to groupings based on phylogenetic analysis and ectopic overexpression studies. We tested the biological function of two of the predicted CLE signaling peptides in the legume Medicago truncatula. These peptides inhibit the activity of the root apical and lateral root meristems in a manner consistent with our functional predictions based on other CLE signaling peptides clustering in the same groups. CONCLUSION: Our analysis provides an identification and classification of a large number of novel potential CLE signaling peptides. The additional motifs we found could lead to future discovery of recognition sites for processing peptidases as well as predictions for receptor binding specificity.
Project description:CLE peptide hormones are critical regulators of many cell proliferation and differentiation mechanisms in plants. These 12-13 amino acid glycosylated peptides play vital roles in a diverse range of plant tissues, including the shoot, root and vasculature. CLE peptides are also involved in controlling legume nodulation. Here, the entire family of CLE peptide-encoding genes was identified in Medicago truncatula (52) and Lotus japonicus (53), including pseudogenes and non-functional sequences that were identified. An array of bioinformatic techniques were used to compare and contrast these complete CLE peptide-encoding gene families with those of fellow legumes, Glycine max and Phaseolus vulgaris, in addition to the model plant Arabidopsis thaliana. This approach provided insight into the evolution of CLE peptide families and enabled us to establish putative M. truncatula and L. japonicus orthologues. This includes orthologues of nodulation-suppressing CLE peptides and AtCLE40 that controls the stem cell population of the root apical meristem. A transcriptional meta-analysis was also conducted to help elucidate the function of the CLE peptide family members. Collectively, our analyses considerably increased the number of annotated CLE peptides in the model legume species, M. truncatula and L. japonicus, and substantially enhanced the knowledgebase of this critical class of peptide hormones.
Project description:Leucine-rich repeat receptor-like kinases (LRR-RLKs) constitute the largest subfamily of receptor-like kinases (RLKs) in plants. They play roles in plant growth and developmental and physiological processes, but less is known about the functions of LRR-RLKs in Medicago truncatula. Our genome-wide analysis revealed 329 LRR-RLK genes in the M.truncatula genome. Phylogenetic and classification analysis suggested that these genes could be classified into 15 groups and 24 subgroups. A total of 321 genes were mapped onto all chromosomes, and 23 tandem duplications (TDs) involving 56 genes were distributed on each chromosome except 4. Twenty-seven M.truncatula LRR-RLK segmental duplication gene pairs were colinearly related. The exon/intron organization, motif composition and arrangements were relatively conserved among members of the same groups or subgroups. Using publicly available RNAseq data and quantitative real-time polymerase chain reaction (qRT-PCR), expression profiling suggested that LRR-RLKs were differentially expressed among different tissues, while some were expressed specifically in the roots and nodules. The expression of LRR-RLKs in A17 and 4 nodule mutants under rhizobial infection showed that 36 LRR-RKLs were highly upregulated in the sickle (skl) mutant [an ethylene (ET)-insensitive, Nod factor-hypersensitive mutant] after 12 h of rhizobium inoculation. Among these LRR-RLKs, six genes were also expressed specifically in the roots and nodules, which might be specific to the Nod factor and involved in autoregulation of the nodulation signal. Our results provide information on the LRR-RLK gene family in M. truncatula and serve as a guide for functional research of the LRR-RLKs.
Project description:Cytokinins are essential for legume plants to establish a nitrogen-fixing symbiosis with rhizobia. Recently, the expression level of cytokinin biosynthesis IPTs (ISOPENTENYLTRANSFERASES) genes was shown to be increased in response to rhizobial inoculation in Lotus japonicus, Medicago truncatula and Pisum sativum. In addition to its well-established positive role in nodule primordium initiation in root cortex, cytokinin negatively regulates infection processes in the epidermis. Moreover, it was reported that shoot-derived cytokinin inhibits the subsequent nodule formation through AON (autoregulation of nodulation) pathway. In L. japonicus, LjIPT3 gene was shown to be activated in the shoot phloem via the components of AON system, negatively affecting nodulation. However, in M. truncatula, the detailed analysis of MtIPTs expression, both in roots and shoots, in response to nodulation has not been performed yet, and the link between IPTs and AON has not been studied so far. In this study, we performed an extensive analysis of MtIPTs expression levels in different organs, focusing on the possible role of MtIPTs in nodule development. MtIPTs expression dynamics in inoculated roots suggest that besides its early established role in the nodule primordia development, cytokinin may be also important for later stages of nodulation. According to expression analysis, MtIPT3, MtIPT4, and MtIPT5 are activated in the shoots in response to inoculation. Among these genes, MtIPT3 is the only one the induction of which was not observed in leaves of the sunn-3 mutant defective in CLV1-like kinase, the key component of AON, suggesting that MtIPT3 is activated in the shoots in an AON-dependent manner. Taken together, our findings suggest that MtIPTs are involved in the nodule development at different stages, both locally in inoculated roots and systemically in shoots, where their expression can be activated in an AON-dependent manner.
Project description:Zinc (Zn) is an essential nutrient for plants that is involved in almost every biological process. This includes symbiotic nitrogen fixation, a process carried out by endosymbiotic bacteria (rhizobia) living within differentiated plant cells of legume root nodules. Zn transport in nodules involves delivery from the root, via the vasculature, release into the apoplast and uptake into nodule cells. Once in the cytosol, Zn can be used directly by cytosolic proteins or delivered into organelles, including symbiosomes of infected cells, by Zn efflux transporters. Medicago truncatula MtMTP2 (Medtr4g064893) is a nodule-induced Zn-efflux protein that was localized to an intracellular compartment in root epidermal and endodermal cells, as well as in nodule cells. Although the MtMTP2 gene is expressed in roots, shoots, and nodules, mtp2 mutants exhibited growth defects only under symbiotic, nitrogen-fixing conditions. Loss of MtMTP2 function resulted in altered nodule development, defects in bacteroid differentiation, and severe reduction of nitrogenase activity. The results presented here support a role of MtMTP2 in intracellular compartmentation of Zn, which is required for effective symbiotic nitrogen fixation in M. truncatula.
Project description:Mitogen-activated protein kinase (MAPK) signaling cascades play critical roles in various cellular events in plants, including stress responses, innate immunity, hormone signaling, and cell specificity. MAPK-mediated stress signaling is also known to negatively regulate nitrogen-fixing symbiotic interactions, but the molecular mechanism of the MAPK signaling cascades underlying the symbiotic nodule development remains largely unknown. We show that the MtMKK5-MtMPK3/6 signaling module negatively regulates the early symbiotic nodule formation, probably upstream of ERN1 (ERF Required for Nodulation 1) and NSP1 (Nod factor Signaling Pathway 1) in Medicago truncatula. The overexpression of MtMKK5 stimulated stress and defense signaling pathways but also reduced nodule formation in M. truncatula roots. Conversely, a MAPK specific inhibitor, U0126, enhanced nodule formation and the expression of an early nodulation marker gene, MtNIN. We found that MtMKK5 directly activates MtMPK3/6 by phosphorylating the TEY motif within the activation loop and that the MtMPK3/6 proteins physically interact with the early nodulation-related transcription factors ERN1 and NSP1. These data suggest that the stress signaling-mediated MtMKK5/MtMPK3/6 module suppresses symbiotic nodule development via the action of early nodulation transcription factors.