Crosstalk between the nodulation signaling pathway and the autoregulation of nodulation in Medicago truncatula

A subset of CLAVATA3/endosperm-surrounding region-related (CLE) peptides are involved in autoregulation of nodulation (AON) in Medicago truncatula (e.g. MtCLE12 and MtCLE13). However, their linkage to other components of the AON pathways downstream of the shoot-derived inhibitor (SDI) is not understood. We have ectopically expressed the putative peptide ligand encoding genes MtCLE12 and MtCLE13 in M. truncatula which abolished nodulation completely in wild-type roots but not in the supernodulating null mutant sunn-4. Further, root growth inhibition was detected when MtCLE12 was ectopically expressed in wild-type roots or synthetic CLE12 peptide was applied exogenously. To identify downstream genes, roots of wild-type and sunn-4 mutant overexpressing MtCLE12 were used for quantitative gene expression analysis. We found that, in 35S:MtCLE12 roots, NODULE INCEPTION (NIN, a central regulator of nodulation) was down-regulated, whereas MtEFD (ethylene response factor required for nodule differentiation) and MtRR8 (a type-A response regulator thought to be involved in the negative regulation of cytokinin signaling), were up-regulated. Moreover, we found that the up-regulation of MtEFD and MtRR8 caused by overexpressing MtCLE12 is SUNN-dependent. Hence, our data link for the first time the pathways for Nod factor signaling, cytokinin perception and AON.     

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.     

Spatiotemporal cytokinin response imaging and ISOPENTENYLTRANSFERASE 3 function in Medicago nodule development

Most legumes can establish a symbiotic association with soil rhizobia that trigger the development of root nodules. These nodules host the rhizobia and allow them to fix nitrogen efficiently. The perception of bacterial lipo-chitooligosaccharides (LCOs) in the epidermis initiates a signaling cascade that allows rhizobial intracellular infection in the root and de-differentiation and activation of cell division that gives rise to the nodule. Thus, nodule organogenesis and rhizobial infection need to be coupled in space and time for successful nodulation. The plant hormone cytokinin (CK) contributes to the coordination of this process, acting as an essential positive regulator of nodule organogenesis. However, the temporal regulation of tissue-specific CK signaling and biosynthesis in response to LCOs or Sinorhizobium meliloti inoculation in Medicago truncatula remains poorly understood. In this study, using a fluorescence-based CK sensor (pTCSn::nls:tGFP), we performed a high-resolution tissue-specific temporal characterization of the sequential activation of CK response during root infection and nodule development in M. truncatula after inoculation with S. meliloti. Loss-of-function mutants of the CK-biosynthetic gene ISOPENTENYLTRANSFERASE 3 (IPT3) showed impairment of nodulation, suggesting that IPT3 is required for nodule development in M. truncatula. Simultaneous live imaging of pIPT3::nls:tdTOMATO and the CK sensor showed that IPT3 induction in the pericycle at the base of nodule primordium contributes to CK biosynthesis, which in turn promotes expression of positive regulators of nodule organogenesis in M. truncatula.

Precise spatial and temporal characterization of cytokinin (CK) responses reveals the function of the CK biosynthesis gene ISOPENTENYLTRANSFERASE 3 during nodule development in Medicago truncatula.     

miR393 and miR164 influence indeterminate but not determinate nodule development

The roles of auxin in the regulation of symbiotic legume nodule formation are unclear. We recently showed that enhanced sensitivity to auxin resulting from overexpression of miR160 inhibits determinate nodule formation in soybean. We examined the roles of miR393 and miR164 in soybean (that forms determinate nodules) and Medicago truncatula (that forms indeterminate nodules). Our results together with previous studies suggest that indeterminate nodule formation requires a higher, but narrow window of auxin sensitivity and that miR164 regulation is not crucial for determinate nodule formation.     

Flavonoids and Auxin Transport Inhibitors Rescue Symbiotic Nodulation in the Medicago truncatula Cytokinin Perception Mutant cre1

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.     

Does SUNN-SYMRK Crosstalk occur in Medicago truncatula for regulating nodule organogenesis?

Recently we reported that overexpression of intracellular kinase domain of Symbiosis Receptor Kinase (SYMRK-kd) hyperactivated spontaneous nodulation in Medicago truncatula indicating the importance of SYMRK ectodomain in restricting nodule number. To clarify whether sunn and sickle pathways were overcome by SYMRK-kd for hyperactivation of nodule organogenesis, we overexpressed SYMRK-kd in these mutants and analyzed for spontaneous nodulation in absence of rhizobia. Spontaneous nodulation in skl/SYMRK-kd roots was 2-fold higher than A17/SYMRK-kd roots indicating nodule organogenesis induced by SYMRK-k_d to be ethylene sensitive. Intriguingly, sunn/SYMRK-kd roots failed to generate any spontaneous nodule which directly indicate the LRR-RLK SUNN to have a role in SYMRK-k_d mediated nodule development under non-symbiotic conditions. We hypothesize a crosstalk between SUNN and SYMRK receptors for activation as well as restriction of nodule development.     

Expression of the Arabidopsis thaliana immune receptor EFR in Medicago truncatula reduces infection by a root pathogenic bacterium,but not nitrogen‐fixing rhizobial symbiosis

Interfamily transfer of plant pattern recognition receptors (PRRs) represents a promising biotechnological approach to engineer broad‐spectrum, and potentially durable, disease resistance in crops. It is however unclear whether new recognition specificities to given pathogen‐associated molecular patterns (PAMPs) affect the interaction of the recipient plant with beneficial microbes. To test this in a direct reductionist approach, we transferred the Brassicaceae‐specific PRR ELONGATION FACTOR‐THERMO UNSTABLE RECEPTOR (EFR), conferring recognition of the bacterial EF‐Tu protein, from Arabidopsis thaliana to the legume Medicago truncatula. Constitutive EFR expression led to EFR accumulation and activation of immune responses upon treatment with the EF‐Tu‐derived elf18 peptide in leaves and roots. The interaction of M. truncatula with the bacterial symbiont Sinorhizobium meliloti is characterized by the formation of root nodules that fix atmospheric nitrogen. Although nodule numbers were slightly reduced at an early stage of the infection in EFR‐Medicago when compared to control lines, nodulation was similar in all lines at later stages. Furthermore, nodule colonization by rhizobia, and nitrogen fixation were not compromised by EFR expression. Importantly, the M. truncatula lines expressing EFR were substantially more resistant to the root bacterial pathogen Ralstonia solanacearum. Our data suggest that the transfer of EFR to M. truncatula does not impede root nodule symbiosis, but has a positive impact on disease resistance against a bacterial pathogen. In addition, our results indicate that Rhizobium can either avoid PAMP recognition during the infection process, or is able to actively suppress immune signaling.     

Genome-Wide Identification and Expression Profiling Analysis of the Aux/IAA Gene Family in Medicago truncatula during the Early Phase of Sinorhizobium meliloti Infection

Background

Auxin/indoleacetic acid (Aux/IAA) genes, coding a family of short-lived nuclear proteins, play key roles in wide variety of plant developmental processes, including root system regulation and responses to environmental stimulus. However, how they function in auxin signaling pathway and symbiosis with rhizobial in Medicago truncatula are largely unknown. The present study aims at gaining deeper insight on distinctive expression and function features of Aux/IAA family genes in Medicago truncatula during nodule formation.

Principal Findings

Using the latest updated draft of the full Medicago truncatula genome, a comprehensive identification and analysis of IAA genes were performed. The data indicated that MtIAA family genes are distributed in all the M. truncatula chromosomes except chromosome 6. Most of MtIAA genes are responsive to exogenous auxin and express in tissues-specific manner. To understand the biological functions of MtIAA genes involved in nodule formation, quantitative real-time polymerase chain reaction (qRT-PCR) was used to test the expression profiling of MtIAA genes during the early phase of Sinorhizobium meliloti (S. meliloti) infection. The expression patterns of most MtIAA genes were down-regulated in roots and up-regulated in shoots by S. meliloti infection. The differences in expression responses between roots and shoots caused by S. meliloti infection were alleviated by 1-NOA application.

Conclusion

The genome-wide identification, evolution and expression pattern analysis of MtIAA genes were performed in this study. The data helps us to understand the roles of MtIAA-mediated auxin signaling in nodule formation during the early phase of S. meliloti infection.     

The involvement of Medicago truncatula non-specific lipid transfer protein N5 in the control of rhizobial infection

Cysteine-rich proteins seem to play important regulatory roles in Medicago truncatula/Sinorhizobium meliloti symbiosis. In particular, a large family of nodule-specific cysteine-rich (NCR) peptides is crucial for the differentiation of nitrogen-fixing bacteroids. The Medicago truncatula N5 protein (MtN5) is currently the only reported non-specific lipid transfer protein necessary for successful rhizobial symbiosis; in addition, MtN5 shares several characteristics with NCR peptides: a small size, a conserved cysteine-rich motif, an N-terminal signal peptide for secretion and antimicrobial activity. Unlike NCR peptides, MtN5 expression is not restricted to the root nodules and is induced during the early phases of symbiosis in root hairs and nodule primordia. Recently, MtN5 was determined to be involved in the regulation of root tissue invasion; while, it was dispensable for nodule primordia formation. Here, we discuss the hypothesis that MtN5 participates in linking the progression of bacterial invasion with restricting the competence of root hairs for infection.     

Different cytokinin histidine kinase receptors regulate nodule initiation as well as later nodule developmental stages in Medicago truncatula

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.     

Effect of Nitrate on Bradyrhizobium — Mungbean (Vigna radiata) Symbiosis in presence of Syringaldehyde

Bradyrhizoblum — mungbean (Vigna radiata) symbiosis was influenced by the application of nitrate in combination with.yrlngaldehyde. Application of nitrate alone at lower concentrations (2, 5 mM) caused a reduction at initial stage (10 DAS) while at later stage (35 DAS–45 DAS) enhanced the nodulation status (nodule number, nodule weight) and nodule efficiency (acetylene reduction activity). Higher concentration of nitrate (10 mM) caused a reduction in nodule number, nodule weight and acetylene reduction activity. Syringaldehyde alone improved the nodulation status and nodule efficiency while it acted synergistically when applied in combination with nitrate. These effects were also reflected in the biomass of plants.     

Enhanced nodulation and symbiotic effectiveness of Medicago truncatula when co-inoculated with Pseudomonas fluorescens WSM3457 and Ensifer (Sinorhizobium) medicae WSM419

Aims

Low numbers of rhizobia in soil or inoculants delay nodulation and decrease symbiotic legume productivity. This study investigated the effect of co-inoculation with a helper bacterium, Pseudomonas fluorescens WSM3457 on the Medicago truncatula - Ensifer (Sinorhizobium) medicae WSM419 symbiosis challenged by a low inoculum dose.

Methods

In a glasshouse experiment the effect of co-inoculation with WSM3457 on the kinetics of nodule initiation and development was assessed 5, 7, 10, 14, 17, 21, and 42 days after inoculation of M. truncatula cv. Caliph with 10³ cells/plant of E. medicae WSM419.

Results

Co-inoculated plants had enhanced rate of nodule initiation and development, greater numbers of larger crown nodules, and by day 42 accumulated more N than plants inoculated with E. medicae WSM419 alone. Nodule development was altered by co-inoculation. Approximately 25% of nodule initials on co-inoculated plants formed in closely associated pairs, young nodules were larger with multiple meristems and developed into cluster-like multi-lobed nodules compared to those on WSM419 inoculated plants. Molecular typing showed WSM3457 occupied a significant proportion of root nodules on co-inoculated plants.

Conclusion

Co-inoculation with P. fluorescens WSM3457 enhanced symbiotic effectiveness of M. truncatula when inoculated with a low inoculum dose of E. medicae WSM419.     

Partner choice in Medicago Truncatula–Sinorhizobium symbiosis

In nitrogen-fixing symbiosis, plant sanctions against ineffective bacteria have been demonstrated in previous studies performed on soybean and yellow bush lupin, both developing determinate nodules with Bradyrhizobium sp. strains. In this study, we focused on the widely studied symbiotic association Medicago truncatula–Sinorhizobium meliloti, which forms indeterminate nodules. Using two strains isolated from the same soil and displaying different nitrogen fixation phenotypes on the same fixed plant line, we analysed the existence of both partner choice and plant sanctions by performing split-root experiments. By measuring different parameters such as the nodule number, the nodule biomass per nodule and the number of viable rhizobia per nodule, we showed that M. truncatula is able to select rhizobia based on recognition signals, both before and after the nitrogen fixation step. However, no sanction mechanism, described as a decrease in rhizobia fitness inside the nodules, was detected. Consequently, even if partner choice seems to be widespread among legumes, sanction of non-effective rhizobia might not be universal.