Structure and antimicrobial activity of NCR169, a nodule-specific cysteine-rich peptide of Medicago truncatula

AbstractA model legume, Medicago truncatula, has over 600 nodule-specific cysteine-rich (NCR) peptides required for symbiosis with rhizobia. Among them, NCR169, an essential factor for establishing symbiosis, has four cysteine residues that are indispensable for its function. However, knowledge of NCR169 structure and mechanism of action is still lacking. In this study, we solved two NMR structures of NCR169 caused by different disulfide linkage patterns. We show that both structures have a consensus C-terminal β-sheet attached to an extended N-terminal region with dissimilar features; one moves widely, whereas the other is relatively stapled. We further revealed that the disulfide bonds of NCR169 contribute to its structural stability and solubility. Regarding the function, one of the NCR169 oxidized forms could bind to negatively charged bacterial phospholipids. Furthermore, the positively charged lysine-rich region of NCR169 may be responsible for its antimicrobial activity against Escherichia coli and Sinorhizobium meliloti. This active region was disordered even in the phospholipid bound state, suggesting that the disordered conformation of this region is key to its function. Morphological observations suggested the mechanism of action of NCR169 on bacteria. The present study on NCR169 provides new insights into the structure and function of NCR peptides.

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Laser Capture Micro-Dissection Coupled to RNA Sequencing: A Powerful Approach Applied to the Model Legume Medicago truncatula in Interaction with Sinorhizobium meliloti

Methods in Molecular Biology - Plant Transcription Factors ◽

10.1007/978-1-4939-8657-6_12 ◽

2018 ◽

pp. 191-224 ◽

Cited By ~ 6

Author(s):

Brice Roux ◽

Nathalie Rodde ◽

Sandra Moreau ◽

Marie-Françoise Jardinaud ◽

Pascal Gamas

Keyword(s):

Rna Sequencing ◽

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Model Legume ◽

Powerful Approach ◽

Laser Capture

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Sinorhizobium meliloti Controls Nitric Oxide–Mediated Post-Translational Modification of a Medicago truncatula Nodule Protein

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-05-15-0118-r ◽

2015 ◽

Vol 28 (12) ◽

pp. 1353-1363 ◽

Cited By ~ 21

Author(s):

Pauline Blanquet ◽

Liliana Silva ◽

Olivier Catrice ◽

Claude Bruand ◽

Helena Carvalho ◽

...

Keyword(s):

Nitric Oxide ◽

Nitrogen Fixation ◽

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Molecular Mechanisms ◽

Root Nodules ◽

Plant Protein ◽

Post Translational Modification ◽

Model Legume ◽

Bacterial Mutants

Nitric oxide (NO) is involved in various plant-microbe interactions. In the symbiosis between soil bacterium Sinorhizobium meliloti and model legume Medicago truncatula, NO is required for an optimal establishment of the interaction but is also a signal for nodule senescence. Little is known about the molecular mechanisms responsible for NO effects in the legume-rhizobium interaction. Here, we investigate the contribution of the bacterial NO response to the modulation of a plant protein post-translational modification in nitrogen-fixing nodules. We made use of different bacterial mutants to finely modulate NO levels inside M. truncatula root nodules and to examine the consequence on tyrosine nitration of the plant glutamine synthetase, a protein responsible for assimilation of the ammonia released by nitrogen fixation. Our results reveal that S. meliloti possesses several proteins that limit inactivation of plant enzyme activity via NO-mediated post-translational modifications. This is the first demonstration that rhizobia can impact the course of nitrogen fixation by modulating the activity of a plant protein.

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Transcription of ENOD8 in Medicago truncatula Nodules Directs ENOD8 Esterase to Developing and Mature Symbiosomes

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-21-4-0404 ◽

2008 ◽

Vol 21 (4) ◽

pp. 404-410 ◽

Cited By ~ 15

Author(s):

Laurent Coque ◽

Purnima Neogi ◽

Catalina Pislariu ◽

Kimberly A. Wilson ◽

Christina Catalano ◽

...

Keyword(s):

Nitrogen Fixation ◽

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Immunoblot Analysis ◽

Nodule Formation ◽

Nitrogenous Compounds ◽

Symbiosome Membrane ◽

The Matrix ◽

And Function ◽

Infected Nodule

In Medicago truncatula nodules, the soil bacterium Sinorhizobium meliloti reduces atmospheric dinitrogen into nitrogenous compounds that the legume uses for its own growth. In nitrogen-fixing nodules, each infected cell contains symbiosomes, which include the rhizobial cell, the symbiosome membrane surrounding it, and the matrix between the bacterium and the symbiosome membrane, termed the symbiosome space. Here, we describe the localization of ENOD8, a nodule-specific esterase. The onset of ENOD8 expression occurs at 4 to 5 days postinoculation, before the genes that support the nitrogen fixation capabilities of the nodule. Expression of an ENOD8 promoter–gusA fusion in nodulated hairy roots of composite transformed M. truncatula plants indicated that ENOD8 is expressed from the proximal end of interzone II to III to the proximal end of the nodules. Confocal immunomicroscopy using an ENOD8-specific antibody showed that the ENOD8 protein was detected in the same zones. ENOD8 protein was localized in the symbiosome membrane or symbiosome space around the bacteroids in the infected nodule cells. Immunoblot analysis of fractionated symbiosomes strongly suggested that ENOD8 protein was found in the symbiosome membrane and symbiosome space, but not in the bacteroid. Determining the localization of ENOD8 protein in the symbiosome is a first step in understanding its role in symbiosome membrane and space during nodule formation and function.

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Specific Host-Responsive Associations Between Medicago truncatula Accessions and Sinorhizobium Strains

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-01-17-0009-r ◽

2017 ◽

Vol 30 (5) ◽

pp. 399-409 ◽

Cited By ~ 16

Author(s):

Théophile Kazmierczak ◽

Marianna Nagymihály ◽

Florian Lamouche ◽

Quentin Barrière ◽

Ibtissem Guefrachi ◽

...

Keyword(s):

Medicago Truncatula ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Nodule Formation ◽

Bacterial Strains ◽

Symbiotic Efficiency ◽

Model Legume ◽

Functional Studies ◽

Multiple Parameters ◽

Nitrogen Fixation Activity

Legume plants interact with rhizobia to form nitrogen-fixing root nodules. Legume-rhizobium interactions are specific and only compatible rhizobia and plant species will lead to nodule formation. Even within compatible interactions, the genotype of both the plant and the bacterial symbiont will impact on the efficiency of nodule functioning and nitrogen-fixation activity. The model legume Medicago truncatula forms nodules with several species of the Sinorhizobium genus. However, the efficiency of these bacterial strains is highly variable. In this study, we compared the symbiotic efficiency of Sinorhizobium meliloti strains Sm1021, 102F34, and FSM-MA, and Sinorhizobium medicae strain WSM419 on the two widely used M. truncatula accessions A17 and R108. The efficiency of the interactions was determined by multiple parameters. We found a high effectiveness of the FSM-MA strain with both M. truncatula accessions. In contrast, specific highly efficient interactions were obtained for the A17-WSM419 and R108-102F34 combinations. Remarkably, the widely used Sm1021 strain performed weakly on both hosts. We showed that Sm1021 efficiently induced nodule organogenesis but cannot fully activate the differentiation of the symbiotic nodule cells, explaining its weaker performance. These results will be informative for the selection of appropriate rhizobium strains in functional studies on symbiosis using these M. truncatula accessions, particularly for research focusing on late stages of the nodulation process.

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Resistance to Subterranean clover mottle virus in Medicago truncatula and genetic mapping of a resistance locus

Crop and Pasture Science ◽

10.1071/cp08373 ◽

2009 ◽

Vol 60 (5) ◽

pp. 480 ◽

Cited By ~ 3

Author(s):

Muhammad Saqib ◽

Simon R. Ellwood ◽

Roger A. C. Jones ◽

Michael G. K. Jones

Keyword(s):

Medicago Truncatula ◽

Recombinant Inbred Lines ◽

Genome Structure ◽

Subterranean Clover ◽

Mottle Virus ◽

Resistance Locus ◽

Hypersensitive Resistance ◽

Phenotypic Data ◽

Model Legume ◽

And Function

Subterranean clover mottle virus (SCMoV), which causes an important disease of annual clover pastures, was inoculated to the annual pasture legume Medicago truncatula, a model legume species used to help understand legume genome structure and function. Two hundred and nine accessions representing the core collection of M. truncatula were inoculated with infective sap containing SCMoV to determine their disease phenotypes. Forty-two of these accessions remained uninfected systemically and so were potentially resistant to the virus. Accession DZA315.16 developed a localised hypersensitive resistance reaction. In an F8 mapping population from a cross between the susceptible parent Jemalong 6/A17 and resistant accession DZA315.16, a total of 166 F8 recombinant inbred lines (RILs) were phenotyped for resistance and susceptibility to SCMoV. Resistant and susceptible lines showed parental phenotypic responses: 84 were susceptible and 82 were resistant, suggesting presence of a single resistance (R) gene. The phenotypic data were combined with genotypic data (76 polymorphic molecular markers) for this RIL population to provide a framework map. Genetic analysis located a single resistance locus termed RSCMoV1 on the long arm of chromosome 6. These results provide a basis for fine mapping the RSCMoV1 gene.

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The LATD Gene of Medicago truncatula Is Required for Both Nodule and Root Development

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi-18-0521 ◽

2005 ◽

Vol 18 (6) ◽

pp. 521-532 ◽

Cited By ~ 67

Author(s):

Lydia J. Bright ◽

Yan Liang ◽

David M. Mitchell ◽

Jeanne M. Harris

Keyword(s):

Medicago Truncatula ◽

Root Development ◽

Sinorhizobium Meliloti ◽

Root Nodules ◽

Lateral Roots ◽

Primary Root ◽

Evolutionary Origin ◽

Nodule Formation ◽

Wild Type ◽

Model Legume

The evolutionary origins of legume root nodules are largely unknown. We have identified a gene,LATD, of the model legume Medicago truncatula, that is required for both nodule and root development, suggesting that these two developmental processes may share a common evolutionary origin. The latd mutant plants initiate nodule formation but do not complete it, resulting in immature, non-nitrogen-fixing nodules. Similarly, lateral roots initiate, but remain shortstumps. The primary root, which initially appears to be wild type, gradually ceases growth and forms an abnormal tipthat resembles that of the mutant lateral roots. Infection by the rhizobial partner, Sinorhizobium meliloti, can occur, although infection is rarely completed. Once inside latd mutant nodules, S. meliloti fails to express rhizobial genes associatedwith the developmental transition from free-living bacterium to endosymbiont, such as bacA and nex38. The infecting rhizobia also fail to express nifH and fix nitrogen. Thus, both plant and bacterial development are blocked in latd mutant roots. Based on the latd mutant phenotype, we propose that the wild-type function of the LATD gene is to maintain root meristems. The strong requirement of both nodules and lateral roots for wild-type LATD gene function supports lateral roots as a possible evolutionary origin for legume nodules.

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Analysis of Medicago truncatula Nodule Expressed Sequence Tags

Molecular Plant-Microbe Interactions ◽

10.1094/mpmi.2000.13.1.62 ◽

2000 ◽

Vol 13 (1) ◽

pp. 62-71 ◽

Cited By ~ 69

Author(s):

János Györgyey ◽

Danièle Vaubert ◽

José I. Jiménez-Zurdo ◽

Celine Charon ◽

Liliane Troussard ◽

...

Keyword(s):

Medicago Truncatula ◽

Expressed Sequence Tags ◽

Sinorhizobium Meliloti ◽

Expression Patterns ◽

Homology Search ◽

Cdna Clones ◽

Systematic Analysis ◽

And Function ◽

Nodulin Genes ◽

Expressed Sequence

Systematic sequencing of expressed sequence tags (ESTs) can give a global picture of the assembly of genes involved in the development and function of organs. Indeterminate nodules representing different stages of the developmental program are especially suited to the study of organogenesis. With the vector λHybriZAP, a cDNA library was constructed from emerging nodules of Medicago truncatula induced by Sinorhizobium meliloti. The 5′ ends of 389 cDNA clones were sequenced, then these ESTs were analyzed both by sequence homology search and by studying their expression in roots and nodules. Two hundred fifty-six ESTs exhibited significant similarities to characterized data base entries and 40 of them represented 26 nodulin genes, while 133 had no similarity to sequences with known function. Only 60 out of the 389 cDNA clones corresponded to previously submitted M. truncatula EST sequences. For 117 cDNAs, reverse Northern (RNA) hybridization with root and nodule RNA probes revealed enhanced expression in the nodule, 48 clones are likely to code for novel nodulins, 33 cDNAs are clones of already known nodulin genes, and 36 clones exhibit similarity to other characterized genes. Thus, systematic analysis of the EST sequences and their expression patterns is a powerful way to identify nodule-specific and nodulation-related genes.

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