Inoculation- and Nitrate-Induced CLE Peptides of Soybean Control NARK-Dependent Nodule Formation

Systemic autoregulation of nodulation in legumes involves a root-derived signal (Q) that is perceived by a CLAVATA1-like leucine-rich repeat receptor kinase (e.g. GmNARK). Perception of Q triggers the production of a shoot-derived inhibitor that prevents further nodule development. We have identified three candidate CLE peptide-encoding genes (GmRIC1, GmRIC2, and GmNIC1) in soybean (Glycine max) that respond to Bradyrhizobium japonicum inoculation or nitrate treatment. Ectopic overexpression of all three CLE peptide genes in transgenic roots inhibited nodulation in a GmNARK-dependent manner. The peptides share a high degree of amino acid similarity in a 12-amino-acid C-terminal domain, deemed to represent the functional ligand of GmNARK. GmRIC1 was expressed early (12 h) in response to Bradyrhizobium-sp.-produced nodulation factor while GmRIC2 was induced later (48 to 72 h) but was more persistent during later nodule development. Neither GmRIC1 nor GmRIC2 were induced by nitrate. In contrast, GmNIC1 was strongly induced by nitrate (2 mM) treatment but not by Bradyrhizobium sp. inoculation and, unlike the other two GmCLE peptides, functioned locally to inhibit nodulation. Grafting demonstrated a requirement for root GmNARK activity for nitrate regulation of nodulation whereas Bradyrhizobium sp.-induced regulation was contingent on GmNARK function in the shoot.

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Nitrate-Induced CLE Peptide Systemically Inhibits Nodulation in Medicago truncatula

Plants ◽

10.3390/plants9111456 ◽

2020 ◽

Vol 9 (11) ◽

pp. 1456

Author(s):

Maria Lebedeva ◽

Mahboobeh Azarakhsh ◽

Yaroslavna Yashenkova ◽

Lyudmila Lutova

Keyword(s):

Medicago Truncatula ◽

Lotus Japonicus ◽

Dependent Manner ◽

Receptor Kinase ◽

Nitrate Treatment ◽

Whole Plant ◽

Cle Peptides ◽

Autoregulation Of Nodulation ◽

Cle Peptide ◽

Plant Level

Legume plants form nitrogen-fixing nodules in symbiosis with soil bacteria rhizobia. The number of symbiotic nodules is controlled at the whole-plant level with autoregulation of nodulation (AON), which includes a shoot-acting CLV1-like receptor kinase and mobile CLE (CLAVATA3/ENDOSPERM SURROUNDING REGION-related) peptides that are produced in the root in response to rhizobia inoculation. In addition to rhizobia-induced CLE peptides, nitrate-induced CLE genes have been identified in Lotus japonicus and Glycine max, which inhibited nodulation when overexpressed. However, nitrate-induced CLE genes that systemically suppress nodulation in AON-dependent manner have not been identified in Medicago truncatula. Here, we found that MtCLE35 expression is activated by both rhizobia inoculation and nitrate treatment in M. truncatula, similarly to L. japonicus CLE genes. Moreover, we found that MtCLE35 systemically suppresses nodulation in AON-dependent manner, suggesting that MtCLE35 may mediate nitrate-induced inhibition of nodulation in M. truncatula.

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Characterisation of Medicago truncatula CLE34 and CLE35 in nodulation control

10.1101/2020.07.31.231605 ◽

2020 ◽

Author(s):

Celine Mens ◽

April H. Hastwell ◽

Huanan Su ◽

Peter M. Gresshoff ◽

Ulrike Mathesius ◽

...

Keyword(s):

Pisum Sativum ◽

Medicago Truncatula ◽

Sequence Homology ◽

Dependent Manner ◽

Legume Species ◽

Mature Peptide ◽

Cle Peptides ◽

Autoregulation Of Nodulation ◽

Cle Peptide ◽

Distinct Role

AbstractLegume plants form a symbiosis with N2-fixing soil rhizobia, resulting in new root organs called nodules that enable N2-fixation. Nodulation is a costly process that is tightly regulated by the host through Autoregulation of Nodulation (AON) and nitrate-dependent regulation of nodulation. Both pathways require legume-specific CLAVATA/ESR-related (CLE) peptides. Nitrogen-induced nodulation-suppressing CLE peptides have not previously been characterised in Medicago truncatula, with only rhizobia-induced MtCLE12 and MtCLE13 identified. Here, we report on novel peptides MtCLE34 and MtCLE35 in nodulation control pathways. The nodulation-suppressing CLE peptides of five legume species were classified into three clades based on sequence homology and phylogeny. This approached identified MtCLE34 and MtCLE35 and four new CLE peptide orthologues of Pisum sativum. Whereas MtCLE12 and MtCLE13 are induced by rhizobia, MtCLE34 and MtCLE35 respond to both rhizobia and nitrate. MtCLE34 was identified as a pseudogene lacking a functional CLE-domain. Overexpression of MtCLE12, MtCLE13 and MtCLE35 inhibits nodulation. Together, our findings indicate that MtCLE12 and MtCLE13 have a distinct role in AON, while MtCLE35 regulates nodule numbers in a rhizobia- and nitrate-dependent manner. MtCLE34 likely had a similar role to MtCLE35 but its function was lost due to a nonsense mutation resulting in the loss of the mature peptide.

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Shoot-derived miR2111 controls legume root and nodule development

10.1101/2020.09.02.278739 ◽

2020 ◽

Cited By ~ 1

Author(s):

Mengbai Zhang ◽

Huanan Su ◽

Peter M. Gresshoff ◽

Brett J. Ferguson

Keyword(s):

Lateral Root ◽

Lateral Roots ◽

Ectopic Expression ◽

Root System Architecture ◽

Negative Regulator ◽

Nodule Development ◽

Wild Type ◽

Root Branching ◽

Autoregulation Of Nodulation ◽

Cle Peptide

AbstractLegumes control their nodule numbers through the Autoregulation Of Nodulation (AON). Rhizobia infection stimulates the production of root-derived CLE peptide hormones that are translocated to the shoot where they regulate a new signal. We used soybean to demonstrate that this shoot-derived signal is miR2111, which is transported via phloem to the root where it targets transcripts of Too Much Love (TML), a negative regulator of nodulation. Shoot perception of rhizobia-induced CLE peptides suppresses miR2111 expression, resulting in TML accumulation in roots and subsequent inhibition of nodule organogenesis. Feeding synthetic mature miR2111 via the petiole increased nodule numbers per plant. Likewise, elevating miR2111 availability by over-expression promoted nodulation, while target mimicry of TML induced the opposite effect on nodule development in wild-type plants and alleviated the supernodulating and stunted root growth phenotypes of AON-defective mutants. Additionally, in non-nodulating wild-type plants, ectopic expression of miR2111 significantly enhanced lateral root emergence with a decrease in lateral root length and average root diameter. In contrast, hairy roots constitutively expressing the target mimic construct exhibited reduced lateral root density. Overall, these findings demonstrate that miR2111 is both the critical shoot-to-root factor that positively regulates root nodule development, and also acts to shape root system architecture via orchestrating the degree of root branching, as well as the length and thickness of lateral roots.

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Rhizobial Synthesized Cytokinins Contribute to But Are Not Essential for the Symbiotic Interaction Between Photosynthetic Bradyrhizobia and Aeschynomene Legumes

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-03-13-0076-r ◽

2013 ◽

Vol 26 (10) ◽

pp. 1232-1238 ◽

Cited By ~ 29

Author(s):

Kateřina Podlešáková ◽

Joel Fardoux ◽

Delphine Patrel ◽

Katia Bonaldi ◽

Ondřej Novák ◽

...

Keyword(s):

Root Nodules ◽

Rna Degradation ◽

Nodule Development ◽

Nodule Formation ◽

Symbiotic Interaction ◽

Extracellular Medium ◽

Exogenous Addition ◽

Bradyrhizobium Sp ◽

Derivatives Of

Cytokinins (CK) play an important role in the formation of nitrogen-fixing root nodules. It has been known for years that rhizobia secrete CK in the extracellular medium but whether they play a role in nodule formation is not known. We have examined this question using the photosynthetic Bradyrhizobium sp. strain ORS285 which is able to nodulate Aeschynomene afraspera and A. indica using a Nod-dependent or Nod-independent symbiotic process, respectively. CK profiling showed that the most abundant CK secreted by Bradyrhizobium sp. strain ORS285 are the 2MeS (2-methylthiol) derivatives of trans-zeatin and isopentenyladenine. In their pure form, these CK can activate legume CK receptors in vitro, and their exogenous addition induced nodule-like structures on host plants. Deletion of the miaA gene showed that transfer RNA degradation is the source of CK production in Bradyrhizobium sp. strain ORS285. In nodulation studies performed with A. indica and A. afraspera, the miaA mutant had a 1-day delay in nodulation and nitrogen fixation. Moreover, A. indica plants formed considerably smaller but more abundant nodules when inoculated with the miaA mutant. These data show that CK produced by Bradyrhizobium sp. strain ORS285 are not the key signal triggering nodule formation during the Nod-independent symbiosis but they contribute positively to nodule development in Aeschynomene plants.

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Characterization of Nops, Nodulation Outer Proteins, Secreted Via the Type III Secretion System of NGR234

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2003.16.9.743 ◽

2003 ◽

Vol 16 (9) ◽

pp. 743-751 ◽

Cited By ~ 91

Author(s):

Corinne Marie ◽

William J. Deakin ◽

Virginie Viprey ◽

Joanna Kopciñska ◽

Wladyslaw Golinowski ◽

...

Keyword(s):

Protein Secretion ◽

Type Iii Secretion ◽

Type Iii Secretion System ◽

Symbiotic Bacterium ◽

Secretion System ◽

Nodule Development ◽

Nodule Formation ◽

Dependent Manner ◽

Double Mutation ◽

Type Iii

The nitrogen-fixing symbiotic bacterium Rhizobium species NGR234 secretes, via a type III secretion system (TTSS), proteins called Nops (nodulation outer proteins). Abolition of TTSS-dependent protein secretion has either no effect or leads to a change in the number of nodules on selected plants. More dramatically, Nops impair nodule development on Crotalaria juncea roots, resulting in the formation of nonfixing pseudonodules. A double mutation of nopX and nopL, which code for two previously identified secreted proteins, leads to a phenotype on Pachyrhizus tuberosus differing from that of a mutant in which the TTSS is not functional. Use of antibodies and a modification of the purification protocol revealed that NGR234 secretes additional proteins in a TTSS-dependent manner. One of them was identified as NopA, a small 7-kDa protein. Single mutations in nopX and nopL were also generated to assess the involvement of each Nop in protein secretion and nodule formation. Mutation of nopX had little effect on NopL and NopA secretion but greatly affected the interaction of NGR234 with many plant hosts tested. NopL was not necessary for the secretion of any Nops but was required for efficient nodulation of some plant species. NopL may thus act as an effector protein whose recognition is dependent upon the hosts' genetic background.

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Developmental Analysis of the GATA Factor HANABA TARANU Mutants in Medicago truncatula Reveals Their Roles in Nodule Formation

Frontiers in Plant Science ◽

10.3389/fpls.2021.616776 ◽

2021 ◽

Vol 12 ◽

Author(s):

Yiteng Xu ◽

Hongfeng Wang ◽

Zhichao Lu ◽

Lizhu Wen ◽

Zhiqun Gu ◽

...

Keyword(s):

Transcription Factors ◽

Medicago Truncatula ◽

Transcriptional Analysis ◽

Nodule Development ◽

Root Tip ◽

Nodule Formation ◽

Loss Of Function ◽

Gata Factor ◽

Nitrate Treatment ◽

Gata Transcription Factors

Formation of nodules on legume roots results from symbiosis with rhizobial bacteria. Here, we identified two GATA transcription factors, MtHAN1 and MtHAN2, in Medicago truncatula, which are the homologs of HANABA TARANU (HAN) and HANABA TARANU LIKE in Arabidopsis thaliana. Our analysis revealed that MtHAN1 and MtHAN2 are expressed in roots and shoots including the root tip and nodule apex. We further show that MtHAN1 and MtHAN2 localize to the nucleus where they interact and that single and double loss-of-function mutants of MtHAN1 and MtHAN2 did not show any obvious phenotype in flower development, suggesting their role is different than their closest Arabidopsis homologues. Investigation of their symbiotic phenotypes revealed that the mthan1 mthan2 double mutant develop twice as many nodules as wild type, revealing a novel biological role for GATA transcription factors. We found that HAN1/2 transcript levels respond to nitrate treatment like their Arabidopsis counterparts. Global gene transcriptional analysis by RNA sequencing revealed different expression genes enriched for several pathways important for nodule development including flavonoid biosynthesis and phytohormones. In addition, further studies suggest that MtHAN1 and MtHAN2 are required for the expression of several nodule-specific cysteine-rich genes, which they may activate directly, and many peptidase and peptidase inhibitor genes. This work expands our knowledge of the functions of MtHANs in plants by revealing an unexpected role in legume nodulation.

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Nitrate inhibits nodule organogenesis through inhibition of cytokinin biosynthesis in Lotus japonicus

10.1101/2020.11.03.366971 ◽

2020 ◽

Author(s):

Jieshun Lin ◽

Yuda Purwana Roswanjaya ◽

Wouter Kohlen ◽

Jens Stougaard ◽

Dugald Reid

Keyword(s):

Nitrogen Fixation ◽

Developmental Process ◽

Root Zone ◽

Lotus Japonicus ◽

Nodule Development ◽

Nodule Formation ◽

Dependent Manner ◽

Concentration Dependent Manner ◽

Cytokinin Biosynthesis ◽

Nitrate Inhibition

AbstractLegumes balance nitrogen acquisition from soil nitrate with symbiotic nitrogen fixation. Nitrogen fixation requires establishment of a new organ, which is a cytokinin dependent developmental process in the root. We found cytokinin biosynthesis is a central integrator, balancing nitrate signalling with symbiotic acquired nitrogen. Low nitrate conditions provide a permissive state for induction of cytokinin by symbiotic signalling and thus nodule development. In contrast, high nitrate is inhibitory to cytokinin accumulation and nodule establishment in the root zone susceptible to nodule formation. This reduction of symbiotic cytokinin accumulation was further exacerbated in cytokinin biosynthesis mutants, which display hypersensitivity to nitrate inhibition of nodule development, maturation and nitrogen fixation. Consistent with this, cytokinin application can rescue nodulation and nitrogen fixation of biosynthesis mutants in a concentration dependent manner. These inhibitory impacts of nitrate on symbiosis occur in a Nlp1 and Nlp4 dependent manner and contrast with the positive influence of nitrate on cytokinin biosynthesis that occurs in non-symbiotic species. Altogether this shows that legumes, as exemplified by Lotus japonicus, have evolved a different cytokinin response to nitrate compared to non-legumes.One sentence summaryCytokinin biosynthesis is suppressed by nitrate in Lotus japonicus, providing a mechanism for nitrate inhibition of symbiotic nodule organogenesis.

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The structure and activity of nodulation-suppressing CLE peptide hormones of legumes

Functional Plant Biology ◽

10.1071/fp14222 ◽

2015 ◽

Vol 42 (3) ◽

pp. 229 ◽

Cited By ~ 18

Author(s):

April H. Hastwell ◽

Peter M. Gresshoff ◽

Brett J. Ferguson

Keyword(s):

Molecular Mechanisms ◽

De Novo ◽

Lotus Japonicus ◽

Peptide Hormones ◽

Negative Feedback Loops ◽

C Terminus ◽

Cle Peptides ◽

Autoregulation Of Nodulation ◽

Cle Peptide ◽

External Stimuli

Legumes form a highly-regulated symbiotic relationship with specific soil bacteria known as rhizobia. This interaction results in the de novo formation of root organs called nodules, in which the rhizobia fix atmospheric di-nitrogen (N2) for the plant. Molecular mechanisms that regulate the nodulation process include the systemic ‘autoregulation of nodulation’ and the local nitrogen-regulation of nodulation pathways. Both pathways are mediated by novel peptide hormones called CLAVATA/ESR-related (CLE) peptides that act to suppress nodulation via negative feedback loops. The mature peptides are 12–13 amino acids in length and are post-translationally modified from the C-terminus of tripartite-domain prepropeptides. Structural redundancy between the prepropeptides exists; however, variations in external stimuli, timing of expression, tissue specificity and presence or absence of key functional domains enables them to act in a specific manner. To date, nodulation-regulating CLE peptides have been identified in Glycine max (L.) Merr., Medicago truncatula Gaertn., Lotus japonicus (Regel) K.Larsen and Phaseolus vulgaris L. One of the L. japonicus peptides, called LjCLE-RS2, has been structurally characterised and found to be an arabinosylated glycopeptide. All of the known nodulation CLE peptides act via an orthologous leucine rich repeat (LRR) receptor kinase. Perception of the peptide results in the production of a novel, unidentified inhibitor signal that acts to suppress further nodulation events. Here, we contrast and compare the various nodulation-suppressing CLE peptides of legumes.

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Transcriptomic analysis of sym28 and sym29 supernodulating mutants of pea (Pisum sativum L.) under complex inoculation with beneficial microorganisms

Biological Communications ◽

10.21638/spbu03.2021.301 ◽

2021 ◽

Vol 66 (3) ◽

Author(s):

Vladimir Zhukov ◽

Evgeny Zorin ◽

Aleksandr Zhernakov ◽

Alexey Afonin ◽

Gulnar Akhtemova ◽

...

Keyword(s):

Pisum Sativum ◽

Soil Microorganisms ◽

Abiotic Factors ◽

Biomass Accumulation ◽

Am Fungi ◽

Nodule Development ◽

Plant Growth Promoting Bacteria ◽

Wild Type ◽

Cle Peptides ◽

Autoregulation Of Nodulation

The garden pea (Pisum sativum L.), like most members of Fabaceae family, is capable of forming symbioses with beneficial soil microorganisms such as nodule bacteria (rhizobia), arbuscular mycorrhizal (AM) fungi and plant growth promoting bacteria (PGPB). The autoregulation of nodulation (AON) system is known to play an important role in controlling both the number of nodules and the level of root colonization by AM via root-to-shoot signaling mediated by CLAVATA/ESR-related (CLE) peptides and their receptors. In the pea, mutations in genes Sym28 (CLV2-like) and Sym29 (CLV1-like), which encode receptors for CLE peptides, lead to the supernodulation phenotype, i.e., excessive nodule formation. The aim of the present study was to analyze the response of pea cv. ‘Frisson’ (wild type) and mutants P64 (sym28) and P88 (sym29) to complex inoculation with rhizobia, AM fungi and PGPB, with regard to biomass accumulation, yield and transcriptomic alterations. The plants were grown in quartz sand for 2 and 4 weeks after inoculation with either rhizobia (Rh) or complex inoculation with Rh + AM, Rh + PGPB, and Rh+AM+PGPB, and the biomass and yield were assessed. Transcriptome sequencing of whole shoots and roots was performed using a modified RNAseq protocol named MACE (Massive Analysis of cDNA Ends). In the experimental conditions, P88 (sym29) plants demonstrated the best biomass accumulation and yield, as compared to the wild type and P64 (sym28) plants, whereas P64 (sym28) had the lowest rate of biomass and seed yield. The transcriptome analysis showed that both supernodulating mutants more actively responded to biotic and abiotic factors than the wild-type plants and demonstrated increased expression of genes characteristic to late stages of nodule development. The roots of P64 (sym28) plants responded to AM+Rh treatment with upregulation of genes encoding plastid proteins, which can be connected with the activation of carotenoid biosynthesis (namely, the non-mevalonate pathway that takes place in root plastids). The more active response to symbionts in P88 (sym29) plants, as compared to cv. ‘Frisson’, was associated with counterregulation of transcripts involved in chloroplast functioning and development in leaves, which accompanies successful plant development in symbiotic conditions. Finally, the effect of retardation of plant aging upon mycorrhization on a transcriptomic level was recorded for cv. ‘Frisson’ but not for P64 (sym28) and P88 (sym29) mutants, which points towards its possible connection with the AON system. The results of this work link the plant’s autoregulation with the responsiveness to inoculation with beneficial soil microorganisms.

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The CLE53–SUNN genetic pathway negatively regulates arbuscular mycorrhiza root colonization in Medicago truncatula

Journal of Experimental Botany ◽

10.1093/jxb/eraa193 ◽

2020 ◽

Vol 71 (16) ◽

pp. 4972-4984 ◽

Cited By ~ 6

Author(s):

Magda Karlo ◽

Clarissa Boschiero ◽

Katrine Gram Landerslev ◽

Gonzalo Sancho Blanco ◽

Jiangqi Wen ◽

...

Keyword(s):

Mycorrhizal Fungi ◽

Phosphorus Uptake ◽

Arbuscular Mycorrhizal ◽

Dependent Manner ◽

High Phosphorus ◽

Peptide Modification ◽

Cle Peptides ◽

Autoregulation Of Nodulation ◽

Low Phosphorus ◽

Phosphorus Levels

Abstract Plants and arbuscular mycorrhizal fungi (AMF) engage in mutually beneficial symbioses based on a reciprocal exchange of nutrients. The beneficial character of the symbiosis is maintained through a mechanism called autoregulation of mycorrhization (AOM). AOM includes root-to-shoot-to-root signaling; however, the molecular details of AOM are poorly understood. AOM shares many features of autoregulation of nodulation (AON) where several genes are known, including the receptor-like kinase SUPER NUMERIC NODULES (SUNN), root-to-shoot mobile CLAVATA3/ENDOSPERM SURROUNDING REGION (ESR)-RELATED (CLE) peptides, and the hydroxyproline O-arabinosyltransferase ROOT DETERMINED NODULATION1 (RDN1) required for post-translational peptide modification. In this work, CLE53 was identified to negatively regulate AMF symbiosis in a SUNN- and RDN1-dependent manner. CLE53 expression was repressed at low phosphorus, while it was induced by AMF colonization and high phosphorus. CLE53 overexpression reduced AMF colonization in a SUNN- and RDN1 dependent manner, while cle53, rdn1, and sunn mutants were more colonized than the wild type. RNA-sequencing identified 700 genes with SUNN-dependent regulation in AMF-colonized plants, providing a resource for future identification of additional AOM genes. Disruption of AOM genes in crops potentially constitutes a novel route for improving AMF-derived phosphorus uptake in agricultural systems with high phosphorus levels.

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