RESUMEN
Bacteroid (name for rhizobia inside nodule cells) differentiation is a prerequisite for successful nitrogen-fixing symbiosis. In certain legumes, under the regulation of host proteins, for example, a large group of NCR (nodule cysteine rich) peptides, bacteroids undergo irreversible terminal differentiation. This process causes them to lose the ability to propagate inside nodule cells while boosting their competency for nitrogen fixation. How host cells maintain the viability of differentiated bacteroids while maximizing their nitrogen-reducing activities remains elusive. Here, through mutant screen, map-based cloning, and genetic complementation, we find that NCR343 is required for the viability of differentiated bacteroids. In Medicago truncatula debino1 mutant, differentiated bacteroids decay prematurely, and NCR343 is proved to be the casual gene for debino1. NCR343 is mainly expressed in the nodule fixation zone, where bacteroids are differentiated. In nodule cells, mature NCR343 peptide is secreted into the symbiosomes. RNA-Seq assay shows that many stress-responsive genes are significantly induced in debino1 bacteroids. Additionally, a group of stress response-related rhizobium proteins are identified as putative interacting partners of NCR343. In summary, our findings demonstrate that beyond promoting bacteroid differentiation, NCR peptides are also required in maintaining the viability of differentiated bacteroids.
Asunto(s)
Medicago truncatula , Rhizobium , Medicago truncatula/genética , Medicago truncatula/metabolismo , Péptidos/metabolismo , Diferenciación Celular , Simbiosis/fisiología , Nitrógeno/metabolismo , Fijación del Nitrógeno/fisiología , Nódulos de las Raíces de las Plantas/metabolismoRESUMEN
The Rhizobium-legume symbiosis is a beneficial interaction in which the bacterium converts atmospheric nitrogen into ammonia and delivers it to the plant in exchange for carbon compounds. This symbiosis implies the adaptation of bacteria to live inside host plant cells. In this work, we apply RP-LC-MS/MS and isobaric tags as relative and absolute quantitation techniques to study the proteomic profile of endosymbiotic cells (bacteroids) induced by Rhizobium leguminosarum bv viciae strain UPM791 in legume nodules. Nitrogenase subunits, tricarboxylic acid cycle enzymes, and stress-response proteins are among the most abundant from over 1000 rhizobial proteins identified in pea (Pisum sativum) bacteroids. Comparative analysis of bacteroids induced in pea and in lentil (Lens culinaris) nodules revealed the existence of a significant host-specific differential response affecting dozens of bacterial proteins, including stress-related proteins, transcriptional regulators, and proteins involved in the carbon and nitrogen metabolisms. A mutant affected in one of these proteins, homologous to a GntR-like transcriptional regulator, showed a symbiotic performance significantly impaired in symbiosis with pea but not with lentil plants. Analysis of the proteomes of bacteroids isolated from both hosts also revealed the presence of different sets of plant-derived nodule-specific cysteine-rich peptides, indicating that the endosymbiotic bacteria find a host-specific cocktail of chemical stressors inside the nodule. By studying variations of the bacterial response to different plant cell environments, we will be able to identify specific limitations imposed by the host that might give us clues for the improvement of rhizobial performance.
Asunto(s)
Proteínas Bacterianas/metabolismo , Lens (Planta)/microbiología , Pisum sativum/microbiología , Rhizobium leguminosarum/metabolismo , Lens (Planta)/genética , Fijación del Nitrógeno , Pisum sativum/genética , Proteínas de Plantas/metabolismo , Proteoma , Rhizobium leguminosarum/genética , SimbiosisRESUMEN
In the indeterminate nodules of a model legume Medicago truncatula, â¼700 nodule-specific cysteine-rich (NCR) peptides with conserved cysteine signature are expressed. NCR peptides are highly diverse in sequence, and some of these cationic peptides exhibit antimicrobial activity in vitro and in vivo. However, there is a lack of knowledge regarding their structural architecture, antifungal activity, and modes of action against plant fungal pathogens. Here, the three-dimensional NMR structure of the 36-amino acid NCR044 peptide was solved. This unique structure was largely disordered and highly dynamic with one four-residue α-helix and one three-residue antiparallel ß-sheet stabilized by two disulfide bonds. NCR044 peptide also exhibited potent fungicidal activity against multiple plant fungal pathogens, including Botrytis cinerea and three Fusarium spp. It inhibited germination in quiescent spores of B. cinerea In germlings, it breached the fungal plasma membrane and induced reactive oxygen species. It bound to multiple bioactive phosphoinositides in vitro. Time-lapse confocal and superresolution microscopy revealed strong fungal cell wall binding, penetration of the cell membrane at discrete foci, followed by gradual loss of turgor, subsequent accumulation in the cytoplasm, and elevated levels in nucleoli of germlings. Spray-applied NCR044 significantly reduced gray mold disease symptoms caused by the fungal pathogen B. cinerea in tomato and tobacco plants, and postharvest products. Our work illustrates the antifungal activity of a structurally unique NCR peptide against plant fungal pathogens and paves the way for future development of this class of peptides as a spray-on fungistat/fungicide.
Asunto(s)
Antifúngicos/farmacología , Péptidos/metabolismo , Péptidos/farmacología , Enfermedades de las Plantas/prevención & control , Proteínas de Plantas/metabolismo , Proteínas de Plantas/farmacología , Simbiosis , Secuencia de Aminoácidos , Botrytis/metabolismo , Membrana Celular/metabolismo , Pared Celular/metabolismo , Cisteína/química , Fusarium/metabolismo , Solanum lycopersicum/metabolismo , Solanum lycopersicum/microbiología , Espectroscopía de Resonancia Magnética , Medicago truncatula/microbiología , Pichia/metabolismo , Enfermedades de las Plantas/microbiología , Nicotiana/metabolismo , Nicotiana/microbiologíaRESUMEN
Legumes in the inverted repeat-lacking clade (IRLC) each produce a unique set of nodule-specific cysteine-rich (NCR) peptides, which act in concert to determine the terminal differentiation of nitrogen-fixing bacteroid. IRLC legumes differ greatly in their numbers of NCR and sequence diversity. This raises the significant question how bacteroid differentiation is collectively controlled by the specific NCR repertoire of an IRLC legume. Astragalus sinicus is an IRLC legume that forms indeterminate nodules with its microsymbiont Mesorhizobium huakuii 7653R. Here, we performed transcriptome analysis of root and nodule samples at 3, 7, 14, 28 days postinoculation with M. huakuii 7653R and its isogenic ∆bacA mutant. BacA is a broad-specificity peptide transporter required for the host-derived NCRs to target rhizobial cells. A total of 167 NCRs were identified in the RNA transcripts. Comparative sequence and electrochemical analysis revealed that A. sinicus NCRs (AsNCRs) are dominated by a unique cationic group (termed subgroup C), whose mature portion is relatively long (>60 amino acids) and phylogenetically distinct and possessing six highly conserved cysteine residues. Subsequent functional characterization showed that a 7653R variant harboring AsNCR083 (a representative of subgroup C AsNCR) displayed significant growth inhibition in laboratory media and formed ineffective white nodules on A. sinicus with irregular symbiosomes. Finally, bacterial two-hybrid analysis led to the identification of GroEL1 and GroEL3 as the molecular targets of AsNCR067 and AsNCR076. Together, our data contribute to a systematic understanding of the NCR repertoire associated with the A. sinicus and M. huakuii symbiosis. [Formula: see text] Copyright © 2022 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license.
Asunto(s)
Cisteína , Fabaceae , Cisteína/química , Cisteína/genética , Cisteína/metabolismo , Fabaceae/microbiología , Nitrógeno/metabolismo , Fijación del Nitrógeno/genética , Péptidos/metabolismo , ARN/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Simbiosis/genética , Transcriptoma/genéticaRESUMEN
The legume-rhizobial symbiosis results in the formation of root nodules that provide an ecological niche for nitrogen-fixing bacteria. However, plant-bacteria genotypic interactions can lead to wide variation in nitrogen fixation efficiency, and it is not uncommon that a bacterial strain forms functional (Fix+) nodules on one plant genotype but nonfunctional (Fix-) nodules on another. Host genetic control of this specificity is unknown. We herein report the cloning of the Medicago truncatula NFS1 gene that regulates the fixation-level incompatibility with the microsymbiont Sinorhizobium meliloti Rm41. We show that NFS1 encodes a nodule-specific cysteine-rich (NCR) peptide. In contrast to the known role of NCR peptides as effectors of endosymbionts' differentiation to nitrogen-fixing bacteroids, we demonstrate that specific NCRs control discrimination against incompatible microsymbionts. NFS1 provokes bacterial cell death and early nodule senescence in an allele-specific and rhizobial strain-specific manner, and its function is dependent on host genetic background.
Asunto(s)
Medicago truncatula , Fijación del Nitrógeno/fisiología , Proteínas de Plantas , Rizoma , Nódulos de las Raíces de las Plantas , Sinorhizobium meliloti/metabolismo , Simbiosis/fisiología , Transaminasas , Medicago truncatula/genética , Medicago truncatula/metabolismo , Medicago truncatula/microbiología , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Rizoma/genética , Rizoma/metabolismo , Rizoma/microbiología , Nódulos de las Raíces de las Plantas/genética , Nódulos de las Raíces de las Plantas/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Transaminasas/genética , Transaminasas/metabolismoRESUMEN
Legumes engage in root nodule symbioses with nitrogen-fixing soil bacteria known as rhizobia. In nodule cells, bacteria are enclosed in membrane-bound vesicles called symbiosomes and differentiate into bacteroids that are capable of converting atmospheric nitrogen into ammonia. Bacteroid differentiation and prolonged intracellular survival are essential for development of functional nodules. However, in the Medicago truncatula-Sinorhizobium meliloti symbiosis, incompatibility between symbiotic partners frequently occurs, leading to the formation of infected nodules defective in nitrogen fixation (Fix-). Here, we report the identification and cloning of the M. truncatula NFS2 gene that regulates this type of specificity pertaining to S. meliloti strain Rm41. We demonstrate that NFS2 encodes a nodule-specific cysteine-rich (NCR) peptide that acts to promote bacterial lysis after differentiation. The negative role of NFS2 in symbiosis is contingent on host genetic background and can be counteracted by other genes encoded by the host. This work extends the paradigm of NCR function to include the negative regulation of symbiotic persistence in host-strain interactions. Our data suggest that NCR peptides are host determinants of symbiotic specificity in M. truncatula and possibly in closely related legumes that form indeterminate nodules in which bacterial symbionts undergo terminal differentiation.
Asunto(s)
Péptidos Catiónicos Antimicrobianos/metabolismo , Bacterias/metabolismo , Medicago truncatula , Fijación del Nitrógeno/fisiología , Proteínas de Plantas/metabolismo , Microbiología del Suelo , Simbiosis/fisiología , Medicago truncatula/metabolismo , Medicago truncatula/microbiologíaRESUMEN
The development and functioning of the nitrogen fixing symbiosis between legume plants and soil bacteria collectively called rhizobia requires continuous chemical dialogue between the partners using different molecules such as flavonoids, lipo-chitooligosaccharides, polysaccharides and peptides. Agrobacterium rhizogenes mediated hairy root transformation of legumes is widely used to study the function of plant genes involved in the process. The identification of transgenic plant tissues is based on antibiotics/herbicide selection and/or the detection of different reporter genes that usually require special equipment such as fluorescent microscopes or destructive techniques and chemicals to visualize enzymatic activity. Here, we developed and efficiently used in hairy root experiments binary vectors containing the MtLAP1 gene driven by constitutive and tissue-specific promoters that facilitate the production of purple colored anthocyanins in transgenic tissues and thus allowing the identification of transformed roots by naked eye. Anthocyanin producing roots were able to establish effective symbiosis with rhizobia. Moreover, it was shown that species-specific allelic variations and a mutation preventing posttranslational acetyl modification of an essential nodule-specific cysteine-rich peptide, NCR169, do not affect the symbiotic interaction of Medicago truncatula cv. Jemalong with Sinorhizobium medicae strain WSM419. Based on the experiments, it could be concluded that it is preferable to use the vectors with tissue-specific promoters that restrict anthocyanin production to the root vasculature for studying biotic interactions of the roots such as symbiotic nitrogen fixation or mycorrhizal symbiosis.
Asunto(s)
Antocianinas/fisiología , Medicago truncatula/fisiología , Fijación del Nitrógeno , Raíces de Plantas/fisiología , Nódulos de las Raíces de las Plantas/crecimiento & desarrollo , Simbiosis , Agrobacterium/genética , Biomarcadores/análisis , Medicago truncatula/genética , Medicago truncatula/crecimiento & desarrollo , Proteínas de Plantas/genética , Proteínas de Plantas/metabolismo , Raíces de Plantas/crecimiento & desarrollo , Raíces de Plantas/microbiología , Plantas Modificadas Genéticamente/genética , Plantas Modificadas Genéticamente/crecimiento & desarrollo , Plantas Modificadas Genéticamente/fisiología , Nódulos de las Raíces de las Plantas/microbiología , Transformación GenéticaRESUMEN
Legume-rhizobium pairs are often observed that produce symbiotic root nodules but fail to fix nitrogen. Using the Sinorhizobium meliloti and Medicago truncatula symbiotic system, we previously described several naturally occurring accessory plasmids capable of disrupting the late stages of nodule development while enhancing bacterial proliferation within the nodule. We report here that host range restriction peptidase (hrrP), a gene found on one of these plasmids, is capable of conferring both these properties. hrrP encodes an M16A family metallopeptidase whose catalytic activity is required for these symbiotic effects. The ability of hrrP to suppress nitrogen fixation is conditioned upon the genotypes of both the host plant and the hrrP-expressing rhizobial strain, suggesting its involvement in symbiotic communication. Purified HrrP protein is capable of degrading a range of nodule-specific cysteine-rich (NCR) peptides encoded by M. truncatula. NCR peptides are crucial signals used by M. truncatula for inducing and maintaining rhizobial differentiation within nodules, as demonstrated in the accompanying article [Horváth B, et al. (2015) Proc Natl Acad Sci USA, 10.1073/pnas.1500777112]. The expression pattern of hrrP and its effects on rhizobial morphology are consistent with the NCR peptide cleavage model. This work points to a symbiotic dialogue involving a complex ensemble of host-derived signaling peptides and bacterial modifier enzymes capable of adjusting signal strength, sometimes with exploitative outcomes.
Asunto(s)
Péptido Hidrolasas/metabolismo , Señales de Clasificación de Proteína , Simbiosis , Datos de Secuencia Molecular , Fijación del Nitrógeno , Péptido Hidrolasas/genética , Proteolisis , Transcripción GenéticaRESUMEN
Contents 411 I. 411 II. 412 III. 412 IV. 413 V. 414 VI. 414 VII. 415 VIII. 415 416 References 416 SUMMARY: Terminal bacteroid differentiation (TBD) is a remarkable case of bacterial cell differentiation that occurs after rhizobia are released intracellularly within plant cells of symbiotic legume organs called nodules. The hallmarks of TBD are cell enlargement, genome amplification and membrane permeabilization. This plant-driven process is governed by a large family of bacteroid-targeted nodule-specific cysteine-rich (NCR) peptides that were until recently thought to be restricted to a specific lineage of the legume family, including the model plant Medicago truncatula. Recently, new plant and bacterial factors involved in TBD have been identified, challenging our view of this phenomenon at mechanistic and evolutionary levels. Here, we review the recent literature and discuss emerging questions about the mechanisms and the role(s) of TBD.
Asunto(s)
Cisteína/metabolismo , Fabaceae/microbiología , Péptidos/metabolismo , Rhizobium/fisiología , Nódulos de las Raíces de las Plantas/microbiología , Simbiosis , Fijación del NitrógenoRESUMEN
The symbiosis of Medicago truncatula with Sinorhizobium meliloti or Sinorhizobium medicae soil bacteria results in the formation of root nodules where bacteria inside the plant cells are irreversibly converted to polyploid, nondividing nitrogen-fixing bacteroids. Bacteroid differentiation is host-controlled and the plant effectors are symbiosis-specific secreted plant peptides. In the M. truncatula genome there are more than 600 symbiotic peptide genes including 500 small genes coding for nodule-specific cysteine-rich (NCR) peptides. While NCR transcripts represent >5% of the nodule transcriptome, the existence of only eight NCR peptides has been demonstrated so far. The predicted NCRs are secreted peptides targeted to the endosymbionts. Correspondingly, all the eight detected peptides were present in the bacteroids. Here, we report on large-scale detection of NCR peptides from nodules and from isolated, semipurified endosymbionts at various stages of their differentiation. In total 138 NCRs were detected in the bacteroids; 38 were cationic while the majority was anionic. The presence of early NCRs in nitrogen-fixing bacteroids indicates their high stability, and their long-term maintenance suggests persisting biological roles in the bacteroids.
Asunto(s)
Medicago truncatula/metabolismo , Medicago truncatula/microbiología , Nódulos de las Raíces de las Plantas/metabolismo , Nódulos de las Raíces de las Plantas/microbiología , Sinorhizobium meliloti/fisiología , Regulación de la Expresión Génica de las Plantas/genética , SimbiosisRESUMEN
Nodule cysteine-rich (NCR) peptides have a major role in the differentiation of endocytosed bacteria into nitrogen-fixing bacteroids. A recent paper by Sankari et al. indicates that NCR247 is essential for the uptake of iron, a mineral nutrient required for nitrogenase activity. Furthermore, the special ability of NCR247 to sequester haem suggests potential applications for human health.
Asunto(s)
Rhizobium , Humanos , Hierro , Simbiosis , Péptidos/metabolismo , Bacterias/metabolismo , Fijación del NitrógenoRESUMEN
Host/symbiont compatibility is a hallmark of the symbiotic nitrogen-fixing interaction between rhizobia and legumes, mediated in part by plant-produced nodule-specific cysteine-rich (NCR) peptides and the bacterial BacA membrane protein that can act as a NCR peptide transporter. In addition, the genetic and metabolic properties supporting symbiotic nitrogen fixation often differ between compatible partners, including those sharing a common partner, highlighting the need for multiple study systems. Here, we report high-quality nodule transcriptome assemblies for Medicago sativa cv. Algonquin and Melilotus officinalis, two legumes able to form compatible symbioses with Sinorhizobium meliloti. The compressed M. sativa and M. officinalis assemblies consisted of 79,978 and 64,593 contigs, respectively, of which 33,341 and 28,278 were assigned putative annotations, respectively. As expected, the two transcriptomes showed broad similarity at a global level. We were particularly interested in the NCR peptide profiles of these plants, as these peptides drive bacterial differentiation during the symbiosis. A total of 412 and 308 NCR peptides were predicted from the M. sativa and M. officinalis transcriptomes, respectively, with approximately 9% of the transcriptome of both species consisting of NCR transcripts. Notably, transcripts encoding highly cationic NCR peptides (isoelectric point > 9.5), which are known to have antimicrobial properties, were â¼2-fold more abundant in M. sativa than in M. officinalis, and â¼27-fold more abundant when considering only NCR peptides in the six-cysteine class. We hypothesize that the difference in abundance of highly cationic NCR peptides explains our previous observation that some rhizobial bacA alleles which can support symbiosis with M. officinalis are unable to support symbiosis with M. sativa.
RESUMEN
Antimicrobial peptides are prominent components of the plant immune system acting against a wide variety of pathogens. Legume plants from the inverted repeat lacking clade (IRLC) have evolved a unique gene family encoding nodule-specific cysteine-rich NCR peptides acting in the symbiotic cells of root nodules, where they convert their bacterial endosymbionts into non-cultivable, polyploid nitrogen-fixing cells. NCRs are usually 30-50 amino acids long peptides having a characteristic pattern of 4 or 6 cysteines and highly divergent amino acid composition. While the function of NCRs is largely unknown, antimicrobial activity has been demonstrated for a few cationic Medicago truncatula NCR peptides against bacterial and fungal pathogens. The advantages of these plant peptides are their broad antimicrobial spectrum, fast killing modes of actions, multiple bacterial targets, and low propensity to develop resistance to them and no or low cytotoxicity to human cells. In the IRLC legumes, the number of NCR genes varies from a few to several hundred and it is possible that altogether hundreds of thousands of different NCR peptides exist. Due to the need for new antimicrobial agents, we investigated the antimicrobial potential of 104 synthetic NCR peptides from M. truncatula, M. sativa, Pisum sativum, Galega orientalis and Cicer arietinum against eight human pathogens, including ESKAPE bacteria. 50 NCRs showed antimicrobial activity with differences in the antimicrobial spectrum and effectivity. The most active peptides eliminated bacteria at concentrations from 0.8 to 3.1 µM. High isoelectric point and positive net charge were important but not the only determinants of their antimicrobial activity. Testing the activity of shorter peptide derivatives against Acinetobacter baumannii and Candida albicans led to identification of regions responsible for the antimicrobial activity and provided insight into their potential modes of action. This work provides highly potent lead molecules without hemolytic activity on human blood cells for novel antimicrobial drugs to fight against pathogens.
RESUMEN
Various legume plants form root nodules in which symbiotic bacteria (rhizobia) fix atmospheric nitrogen after differentiation into a symbiotic form named bacteroids. In some legume species, bacteroid differentiation is promoted by defensin-like nodule-specific cysteine-rich (NCR) peptides. NCR peptides have best been studied in the model legume Medicago truncatula Gaertn., while in many other legumes relevant information is still fragmentary. Here, we characterize the NCR gene family in pea (Pisum sativum L.) using genomic and transcriptomic data. We found 360 genes encoding NCR peptides that are expressed in nodules. The sequences of pea NCR genes and putative peptides are highly variable and differ significantly from NCR sequences of M. truncatula. Indeed, only one pair of orthologs (PsNCR47-MtNCR312) has been identified. The NCR genes in the pea genome are located in clusters, and the expression patterns of NCR genes from one cluster tend to be similar. These data support the idea of independent evolution of NCR genes by duplication and diversification in related legume species. We also described spatiotemporal expression profiles of NCRs and identified specific transcription factor (TF) binding sites in promoters of "early" and "late" NCR genes. Further, we studied the expression of NCR genes in nodules of Fix- mutants and predicted potential regulators of NCR gene expression, one among them being the TF ERN1 involved in the early steps of nodule organogenesis. In general, this study contributes to understanding the functions of NCRs in legume nodules and contributes to understanding the diversity and potential antibiotic properties of pea nodule-specific antimicrobial molecules.
RESUMEN
Symbiotic nitrogen fixation (SNF) has a high energetic cost for legume plants; legumes thus reduce SNF when soil N is available. The present study aimed to increase our understanding regarding the impacts of the two principal forms of available N in soils (ammonium and nitrate) on SNF. We continuously measured the SNF of Medicago truncatula under controlled conditions. This permitted nodule sampling for comparative transcriptome profiling at points connected to the nodules' reaction following ammonium or nitrate applications. The N component of both ions systemically induced a rhythmic pattern of SNF, while the activity in control plants remained constant. This rhythmic activity reduced the per-day SNF. The nitrate ion had additional local effects; the more pronounced were a strong downregulation of leghaemoglobin, nodule cysteine-rich (NCR) peptides and nodule-enhanced nicotianamine synthase (neNAS). The neNAS has proven to be of importance for nodule functioning. Although other physiological impacts of nitrate on nodules were observed (e.g. nitrosylation of leghaemoglobin), the main effect was a rapid ion-specific and organ-specific change in gene expression levels. Contrastingly, during the first hours after ammonium applications, the transcriptome remained virtually unaffected. Therefore, nitrate-induced genes could be key for increasing the nitrate tolerance of SNF.
Asunto(s)
Compuestos de Amonio/metabolismo , Medicago truncatula/fisiología , Nitratos/metabolismo , Fijación del Nitrógeno , Nódulos de las Raíces de las Plantas/fisiología , Simbiosis , Medicago truncatula/microbiología , Nódulos de las Raíces de las Plantas/microbiologíaRESUMEN
Legume-rhizobium symbiosis represents one of the most successfully co-evolved mutualisms. Within nodules, the bacterial cells undergo distinct metabolic and morphological changes and differentiate into nitrogen-fixing bacteroids. Legumes in the inverted repeat lacking clade (IRLC) employ an array of defensin-like small secreted peptides (SSPs), known as nodule-specific cysteine-rich (NCR) peptides, to regulate bacteroid differentiation and activity. While most NCRs exhibit bactericidal effects in vitro, studies confirm that inside nodules they target the bacterial cell cycle and other cellular pathways to control and extend rhizobial differentiation into an irreversible (or terminal) state where the host gains control over bacteroids. While NCRs are well established as positive regulators of effective symbiosis, more recent findings also suggest that NCRs affect partner compatibility. The extent of bacterial differentiation has been linked to species-specific size and complexity of the NCR gene family that varies even among closely related species, suggesting a more recent origin of NCRs followed by rapid expansion in certain species. NCRs have diversified functionally, as well as in their expression patterns and responsiveness, likely driving further functional specialisation. In this review, we evaluate the functions of NCR peptides and their role as a driving force underlying the outcome of rhizobial symbiosis, where the plant is able to determine the outcome of rhizobial interaction in a temporal and spatial manner.
Asunto(s)
Cisteína/química , Medicago truncatula/metabolismo , Fragmentos de Péptidos/metabolismo , Proteínas de Plantas/metabolismo , Rhizobium/fisiología , Nódulos de las Raíces de las Plantas/metabolismo , Simbiosis , Regulación de la Expresión Génica de las Plantas , Medicago truncatula/microbiología , Nódulos de las Raíces de las Plantas/microbiologíaRESUMEN
The symbiosis specific NCR247 and NCR335 cationic plant peptides of Medicago truncatula have been shown to exert antimicrobial activity against a wide range of microbes. However, their antimicrobial efficiency is clearly limited by divalent cations. Here, the antibacterial and antifungal activities of NCR247 and NCR335 peptides were compared to those of the well-characterized peptide antibiotics polymyxin B and the aminoglycoside streptomycin on three model microbes, Escherichia coli, Bacillus subtilis and Saccharomyces cerevisiae as representatives of Gram-negative and Gram-positive bacteria as well as eukaryotic fungi. The aim of the study was to assess how the killing efficiency of these peptides depends on various, widely used antimicrobial susceptibility assays. Validated resazurin microdilution assay was used to determine minimal growth inhibitory concentrations in three general test media (MHB, MHBII and low-salt medium LSM). Bactericidal/fungicidal activities were determined by the commonly used drop plate assay. The natural plant peptides showed distinct characteristics, NCR247 had a generally high sensitivity for Ca2+ and Mg2+ in the medium, while NCR335 proved to be a robust and strong antimicrobial agent with comparable efficiency values to polymyxin B. Activity data were confirmed visually, both NCR247 and NCR335 treatments at minimal bactericidal concentrations induced complete disruption of the membranes and provoked cell lysis on all tested microorganisms as observed by scanning electron microscopy.