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C-terminally encoded peptides (CEPs) are small secreted signaling peptides that promote nitrogen-fixing root nodulation symbiosis in legumes, depending on soil mineral nitrogen availability. 1 In Medicago truncatula, their action is mediated by the leucine-rich repeat receptor-like protein kinase COMPACT ROOT ARCHITECTURE 2 (CRA2). 2 , 3 , 4 Like most land plants, under inorganic phosphate limitation, M. truncatula establishes another root endosymbiotic interaction with arbuscular fungi, the arbuscular mycorrhizal symbiosis (AMS). Because this interaction is beneficial for the plant but has a high energetic cost, it is tightly controlled by host plants to limit fungal infections mainly depending on phosphate availability. 5 We show in this study that the expression of a subset of CEP-encoding genes is enhanced in the low-phosphate conditions and that overexpression of the low-phosphate-induced MtCEP1 gene, previously shown to promote the nitrogen-fixing root nodulation symbiosis, enhances AMS from the initial entry point of the fungi. Conversely, a loss-of-function mutation of the CRA2 receptor required for mediating CEP peptide action 2 decreases the endomycorrhizal interaction from the same initial fungal entry stage. Transcriptomic analyses revealed that the cra2 mutant is negatively affected in the regulation of key phosphate transport and response genes as well as in the biosynthesis of strigolactone hormones that are required for establishing AMS. Accordingly, strigolactone contents were drastically decreased in cra2 mutant roots. Overall, we showed that the CEP/CRA2 pathway promotes both root nodulation and AMS in legume plants, depending on soil mineral nutrient availability., Competing Interests: Declaration of interests The authors declare no competing interests, (Copyright © 2024. Published by Elsevier Inc.)

Legume plants can acquire mineral nitrogen (N) either through their roots or via a symbiotic interaction with N-fixing rhizobia bacteria housed in root nodules. To identify shoot-to-root systemic signals acting in Medicago truncatula plants at N deficit or N satiety, plants were grown in a split-root experimental design in which either high or low N was provided to half of the root system, allowing the analysis of systemic pathways independently of any local N response. Among the plant hormone families analyzed, the cytokinin trans-zeatin accumulated in plants at N satiety. Cytokinin application by petiole feeding led to inhibition of both root growth and nodulation. In addition, an exhaustive analysis of miRNAs revealed that miR2111 accumulates systemically under N deficit in both shoots and non-treated distant roots, whereas a miRNA related to inorganic phosphate (Pi) acquisition, miR399, accumulates in plants grown under N satiety. These two accumulation patterns are dependent on Compact Root Architecture 2 (CRA2), a receptor required for C-terminally Encoded Peptide (CEP) signaling. Constitutive ectopic expression of miR399 reduced nodule numbers and root biomass depending on Pi availability, suggesting that the miR399-dependent Pi-acquisition regulatory module controlled by N availability affects the development of the whole legume plant root system., (© The Author(s) 2024. Published by Oxford University Press on behalf of the Society for Experimental Biology. All rights reserved. For commercial re-use, please contact reprints@oup.com for reprints and translation rights for reprints. All other permissions can be obtained through our RightsLink service via the Permissions link on the article page on our site—for further information please contact journals.permissions@oup.com.)

C-terminally encoded peptides (CEP) signaling peptides are drivers of systemic pathways regulating nitrogen (N) acquisition in different plants, from Arabidopsis to legumes, depending on mineral N availability (e.g. nitrate) and on the whole plant N demand. Recent studies in the Medicago truncatula model legume revealed how root-produced CEP peptides control the root competence for endosymbiosis with N fixing rhizobia soil bacteria through the activity of the Compact Root Architecture 2 (CRA2) CEP receptor in shoots. Among CEP genes, MtCEP7 was shown to be tightly linked to nodulation, and the dynamic temporal regulation of its expression reflects the plant ability to maintain a different symbiotic root competence window depending on the symbiotic efficiency of the rhizobium strain, as well as to reinitiate a new window of root competence for nodulation., (© 2023 The Authors New Phytologist © 2023 New Phytologist Foundation.)

In mature symbiotic root nodules, differentiated rhizobia fix atmospheric dinitrogen and provide ammonium to fulfill the plant nitrogen (N) demand. The plant enables this process by providing photosynthates to the nodules. The symbiosis is adjusted to the whole plant N demand thanks to systemic N signaling controlling nodule development. Symbiotic plants under N deficit stimulate nodule expansion and activate nodule senescence under N satiety. Besides, nodules are highly sensitive to drought. Here, we used split-root systems to characterize the systemic responses of symbiotic plants to a localized osmotic stress. We showed that polyéthylène glycol (PEG) application rapidly inhibited the symbiotic dinitrogen fixation activity of nodules locally exposed to the treatment, resulting to the N limitation of the plant supplied exclusively by symbiotic dinitrogen fixation. The localized PEG treatment triggered systemic signaling stimulating nodule development in the distant untreated roots. This response was associated with an enhancement of the sucrose allocation. Our analyses showed that transcriptomic reprogramming associated with PEG and N deficit systemic signaling(s) shared many targets transcripts. Altogether, our study suggests that systemic N signaling is a component of the adaptation of the symbiotic plant to the local variations of its edaphic environment., Competing Interests: The authors declare that the research was conducted in the absence of any commercial or financial relationships that could be construed as a potential conflict of interest., (Copyright © 2023 Martin, Pervent, Lambert, Colella, Tancelin, Severac, Clément, Tillard, Frugier and Lepetit.)

Legumes acquire soil nutrients through nitrogen-fixing root nodules and lateral roots. To balance the costs and benefits of nodulation, legumes negatively control root nodule number by autoregulatory and hormonal pathways. How legumes simultaneously coordinate root nodule and lateral root development to procure nutrients remains poorly understood. In Medicago (Medicago truncatula), a subset of mature C-TERMINALLY ENCODED PEPTIDE (CEP) hormones can systemically promote nodule number, but all CEP hormones tested to date negatively regulate lateral root number. Here we showed that Medicago CEP7 produces a mature peptide, SymCEP7, that promotes nodulation from the shoot without compromising lateral root number. Rhizobial inoculation induced CEP7 in the susceptible root nodulation zone in a Nod factor-dependent manner, and, in contrast to other CEP genes, its transcription level was elevated in the ethylene signaling mutant sickle. Using mass spectrometry, fluorescence microscopy and expression analysis, we demonstrated that SymCEP7 activity requires the COMPACT ROOT ARCHITECTURE 2 receptor and activates the shoot-to-root systemic effector, miR2111. Shoot-applied SymCEP7 rapidly promoted nodule number in the pM to nM range at concentrations up to five orders of magnitude lower than effects mediated by root-applied SymCEP7. Shoot-applied SymCEP7 also promoted nodule number in White Clover (Trifolium repens) and Lotus (Lotus japonicus), which suggests that this biological function may be evolutionarily conserved. We propose that SymCEP7 acts in the Medicago shoot to counter balance the autoregulation pathways induced rapidly by rhizobia to enable nodulation without compromising lateral root growth, thus promoting the acquisition of nutrients other than nitrogen to support their growth., Competing Interests: Conflict of interest statement. None declared., (© American Society of Plant Biologists 2023. All rights reserved. For permissions, please e-mail: journals.permissions@oup.com.)

The complex and dynamic three-dimensional organization of chromatin within the nucleus makes understanding the control of gene expression challenging, but also opens up possible ways to epigenetically modulate gene expression. Because plants are sessile, they evolved sophisticated ways to rapidly modulate gene expression in response to environmental stress, that are thought to be coordinated by changes in chromatin conformation to mediate specific cellular and physiological responses. However, to what extent and how stress induces dynamic changes in chromatin reorganization remains poorly understood. Here, we comprehensively investigated genome-wide chromatin changes associated with transcriptional reprogramming response to heat stress in tomato. Our data show that heat stress induces rapid changes in chromatin architecture, leading to the transient formation of promoter-enhancer contacts, likely driving the expression of heat-stress responsive genes. Furthermore, we demonstrate that chromatin spatial reorganization requires HSFA1a, a transcription factor (TF) essential for heat stress tolerance in tomato. In light of our findings, we propose that TFs play a key role in controlling dynamic transcriptional responses through 3D reconfiguration of promoter-enhancer contacts., (© 2023. The Author(s).)

Medicago truncatula is a model legume species that has been studied for decades to understand the symbiotic relationship between legumes and soil bacteria collectively named rhizobia. This symbiosis called nodulation is initiated in roots with the infection of root hair cells by the bacteria, as well as the initiation of nodule primordia from root cortical, endodermal, and pericycle cells, leading to the development of a new root organ, the nodule, where bacteria fix and assimilate the atmospheric dinitrogen for the benefit of the plant. Here, we report the isolation and use of the nuclei from mock and rhizobia-inoculated roots for the single nuclei RNA-seq (sNucRNA-seq) profiling to gain a deeper understanding of early responses to rhizobial infection in Medicago roots. A gene expression map of the Medicago root was generated, comprising 25 clusters, which were annotated as specific cell types using 119 Medicago marker genes and orthologs to Arabidopsis cell-type marker genes. A focus on root hair, cortex, endodermis, and pericycle cell types, showing the strongest differential regulation in response to a short-term (48 h) rhizobium inoculation, revealed not only known genes and functional pathways, validating the sNucRNA-seq approach, but also numerous novel genes and pathways, allowing a comprehensive analysis of early root symbiotic responses at a cell type-specific level., (Copyright © 2022 The Author. Published by Elsevier Inc. All rights reserved.)

Senescence determines plant organ lifespan depending on aging and environmental cues. During the endosymbiotic interaction with rhizobia, legume plants develop a specific organ, the root nodule, which houses nitrogen (N)-fixing bacteria. Unlike earlier processes of the legume-rhizobium interaction (nodule formation, N fixation), mechanisms controlling nodule senescence remain poorly understood. To identify nodule senescence-associated genes, we performed a dual plant-bacteria RNA sequencing approach on Medicago truncatula-Sinorhizobium meliloti nodules having initiated senescence either naturally (aging) or following an environmental trigger (nitrate treatment or salt stress). The resulting data allowed the identification of hundreds of plant and bacterial genes differentially regulated during nodule senescence, thus providing an unprecedented comprehensive resource of new candidate genes associated with this process. Remarkably, several plant and bacterial genes related to the cell cycle and stress responses were regulated in senescent nodules, including the rhizobial RpoE2-dependent general stress response. Analysis of selected core nodule senescence plant genes allowed showing that MtNAC969 and MtS40, both homologous to leaf senescence-associated genes, negatively regulate the transition between N fixation and senescence. In contrast, overexpression of a gene involved in the biosynthesis of cytokinins, well-known negative regulators of leaf senescence, may promote the transition from N fixation to senescence in nodules., (© 2022 John Wiley & Sons Ltd.)

Legume plants form nitrogen (N)-fixing symbiotic nodules when mineral N is limiting in soils. As N fixation is energetically costly compared to mineral N acquisition, these N sources, and in particular nitrate, inhibit nodule formation and N fixation. Here, in the model legume Medicago truncatula, we characterized a CLAVATA3-like (CLE) signaling peptide, MtCLE35, the expression of which is upregulated locally by high-N environments and relies on the Nodule Inception-Like Protein (NLP) MtNLP1. MtCLE35 inhibits nodule formation by affecting rhizobial infections, depending on the Super Numeric Nodules (MtSUNN) receptor. In addition, high N or the ectopic expression of MtCLE35 represses the expression and accumulation of the miR2111 shoot-to-root systemic effector, thus inhibiting its positive effect on nodulation. Conversely, ectopic expression of miR2111 or downregulation of MtCLE35 by RNA interference increased miR2111 accumulation independently of the N environment, and thus partially bypasses the nodulation inhibitory action of nitrate. Overall, these results demonstrate that the MtNLP1-dependent, N-induced MtCLE35 signaling peptide acts through the MtSUNN receptor and the miR2111 systemic effector to inhibit nodulation., (© American Society of Plant Biologists 2021. All rights reserved. For permissions, please email: journals.permissions@oup.com.)

Plant nutrient acquisition is tightly regulated by resource availability and metabolic needs, implying the existence of communication between roots and shoots to ensure their integration at the whole-plant level. Here, we focus on systemic signaling pathways controlling nitrogen (N) nutrition, achieved both by the root import of mineral N and, in legume plants, through atmospheric N fixation by symbiotic bacteria inside dedicated root nodules. We explore features conserved between systemic pathways repressing or enhancing symbiotic N fixation and the regulation of mineral N acquisition by roots, as well as their integration with other environmental factors, such as phosphate, light, and CO 2 availability., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

The quest for signatures of selection using single nucleotide polymorphism (SNP) data has proven efficient to uncover genes involved in conserved and/or adaptive molecular functions, but none of the statistical methods were designed to identify interacting alleles as targets of selective processes. Here, we propose a statistical test aimed at detecting epistatic selection, based on a linkage disequilibrium (LD) measure accounting for population structure and heterogeneous relatedness between individuals. SNP-based ([Formula: see text]) and window-based ([Formula: see text]) statistics fit a Student distribution, allowing to test the significance of correlation coefficients. As a proof of concept, we use SNP data from the Medicago truncatula symbiotic legume plant and uncover a previously unknown gene coadaptation between the MtSUNN (Super Numeric Nodule) receptor and the MtCLE02 (CLAVATA3-Like) signaling peptide. We also provide experimental evidence supporting a MtSUNN-dependent negative role of MtCLE02 in symbiotic root nodulation. Using human HGDP-CEPH SNP data, our new statistical test uncovers strong LD between SLC24A5 (skin pigmentation) and EDAR (hairs, teeth, sweat glands development) world-wide, which persists after correction for population structure and relatedness in Central South Asian populations. This result suggests that epistatic selection or coselection could have contributed to the phenotypic make-up in some human populations. Applying this approach to genome-wide SNP data will facilitate the identification of coadapted gene networks in model or non-model organisms.

Because of the large amount of energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system, and the developmental coordination of these organs under conditions of reduced nitrogen (N) availability remains elusive. We show that the Medicago truncatula COMPACT ROOT ARCHITECTURE2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low N. C-TERMINALLY ENCODED PEPTIDE (MtCEP1) peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of YUCCA2 ( MtYUC2 ) auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by overexpressing MtYUC2 , and conversely, a treatment with YUC inhibitors or an MtYUC2 knockout rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser 643 and Ser 924 , preventing its cleavage and thereby repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement with this interaction, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions., (© 2020 American Society of Plant Biologists. All rights reserved.)

Nitrogen-fixing root nodulation in legumes challenged with nitrogen-limiting conditions requires infection of the root hairs by soil symbiotic bacteria, collectively referred to as rhizobia, and the initiation of cell divisions in the root cortex. Cytokinin hormones are critical for early nodulation to coordinate root nodule organogenesis and the progression of bacterial infections. Cytokinin signaling involves regulation of the expression of cytokinin primary response genes by type-B response regulator (RRB) transcription factors. RNA interference or mutation of MtRRB3 , the RRB-encoding gene most strongly expressed in Medicago truncatula roots and nodules, significantly decreased the number of nodules formed, indicating a function of this RRB in nodulation initiation. Fewer infection events were also observed in rrb3 mutant roots associated with a reduced Nod factor induction of the Early Nodulin 11 ( MtENOD11 ) infection marker, and of the cytokinin-regulated Nodulation Signaling Pathway 2 ( Mt NSP2 ) gene. Rhizobial infections correlate with an expansion of the nuclear area, suggesting the activation of endoreduplication cycles linked to the cytokinin-regulated Cell Cycle Switch 52A ( Mt CCS52A ) gene. Although no significant difference in nucleus size and endoreduplication were detected in rhizobia-infected rrb3 mutant roots, expression of the MtCCS52A endoreduplication marker was reduced. As the MtRRB3 expression pattern overlaps with those of MtNSP2 and MtCCS52A in roots and nodule primordia, chromatin immunoprecipitation-quantitative PCR and protoplast trans-activation assays were used to show that MtRRB3 can interact with and trans-activate MtNSP2 and MtCCS52A promoters. Overall, we highlight that the MtRRB3 cytokinin signaling transcription factor coordinates the expression of key early nodulation genes., (© 2020 American Society of Plant Biologists. All Rights Reserved.)

Because of the high energy consumed during symbiotic nitrogen fixation, legumes must balance growth and symbiotic nodulation. Both lateral roots and nodules form on the root system and the developmental coordination of these organs according to reduced nitrogen (N) availability remains elusive. We show that the Compact Root Architecture 2 (MtCRA2) receptor-like kinase is essential to promote the initiation of early symbiotic nodulation and to inhibit root growth in response to low-N. MtCEP1 peptides can activate MtCRA2 under N-starvation conditions, leading to a repression of MtYUC2 auxin biosynthesis gene expression, and therefore of auxin root responses. Accordingly, the compact root architecture phenotype of cra2 can be mimicked by an auxin treatment or by over-expressing MtYUC2, and conversely, a treatment with YUC inhibitors or a MtYUC2 knock-out rescues the cra2 root phenotype. The MtCEP1-activated CRA2 can additionally interact with and phosphorylate the MtEIN2 ethylene signaling component at Ser643 and Ser924, preventing its cleavage and therefore repressing ethylene responses, thus locally promoting the root susceptibility to rhizobia. In agreement, the cra2 low nodulation phenotype is rescued by an ein2 mutation. Overall, by reducing auxin biosynthesis and inhibiting ethylene signaling, the MtCEP1/MtCRA2 pathway balances root and nodule development under low-N conditions., (© 2020 American Society of Plant Biologists. All rights reserved.)

Legumes tightly regulate nodule number to balance the cost of supporting symbiotic rhizobia with the benefits of nitrogen fixation. C-terminally Encoded Peptides (CEPs) and CLAVATA3-like (CLE) peptides positively and negatively regulate nodulation, respectively, through independent systemic pathways, but how these regulations are coordinated remains unknown. Here, we show that rhizobia, Nod Factors, and cytokinins induce a symbiosis-specific CEP gene, MtCEP7, which positively regulates rhizobial infection. Via grafting and split root studies, we reveal that MtCEP7 increases nodule number systemically through the MtCRA2 receptor. MtCEP7 and MtCLE13 expression in rhizobia-inoculated roots rely on the MtCRE1 cytokinin receptor and on the MtNIN transcription factor. MtNIN binds and transactivates MtCEP7 and MtCLE13, and a NIN Binding Site (NBS) identified within the proximal MtCEP7 promoter is required for its symbiotic activation. Overall, these results demonstrate that a cytokinin-MtCRE1-MtNIN regulatory module coordinates the expression of two antagonistic, symbiosis-related, peptide hormones from different families to fine-tune nodule number.

Root system architecture (RSA) influences the effectiveness of resources acquisition from soils but the genetic networks that control RSA remain largely unclear. We used rhizoboxes, X-ray computed tomography, grafting, auxin transport measurements and hormone quantification to demonstrate that Arabidopsis and Medicago CEP (C-TERMINALLY ENCODED PEPTIDE)-CEP RECEPTOR signalling controls RSA, the gravitropic set-point angle (GSA) of lateral roots (LRs), auxin levels and auxin transport. We showed that soil-grown Arabidopsis and Medicago CEP receptor mutants have a narrower RSA, which results from a steeper LR GSA. Grafting showed that CEPR1 in the shoot controls GSA. CEP receptor mutants exhibited an increase in rootward auxin transport and elevated shoot auxin levels. Consistently, the application of auxin to wild-type shoots induced a steeper GSA and auxin transport inhibitors counteracted the CEP receptor mutant's steep GSA phenotype. Concordantly, CEP peptides increased GSA and inhibited rootward auxin transport in wild-type but not in CEP receptor mutants. The results indicated that CEP-CEP receptor-dependent signalling outputs in Arabidopsis and Medicago control overall RSA, LR GSA, shoot auxin levels and rootward auxin transport. We propose that manipulating CEP signalling strength or CEP receptor downstream targets may provide means to alter RSA., (© 2020 The Authors. New Phytologist © 2020 New Phytologist Trust.)

Nitrogen-deprived legume plants form new root organs, the nodules, following a symbiosis with nitrogen-fixing rhizobial bacteria [1]. Because this interaction is beneficial for the plant but has a high energetic cost, nodulation is tightly controlled by host plants through systemic pathways (acting at long distance) to promote or limit rhizobial infections and nodulation depending on earlier infections and on nitrogen availability [2]. In the Medicago truncatula model legume, CLE12 (Clavata3/Embryo surrounding region 12) and CLE13 signaling peptides produced in nodulated roots act in shoots through the SUNN (Super Numeric Nodule) receptor to negatively regulate nodulation and therefore autoregulate nodule number [3-5]. Conversely, CEP (C-terminally Encoded Peptide) signaling peptides produced in nitrogen-starved roots act in shoots through the CRA2 (Compact Root Architecture 2) receptor to promote nodulation already in the absence of rhizobia [6-9]. We show in this study that a downstream shoot-to-root signaling effector of these systemic pathways is the shoot-produced miR2111 microRNA [10] that negatively regulates TML1 (Too Much Love 1) and TML2 [11] transcripts accumulation in roots, ultimately promoting nodulation. Low nitrogen conditions and CEP1 signaling peptides induce in the absence of rhizobia the production of miR2111 depending on CRA2 activity in shoots, thus favoring root competence for nodulation. Together with the SUNN pathway negatively regulating the same miR2111 systemic effector when roots are nodulated, this allows a dynamic fine-tuning of the nodulation capacity of legume roots by nitrogen availability and rhizobial cues., Competing Interests: Declaration of Interests The authors declare no competing interests., (Copyright © 2020 Elsevier Ltd. All rights reserved.)

Background: Legumes can establish on nitrogen-deprived soils a symbiotic interaction with Rhizobia bacteria, leading to the formation of nitrogen-fixing root nodules. Cytokinin phytohormones are critical for triggering root cortical cell divisions at the onset of nodule initiation. Cytokinin signaling is based on a Two-Component System (TCS) phosphorelay cascade, involving successively Cytokinin-binding Histidine Kinase receptors, phosphorelay proteins shuttling between the cytoplasm and the nucleus, and Type-B Response Regulator (RRB) transcription factors activating the expression of cytokinin primary response genes. Among those, Type-A Response Regulators (RRA) exert a negative feedback on the TCS signaling. To determine whether the legume plant nodulation capacity is linked to specific features of TCS proteins, a genome-wide identification was performed in six legume genomes (Cajanus cajan, pigeonpea; Cicer arietinum, chickpea; Glycine max, soybean; Phaseolus vulgaris, common bean; Lotus japonicus; Medicago truncatula). The diversity of legume TCS proteins was compared to the one found in two non-nodulating species, Arabidopsis thaliana and Vitis vinifera, which are references for functional analyses of TCS components and phylogenetic analyses, respectively., Results: A striking expansion of non-canonical RRBs was identified, notably leading to the emergence of proteins where the conserved phosphor-accepting aspartate residue is replaced by a glutamate or an asparagine. M. truncatula genome-wide expression datasets additionally revealed that only a limited subset of cytokinin-related TCS genes is highly expressed in different organs, namely MtCHK1/MtCRE1, MtHPT1, and MtRRB3, suggesting that this "core" module potentially acts in most plant organs including nodules., Conclusions: Further functional analyses are required to determine the relevance of these numerous non-canonical TCS RRBs in symbiotic nodulation, as well as of canonical MtHPT1 and MtRRB3 core signaling elements.

Plant systemic signaling pathways allow the integration and coordination of shoot and root organ metabolism and development at the whole-plant level depending on nutrient availability. In legumes, two systemic pathways have been reported in the Medicago truncatula model to regulate root nitrogen-fixing symbiotic nodulation. Both pathways involve leucine-rich repeat receptor-like kinases acting in shoots and proposed to perceive signaling peptides produced in roots depending on soil nutrient availability. In this study, we characterized in the M. truncatula Jemalong A17 genotype a mutant allelic series affecting the Compact Root Architecture2 (CRA2) receptor. These analyses revealed that this pathway acts systemically from shoots to positively regulate nodulation and is required for the activity of carboxyl-terminally encoded peptides (CEPs). In addition, we generated a double mutant to test genetic interactions of the CRA2 systemic pathway with the CLAVATA3/EMBRYO SURROUNDING REGION peptide (CLE)/Super Numeric Nodule (SUNN) receptor systemic pathway negatively regulating nodule number from shoots, which revealed an intermediate nodule number phenotype close to the wild type. Finally, we showed that the nitrate inhibition of nodule numbers was observed in cra2 mutants but not in sunn and cra2 sunn mutants. Overall, these results suggest that CEP/CRA2 and CLE/SUNN systemic pathways act independently from shoots to regulate nodule numbers., (© 2019 American Society of Plant Biologists. All Rights Reserved.)

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., (© The Author(s) 2019. Published by Oxford University Press on behalf of the Society for Experimental Biology.)

The root system displays a remarkable plasticity that enables plants to adapt to changing environmental conditions. This plasticity is tightly linked to the activity of root apical meristems (RAMs) and to the formation of lateral roots, both controlled by related hormonal crosstalks. In Arabidopsis thaliana, gibberellins (GAs) were shown to positively control RAM growth and the formation of lateral roots. However, we showed in Medicago truncatula that GAs negatively regulate root growth and RAM size as well as the number of lateral roots depending at least on the MtDELLA1 protein. By using confocal microscopy and molecular analyses, we showed that GAs primarily regulate RAM size by affecting cortical cell expansion and additionally negatively regulate a subset of cytokinin-induced root expansin encoding genes. Moreover, GAs reduce the number of cortical cell layers, resulting in the formation of both shorter and thinner roots. These results suggest contrasting effects of GA regulations on the root system architecture depending on plant species.

Advances in deciphering the functional architecture of eukaryotic genomes have been facilitated by recent breakthroughs in sequencing technologies, enabling a more comprehensive representation of genes and repeat elements in genome sequence assemblies, as well as more sensitive and tissue-specific analyses of gene expression. Here we show that PacBio sequencing has led to a substantially improved genome assembly of Medicago truncatula A17, a legume model species notable for endosymbiosis studies 1 , and has enabled the identification of genome rearrangements between genotypes at a near-base-pair resolution. Annotation of the new M. truncatula genome sequence has allowed for a thorough analysis of transposable elements and their dynamics, as well as the identification of new players involved in symbiotic nodule development, in particular 1,037 upregulated long non-coding RNAs (lncRNAs). We have also discovered that a substantial proportion (~35% and 38%, respectively) of the genes upregulated in nodules or expressed in the nodule differentiation zone colocalize in genomic clusters (270 and 211, respectively), here termed symbiotic islands. These islands contain numerous expressed lncRNA genes and display differentially both DNA methylation and histone marks. Epigenetic regulations and lncRNAs are therefore attractive candidate elements for the orchestration of symbiotic gene expression in the M. truncatula genome.

Gibberellins (GAs) and cytokinins (CKs) are hormones that play antagonistic roles in several developmental processes in plants. However, there has been little exploration of their reciprocal interactions. Recent work in Medicago truncatula has revealed that GA signalling can regulate CK levels and response in roots. Here, we examine the reciprocal interaction, by assessing how CKs and the CRE1 (Cytokinin Response 1) CK receptor may influence endogenous GA levels. Real-Time RT-PCR analyses revealed that the expression of key GA biosynthesis genes is regulated in response to a short-term CK treatment and requires the CRE1 receptor. Similarly, GA quantifications indicated that a short-term CK treatment decreases the GA 1 pool in wild-type plants and that GA levels are increased in the cre1 mutant compared to the wild-type. These data suggest that the M. truncatula CRE1-dependent CK signaling pathway negatively regulates bioactive GA levels.

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.

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., (© 2017 American Society of Plant Biologists. All Rights Reserved.)

Substantial progress has been made in the understanding of early stages of the symbiotic interaction between legume plants and rhizobium bacteria. Those include the specific recognition of symbiotic partners, the initiation of bacterial infection in root hair cells, and the inception of a specific organ in the root cortex, the nodule. Increasingly complex regulatory networks have been uncovered in which cytokinin (CK) phytohormones play essential roles in different aspects of early symbiotic stages. Intriguingly, these roles can be either positive or negative, cell autonomous or non-cell autonomous, and vary, depending on time, root tissues, and possibly legume species. Recent developments on CK symbiotic functions and interconnections with other signaling pathways during nodule initiation are the focus of this review., (Copyright © 2017 Elsevier Ltd. All rights reserved.)

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.

We investigated the role of KNOX genes in legume root nodule organogenesis. Class 1 KNOX homeodomain transcription factors (TFs) are involved in plant shoot development and leaf shape diversity. Class 2 KNOX genes are less characterized, even though an antagonistic function relative to class 1 KNOXs was recently proposed. In silico expression data and further experimental validation identified in the Medicago truncatula model legume three class 2 KNOX genes, belonging to the KNAT3/4/5-like subclass (Mt KNAT3/4/5-like), as expressed during nodulation from early stages. RNA interference (RNAi)-mediated silencing and overexpression studies were used to unravel a function for KNOX TFs in nodule development. Mt KNAT3/4/5-like genes encoded four highly homologous proteins showing overlapping expression patterns during nodule organogenesis, suggesting functional redundancy. Simultaneous reduction of Mt KNAT3/4/5-like genes indeed led to an increased formation of fused nodule organs, and decreased the expression of the MtEFD (Ethylene response Factor required for nodule Differentiation) TF and its direct target MtRR4, a cytokinin response gene. Class 2 KNOX TFs therefore regulate legume nodule development, potentially through the MtEFD/MtRR4 cytokinin-related regulatory module, and may control nodule organ boundaries and shape like class 2 KNOX function in leaf development., (© 2016 The Authors. New Phytologist © 2016 New Phytologist Trust.)

DELLA proteins, acting as integrators of gibberellin (GA) action, are emerging as key regulators of root system architecture. Recent studies have revealed how they dictate the dynamics of root growth and are required for the establishment of root endosymbioses with rhizobial bacteria and mycorrhizal fungi. Like conductors, DELLAs can thereby harmonize root development depending on soil environments., (Copyright © 2016 Elsevier Ltd. All rights reserved.)

Cold acclimation is an important process by which plants respond to low temperature and enhance their winter hardiness. C-REPEAT BINDING FACTOR1 (CBF1), CBF2, and CBF3 genes were shown previously to participate in cold acclimation in Medicago truncatula In addition, MtCBF4 is transcriptionally induced by salt, drought, and cold stresses. We show here that MtCBF4, shown previously to enhance drought and salt tolerance, also positively regulates cold acclimation and freezing tolerance. To identify molecular factors acting upstream and downstream of the MtCBF4 transcription factor (TF) in cold responses, we first identified genes that are differentially regulated upon MtCBF4 overexpression using RNAseq Digital Gene Expression Profiling. Among these, we showed that MtCBF4 directly activates the transcription of the COLD ACCLIMATION SPECIFIC15 (MtCAS15) gene. To gain insights into how MtCBF4 is transcriptionally regulated in response to cold, an R2R3-MYB TF, MtMYB3, was identified based on a yeast one-hybrid screen as binding directly to MYB cis-elements in the MtCBF4 promoter, leading to the inhibition of MtCBF4 expression. In addition, another MYB TF, MtMYB61, identified as an interactor of MtMYB3, can relieve the inhibitory effect of MtMYB3 on MtCBF4 transcription. This study, therefore, supports a model describing how MtCBF4 is regulated by antagonistic MtMYB3/MtMYB61 TFs, leading to the up-regulation of downstream targets such as MtCAS15 acting in cold acclimation in M. truncatula., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Legume plants adapt to low nitrogen by developing an endosymbiosis with nitrogen-fixing soil bacteria to form a new specific organ: the nitrogen-fixing nodule. In the Medicago truncatula model legume, the MtCRE1 cytokinin receptor is essential for this symbiotic interaction. As three other putative CHASE-domain containing histidine kinase (CHK) cytokinin receptors exist in M. truncatula, we determined their potential contribution to this symbiotic interaction. The four CHKs have extensive redundant expression patterns at early nodulation stages but diverge in differentiated nodules, even though MtCHK1/MtCRE1 has the strongest expression at all stages. Mutant and knock-down analyses revealed that other CHKs than MtCHK1/CRE1 are positively involved in nodule initiation, which explains the delayed nodulation phenotype of the chk1/cre1 mutant. In addition, cre1 nodules exhibit an increased growth, whereas other chk mutants have no detectable phenotype, and the maintained nitrogen fixation capacity in cre1 requires other CHK genes. Interestingly, an AHK4/CRE1 genomic locus from the aposymbiotic Arabidopsis plant rescues nodule initiation but not the nitrogen fixation capacity. This indicates that different CHK cytokinin signalling pathways regulate not only nodule initiation but also later developmental stages, and that legume-specific determinants encoded by the MtCRE1 gene are required for later nodulation stages than initiation., (© 2016 John Wiley & Sons Ltd.)

Legumes develop symbiotic interactions with rhizobial bacteria to form nitrogen-fixing nodules. Bacterial Nod factors (NFs) and plant regulatory pathways modulating NF signalling control rhizobial infections and nodulation efficiency. Here we show that gibberellin (GA) signalling mediated by DELLA proteins inhibits rhizobial infections and controls the NF induction of the infection marker ENOD11 in Medicago truncatula. Ectopic expression of a constitutively active DELLA protein in the epidermis is sufficient to promote ENOD11 expression in the absence of symbiotic signals. We show using heterologous systems that DELLA proteins can interact with the nodulation signalling pathway 2 (NSP2) and nuclear factor-YA1 (NF-YA1) transcription factors that are essential for the activation of NF responses. Furthermore, MtDELLA1 can bind the ERN1 (ERF required for nodulation 1) promoter and positively transactivate its expression. Overall, we propose that GA-dependent action of DELLA proteins may directly regulate the NSP1/NSP2 and NF-YA1 activation of ERN1 transcription to regulate rhizobial infections.

A large range of microorganisms can associate with plants, resulting in neutral, friendly or hostile interactions. The ability of plants to recognize compatible and incompatible microorganisms and to limit or promote their colonization is therefore crucial for their survival. Elaborated communication networks determine the degree of association between the host plant and the invading microorganism. Central to these regulations of plant microbe interactions, phytohormones modulate microorganism plant associations and coordinate cellular and metabolic responses associated to the progression of microorganisms across different plant tissues. We review here hormonal regulations, focusing on auxin and cytokinin phytohormones, involved in the interactions between plant roots and soil microorganisms, including bacterial and fungi associations, either beneficial (symbiotic) or detrimental (pathogenic). The aim is to highlight similarities and differences in cytokinin/auxin functions amongst various compatible versus incompatible associations.

C-TERMINALLY ENCODED PEPTIDEs (CEPs) control root system architecture in a non-cell-autonomous manner. In Medicago truncatula, MtCEP1 affects root development by increasing nodule formation and inhibiting lateral root emergence by unknown pathways. Here, we show that the MtCEP1 peptide-dependent increase in nodulation requires the symbiotic signaling pathway and ETHYLENE INSENSITIVE2 (EIN2)/SICKLE (SKL), but acts independently of SUPER NUMERIC NODULES. MtCEP1-dependent inhibition of lateral root development acts through an EIN2-independent mechanism. MtCEP1 increases nodulation by promoting rhizobial infections, the developmental competency of roots for nodulation, the formation of fused nodules, and an increase in frequency of nodule development that initiates at proto-phloem poles. These phenotypes are similar to those of the ein2/skl mutant and support that MtCEP1 modulates EIN2-dependent symbiotic responses. Accordingly, MtCEP1 counteracts the reduction in nodulation induced by increasing ethylene precursor concentrations, and an ethylene synthesis inhibitor treatment antagonizes MtCEP1 root phenotypes. MtCEP1 also inhibits the development of EIN2-dependent pseudonodule formation. Finally, mutants affecting the COMPACT ROOT ARCHITECTURE2 (CRA2) receptor, which is closely related to the Arabidopsis CEP Receptor1, are unresponsive to MtCEP1 effects on lateral root and nodule formation, suggesting that CRA2 is a CEP peptide receptor mediating both organogenesis programs. In addition, an ethylene inhibitor treatment counteracts the cra2 nodulation phenotype. These results indicate that MtCEP1 and its likely receptor, CRA2, mediate nodulation and lateral root development through different pathways., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Nod factors (NFs) are lipochitooligosaccharidic signal molecules produced by rhizobia, which play a key role in the rhizobium-legume symbiotic interaction. In this study, we analyzed the gene expression reprogramming induced by purified NF (4 and 24 h of treatment) in the root epidermis of the model legume Medicago truncatula Tissue-specific transcriptome analysis was achieved by laser-capture microdissection coupled to high-depth RNA sequencing. The expression of 17,191 genes was detected in the epidermis, among which 1,070 were found to be regulated by NF addition, including previously characterized NF-induced marker genes. Many genes exhibited strong levels of transcriptional activation, sometimes only transiently at 4 h, indicating highly dynamic regulation. Expression reprogramming affected a variety of cellular processes, including perception, signaling, regulation of gene expression, as well as cell wall, cytoskeleton, transport, metabolism, and defense, with numerous NF-induced genes never identified before. Strikingly, early epidermal activation of cytokinin (CK) pathways was indicated, based on the induction of CK metabolic and signaling genes, including the CRE1 receptor essential to promote nodulation. These transcriptional activations were independently validated using promoter:β-glucuronidase fusions with the MtCRE1 CK receptor gene and a CK response reporter (TWO COMPONENT SIGNALING SENSOR NEW). A CK pretreatment reduced the NF induction of the EARLY NODULIN11 (ENOD11) symbiotic marker, while a CK-degrading enzyme (CYTOKININ OXIDASE/DEHYDROGENASE3) ectopically expressed in the root epidermis led to increased NF induction of ENOD11 and nodulation. Therefore, CK may play both positive and negative roles in M. truncatula nodulation., (© 2016 American Society of Plant Biologists. All Rights Reserved.)

Initiation of symbiotic nodules in legumes requires cytokinin signaling, but its mechanism of action is largely unknown. Here, we tested whether the failure to initiate nodules in the Medicago truncatula cytokinin perception mutant cre1 (cytokinin response1) is due to its altered ability to regulate auxin transport, auxin accumulation, and induction of flavonoids. We found that in the cre1 mutant, symbiotic rhizobia cannot locally alter acro- and basipetal auxin transport during nodule initiation and that these mutants show reduced auxin (indole-3-acetic acid) accumulation and auxin responses compared with the wild type. Quantification of flavonoids, which can act as endogenous auxin transport inhibitors, showed a deficiency in the induction of free naringenin, isoliquiritigenin, quercetin, and hesperetin in cre1 roots compared with wild-type roots 24 h after inoculation with rhizobia. Coinoculation of roots with rhizobia and the flavonoids naringenin, isoliquiritigenin, and kaempferol, or with the synthetic auxin transport inhibitor 2,3,5,-triiodobenzoic acid, rescued nodulation efficiency in cre1 mutants and allowed auxin transport control in response to rhizobia. Our results suggest that CRE1-dependent cytokinin signaling leads to nodule initiation through the regulation of flavonoid accumulation required for local alteration of polar auxin transport and subsequent auxin accumulation in cortical cells during the early stages of nodulation., (© 2015 American Society of Plant Biologists. All rights reserved.)

Cytokinins are phytohormones that regulate many developmental and environmental responses. The Medicago truncatula cytokinin receptor MtCRE1 (Cytokinin Response 1) is required for the nitrogen-fixing symbiosis with rhizobia. As several cytokinin signaling genes are modulated in roots depending on different biotic and abiotic conditions, we assessed potential involvement of this pathway in various root environmental responses. Phenotyping of cre1 mutant roots infected by the Gigaspora margarita arbuscular mycorrhizal (AM) symbiotic fungus, the Aphanomyces euteiches root oomycete, or subjected to an abiotic stress (salt), were carried out. Detailed histological analysis and quantification of cre1 mycorrhized roots did not reveal any detrimental phenotype, suggesting that MtCRE1 does not belong to the ancestral common symbiotic pathway shared by rhizobial and AM symbioses. cre1 mutants formed an increased number of emerged lateral roots compared to wild-type plants, a phenotype which was also observed under non-stressed conditions. In response to A. euteiches, cre1 mutants showed reduced disease symptoms and an increased plant survival rate, correlated to an enhanced formation of lateral roots, a feature previously linked to Aphanomyces resistance. Overall, we showed that the cytokinin CRE1 pathway is not only required for symbiotic nodule organogenesis but also affects both root development and resistance to abiotic and biotic environmental stresses.

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.

RNA-dependent RNA polymerase 6 (RDR6) and suppressor of gene silencing 3 (SGS3) act together in post-transcriptional transgene silencing mediated by small interfering RNAs (siRNAs) and in biogenesis of various endogenous siRNAs including the tasiARFs, known regulators of auxin responses and plant development. Legumes, the third major crop family worldwide, has been widely improved through transgenic approaches. Here, we isolated rdr6 and sgs3 mutants in the model legume Medicago truncatula. Two sgs3 and one rdr6 alleles led to strong developmental defects and impaired biogenesis of tasiARFs. In contrast, the rdr6.1 homozygous plants produced sufficient amounts of tasiARFs to ensure proper development. High throughput sequencing of small RNAs from this specific mutant identified 354 potential MtRDR6 substrates, for which siRNA production was significantly reduced in the mutant. Among them, we found a large variety of novel phased loci corresponding to protein-encoding genes or transposable elements. Interestingly, measurement of GFP expression revealed that post-transcriptional transgene silencing was reduced in rdr6.1 roots. Hence, this novel mis-sense mutation, affecting a highly conserved amino acid residue in plant RDR6s, may be an interesting tool both to analyse endogenous pha-siRNA functions and to improve transgene expression, at least in legume species., (© 2014 Society for Experimental Biology, Association of Applied Biologists and John Wiley & Sons Ltd.)

Background: Legume roots show a remarkable plasticity to adapt their architecture to biotic and abiotic constraints, including symbiotic interactions. However, global analysis of miRNA regulation in roots is limited, and a global view of the evolution of miRNA-mediated diversification in different ecotypes is lacking., Results: In the model legume Medicago truncatula, we analyze the small RNA transcriptome of roots submitted to symbiotic and pathogenic interactions. Genome mapping and a computational pipeline identify 416 miRNA candidates, including known and novel variants of 78 miRNA families present in miRBase. Stringent criteria of pre-miRNA prediction yield 52 new mtr-miRNAs, including 27 miRtrons. Analyzing miRNA precursor polymorphisms in 26 M. truncatula ecotypes identifies higher sequence polymorphism in conserved rather than Medicago-specific miRNA precursors. An average of 19 targets, mainly involved in environmental responses and signalling, is predicted per novel miRNA. We identify miRNAs responsive to bacterial and fungal pathogens or symbionts as well as their related Nod and Myc-LCO symbiotic signals. Network analyses reveal modules of new and conserved co-expressed miRNAs that regulate distinct sets of targets, highlighting potential miRNA-regulated biological pathways relevant to pathogenic and symbiotic interactions., Conclusions: We identify 52 novel genuine miRNAs and large plasticity of the root miRNAome in response to the environment, and also in response to purified Myc/Nod signaling molecules. The new miRNAs identified and their sequence variation across M. truncatula ecotypes may be crucial to understand the adaptation of root growth to the soil environment, notably in the agriculturally important legume crops.

• A plant-microbe dual biological system was set up involving the model legume Medicago truncatula and two bacteria, the soil-borne root pathogen Ralstonia solanacearum and the beneficial symbiont Sinorhizobium meliloti. • Comparison of transcriptomes under symbiotic and pathogenic conditions highlighted the transcription factor MtEFD (Ethylene response Factor required for nodule Differentiation) as being upregulated in both interactions, together with a set of cytokinin-related transcripts involved in metabolism, signaling and response. MtRR4 (Response Regulator), a cytokinin primary response gene negatively regulating cytokinin signaling and known as a target of MtEFD in nodulation processes, was retrieved in this set of transcripts. • Refined studies of MtEFD and MtRR4 expression during M. truncatula and R. solanacearum interaction indicated differential kinetics of induction and requirement of central regulators of bacterial pathogenicity, HrpG and HrpB. Similar to MtRR4, MtEFD upregulation during the pathogenic interaction was dependent on cytokinin perception mediated by the MtCRE1 (Cytokinin REsponse 1) receptor. • The use of M. truncatula efd-1 and cre1-1 mutants evidenced MtEFD and cytokinin perception as positive factors for bacterial wilt development. These factors therefore play an important role in both root nodulation and root disease development., (© 2013 The Authors. New Phytologist © 2013 New Phytologist Trust.)

PRAF proteins are present in all plants, but their functions remain unclear. We investigated the role of one member of the PRAF family, MtZR1, on the development of roots and nitrogen-fixing nodules in Medicago truncatula. We found that MtZR1 was expressed in all M. truncatula organs. Spatiotemporal analysis showed that MtZR1 expression in M. truncatula roots was mostly limited to the root meristem and the vascular bundles of mature nodules. MtZR1 expression in root nodules was down-regulated in response to various abiotic stresses known to affect nitrogen fixation efficiency. The down-regulation of MtZR1 expression by RNA interference in transgenic roots decreased root growth and impaired nodule development and function. MtZR1 overexpression resulted in longer roots and significant changes to nodule development. Our data thus indicate that MtZR1 is involved in the development of roots and nodules. To our knowledge, this work provides the first in vivo experimental evidence of a biological role for a typical PRAF protein in plants., (© 2013 John Wiley & Sons Ltd.)

Some bacterial group II introns are widely used for genetic engineering in bacteria, because they can be reprogrammed to insert into the desired DNA target sites. There is considerable interest in developing this group II intron gene targeting technology for use in eukaryotes, but nuclear genomes present several obstacles to the use of this approach. The nuclear genomes of eukaryotes do not contain group II introns, but these introns are thought to have been the progenitors of nuclear spliceosomal introns. We investigated the expression and subcellular localization of the bacterial RmInt1 group II intron-encoded protein (IEP) in Arabidopsis thaliana protoplasts. Following the expression of translational fusions of the wild-type protein and several mutant variants with EGFP, the full-length IEP was found exclusively in the nucleolus, whereas the maturase domain alone targeted EGFP to nuclear speckles. The distribution of the bacterial RmInt1 IEP in plant cell protoplasts suggests that the compartmentalization of eukaryotic cells into nucleus and cytoplasm does not prevent group II introns from invading the host genome. Furthermore, the trafficking of the IEP between the nucleolus and the speckles upon maturase inactivation is consistent with the hypothesis that the spliceosomal machinery evolved from group II introns.

Bacteria present in the rhizosphere of plants often synthesize phytohormones, and these signals can consequently affect root system development. In legumes, plants adapt to nitrogen starvation by forming lateral roots as well as a new organ, the root nodule, following a symbiotic interaction with bacteria collectively referred to as rhizobia. As cytokinin (CK) phytohormones were shown to be necessary and sufficient to induce root nodule organogenesis, the relevance of CK production by symbiotic rhizobia was questioned. In this study, we analyzed quantitatively, by liquid chromatography-tandem mass spectrometry, the production of 25 forms of CK in nine rhizobia strains belonging to four different species. All bacterial strains were able to synthesize a mix of CK, and bioactive forms of CK, such as iP, were notably found to be secreted in bacterial culture supernatants. Use of a mutant affected in extracellular polysaccharide (EPS) production revealed a negative correlation of EPS production with the ability to secrete CK. In addition, analysis of a nonnodulating Sinorhizobium meliloti strain revealed a similar pattern of CK production and secretion when compared with a related nodulating strain. This indicates that bacterially produced CK are not sufficient to induce symbiotic nodulation.

In the past decade, hundreds of non-coding RNAs (small and long RNAs) have been identified as crucial elements in developmental processes and stress response in plants. Among small RNAs, the microRNAs or miRNAs control levels of specific mRNA by inhibiting translation or reducing the stability of their mRNA targets through integration into different ribonucleoproteins (RNP). Spatio-temporal expression of small and long RNAs, using reporter genes or in situ hybridization, is essential to understand their functions. We are interested in understanding the role of various non-coding RNAs (including miRNAs) in the regulation of root and nodule development in legumes, which are agriculturally important crops. Here, we present the protocol we are currently using for detection of small and long RNA in model legume plants and tissues, like nodules and roots. The probe selection, as well as the fixation and permeabilization steps allowing to preserve tissues and cell integrity and to increase accessibility to RNA targets, will be specifically discussed.

Proteins are distributed in different cellular compartments. Our group studies the role of non-coding RNAs and associated RNPs in the development and stress response in legumes. Ribonucleoproteins (RNPs) are RNA-protein complexes that play different roles in many cellular processes. Long and small non-coding RNAs determine the specificity of action of several RNPs as the RNA Induced Silencing Complex (RISC), or affect mRNA translation, splicing and stability by interacting with other RNPs such as P-bodies, spliceosome or polysomes. Together with small and long RNAs (Chapter 20), the precise localization of the associated RNPs or the translational products regulated by small RNAs (ie target proteins regulated by miRNAs, or translationally-regulated products) by immunocytochemistry could bring novel insights into these regulatory processes. The protocol described is currently used for detection of RNP associated proteins in nodules and roots of Medicago truncatula but could be extended to any other protein. The critical points, as the choice of the antibody and the fixation and permeabilization steps, that allow preservation of tissue and cell integrity and increase the accessibility to epitopes, will be discussed.