Nodule-specific Cysteine-rich Peptides Research Articles

Plant-Derived, Nodule-Specific Cysteine-Rich Peptides as a Novel Source of Biopesticides for Controlling Citrus Greening Disease.

Nodule-specific cysteine-rich (NCR) peptides, encoded in the genome of the Mediterranean legume Medicago truncatula (barrelclover), are known to regulate plant-microbe interactions. A subset of computationally derived 20-mer peptide fragments from 182 NCR peptides was synthesized to identify those with activity against the unculturable vascular pathogen associated with citrus greening disease, 'Candidatus Liberibacter asiaticus' (CLas). Grounded in a design of experiments framework, we evaluated the peptides in a screening pipeline involving three distinct assays: a bacterial culture assay with Liberibacter crescens, a CLas-infected excised citrus leaf assay, and an assay to evaluate effects on bacterial acquisition by the nymphal stage of hemipteran vector Diaphorina citri. A subset of the 20-mer NCR peptide fragments inhibits both CLas growth in citrus leaves and CLas acquisition by D. citri. Two peptides induced higher levels of D. citri mortality. These findings reveal 20-mer NCR peptides as a new class of plant-derived biopesticide molecules to control citrus greening disease.

Targeted mutagenesis of Medicago truncatula Nodule-specific Cysteine-Rich (NCR) genes using the Agrobacterium rhizogenes-mediated CRISPR/Cas9 system

The host-produced nodule specific cysteine-rich (NCR) peptides control the terminal differentiation of endosymbiotic rhizobia in the nodules of IRLC legumes. Although the Medicago truncatula genome encodes about 700 NCR peptides, only few of them have been proven to be crucial for nitrogen-fixing symbiosis. In this study, we applied the CRISPR/Cas9 gene editing technology to generate knockout mutants of NCR genes for which no genetic or functional data were previously available. We have developed a workflow to analyse the mutation and the symbiotic phenotype of individual nodules formed on Agrobacterium rhizogenes-mediated transgenic hairy roots. The selected NCR genes were successfully edited by the CRISPR/Cas9 system and nodules formed on knockout hairy roots showed wild type phenotype indicating that peptides NCR068, NCR089, NCR128 and NCR161 are not essential for symbiosis between M. truncatula Jemalong and Sinorhizobium medicae WSM419. We regenerated stable mutants edited for the NCR068 from hairy roots obtained by A. rhizogenes-mediated transformation. The analysis of the symbiotic phenotype of stable ncr068 mutants showed that peptide NCR068 is not required for symbiosis with S. meliloti strains 2011 and FSM-MA either. Our study reports that gene editing can help to elicit the role of certain NCRs in symbiotic nitrogen fixation.

Nodule-specific cysteine-rich peptide 343 is required for symbiotic nitrogen fixation in Medicago truncatula.

Symbiotic interactions between legumes and rhizobia lead to the development of root nodules and nitrogen fixation by differentiated bacteroids within nodules. Differentiation of the endosymbionts is reversible or terminal, determined by plant effectors. In inverted repeat lacking clade legumes, nodule-specific cysteine-rich (NCR) peptides control the terminal differentiation of bacteroids. Medicago truncatula contains ∼700 NCR-coding genes. However, the role of few NCR peptides has been demonstrated. Here, we report characterization of fast neutron 2106 (FN2106), a symbiotic nitrogen fixation defective (fix-) mutant of M. truncatula. Using a transcript-based approach, together with linkage and complementation tests, we showed that loss-of-function of NCR343 results in impaired bacteroid differentiation and/or maintenance and premature nodule senescence of the FN2106 mutant. NCR343 was specifically expressed in nodules. Subcellular localization studies showed that the functional NCR343-YFP fusion protein colocalizes with bacteroids in symbiosomes in infected nodule cells. Transcriptomic analyses identified senescence-, but not defense-related genes, as being significantly upregulated in ncr343 (FN2106) nodules. Taken together, results from our phenotypic and transcriptomic analyses of a loss-of-function ncr343 mutant demonstrate an essential role of NCR343 in bacteroid differentiation and/or maintenance required for symbiotic nitrogen fixation.

The Medicago truncatula nodule-specific cysteine-rich peptides, NCR343 and NCR-new35 are required for the maintenance of rhizobia in nitrogen-fixing nodules.

In the nodules of IRLC legumes, including Medicago truncatula, nitrogen-fixing rhizobia undergo terminal differentiation resulting in elongated and endoreduplicated bacteroids specialized for nitrogen fixation. This irreversible transition of rhizobia is mediated by host produced nodule-specific cysteine-rich (NCR) peptides, of which c. 700 are encoded in the M.truncatula genome but only few of them have been proved to be essential for nitrogen fixation. We carried out the characterization of the nodulation phenotype of three ineffective nitrogen-fixing M. truncatula mutants using confocal and electron microscopy, monitored the expression of defence and senescence-related marker genes, and analysed the bacteroid differentiation with flow cytometry. Genetic mapping combined with microarray- or transcriptome-based cloning was used to identify the impaired genes. Mtsym19 and Mtsym20 mutants are defective in the same peptide NCR-new35 and the lack of NCR343 is responsible for the ineffective symbiosis of NF-FN9363. We found that theexpression of NCR-new35 is significantly lower and limited to the transition zone of the nodule compared with other crucial NCRs. The fluorescent protein-tagged version of NCR343 and NCR-new35 localized to the symbiotic compartment. Our discovery added two additional members to the group of NCR genes essential for nitrogen-fixing symbiosis in M. truncatula.

Enhanced Antibacterial Activity of Substituted Derivatives of NCR169C Peptide.

Medicago truncatula in symbiosis with its rhizobial bacterium partner produces more than 700 nodule-specific cysteine-rich (NCR) peptides with diverse physicochemical properties. Most of the cationic NCR peptides have antimicrobial activity and the potential to tackle antimicrobial resistance with their novel modes of action. This work focuses on the antibacterial activity of the NCR169 peptide derivatives as we previously demonstrated that the C-terminal sequence of NCR169 (NCR169C17-38) has antifungal activity, affecting the viability, morphology, and biofilm formation of various Candida species. Here, we show that NCR169C17-38 and its various substituted derivatives are also able to kill ESKAPE pathogens such as Enterococcus faecalis, Staphylococcus aureus, Klebsiella pneumoniae, Acinetobacter baumannii, Pseudomonas aeruginosa, and Escherichia coli. The replacement of the two cysteines with serines enhanced the antimicrobial activity against most of the tested bacteria, indicating that the formation of a disulfide bridge is not required. As tryptophan can play role in the interaction with bacterial membranes and thus in antibacterial activity, we replaced the tryptophans in the NCR169C17-38C12,17/S sequence with various modified tryptophans, namely 5-methyl tryptophan, 5-fluoro tryptophan, 6-fluoro tryptophan, 7-aza tryptophan, and 5-methoxy tryptophan, in the synthesis of NCR169C17-38C12,17/S analogs. The results demonstrate that the presence of modified fluorotryptophans can significantly enhance the antimicrobial activity without notable hemolytic effect, and this finding could be beneficial for the further development of new AMPs from the members of the NCR peptide family.

Fine-Tuned Immune Antagonism and Nodule-Specific Cysteine-Rich Peptides Govern the Symbiotic Specificity Between Alfalfa Cultivars and Ensifer meliloti

Alfalfa expresses significantly distinct sets of genes in response to infection with different rhizobial strains at the below species level (i.e., biotype or strain). However, differences in the transcriptomic profiles of two alfalfa cultivars nodulated by a single rhizobium strain have been largely unexamined. In this study, comparative RNA-seq analysis of two alfalfa cultivars, Medicago sativa cv. Gannong No. 3 (G3) and cv. Gannong No. 9 (G9) inoculated with one Ensifer meliloti strain LL2, with varying symbiotic performance, was conducted, followed by hub gene interaction network construction based on weighted gene co-expression network analysis (WGCNA). The G9-LL2 symbiotic system showed better nodule formation, nitrogen fixation, and growth characteristics than the G3-LL2 system. Compared with the non-inoculated control, the LL2-inoculated G9 plants (10,053) produced more differentially expressed genes (DEGs) than the LL2-inoculated G3 plants (7112). A group of 227 genes displayed completely distinguished expression in G9 (6.63 < log2(FC) < 15.45) and G3 (‒ 3.05 < log2(FC) < 12.05), which are primarily involved in encoding nodule-specific cysteine-rich peptides (NCRs), nodulin, and leghemoglobin. Although genes with predicted roles in nitrogen metabolism were primarily upregulated and almost all of those in ubiquitin-mediated proteolysis and plant–pathogen interaction were suppressed, interestingly, a consistently higher expression level measured by log2(FC) was observed in G9 plants. Hub gene interaction networks showed that NCRs, late nodulin, and genes related to plant immunity (TIR-NBS-LRR, defensin, thioredoxin, thionine, and polygalacturonase) regulate other genes at the source node positions. After successful initiation of nodulation in both alfalfa cultivars G3 and G9 by E. meliloti strain LL2, G9 achieved preferable outcomes of rhizobia–alfalfa symbiosis by equilibrating the antagonism and compatibility of plant immunity. It elevated PTI, suppressed defense and ETI, and enhanced nitrogen fixation and utilization efficiency by inducing the expression of genes encoding NIN, NFH1, LysM-RLK, LRP, NCRs, nodulin, and leghemoglobin. Hub genes were predominantly associated with highly specific rhizobia–alfalfa symbiosis positively governed by NCRs and fine-tuned immune antagonism, comprising NCRs, late nodulin, and TIR-NBS-LRR. These findings provide insights into the genetic mechanisms underlying the modification and efficient utilization of semi-compatible and incompatible rhizobial resources.

A variable gene family encoding nodule-specific cysteine-rich peptides in pea (Pisum sativum L.)

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.

Legume NCRs and nodule-specific defensins of actinorhizal plants—Do they share a common origin?

The actinorhizal plant Datisca glomerata (Datiscaceae, Cucurbitales) establishes a root nodule symbiosis with actinobacteria from the earliest branching symbiotic Frankia clade. A subfamily of a gene family encoding nodule-specific defensin-like cysteine-rich peptides is highly expressed in D. glomerata nodules. Phylogenetic analysis of the defensin domain showed that these defensin-like peptides share a common evolutionary origin with nodule-specific defensins from actinorhizal Fagales and with nodule-specific cysteine-rich peptides (NCRs) from legumes. In this study, the family member with the highest expression levels, DgDef1, was characterized. Promoter-GUS studies on transgenic hairy roots showed expression in the early stage of differentiation of infected cells, and transient expression in the nodule apex. DgDef1 contains an N-terminal signal peptide and a C-terminal acidic domain which are likely involved in subcellular targeting and do not affect peptide activity. In vitro studies with E. coli and Sinorhizobium meliloti 1021 showed that the defensin domain of DgDef1 has a cytotoxic effect, leading to membrane disruption with 50% lethality for S. meliloti 1021 at 20.8 μM. Analysis of the S. meliloti 1021 transcriptome showed that, at sublethal concentrations, DgDef1 induced the expression of terminal quinol oxidases, which are associated with the oxidative stress response and are also expressed during symbiosis. Overall, the changes induced by DgDef1 are reminiscent of those of some legume NCRs, suggesting that nodule-specific defensin-like peptides were part of the original root nodule toolkit and were subsequently lost in most symbiotic legumes, while being maintained in the actinorhizal lineages.

Reference nodule transcriptomes for Melilotus officinalis and Medicago sativa cv. Algonquin.

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 ∼2‐fold more abundant in M. sativa than in M. officinalis , and ∼27‐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 .

Negotiating mutualism: A locus for exploitation by rhizobia has a broad effect size distribution and context-dependent effects on legume hosts.

In mutualisms, variation at genes determining partner fitness provides the raw material upon which coevolutionary selection acts, setting the dynamics and pace of coevolution. However, we know little about variation in the effects of genes that underlie symbiotic fitness in natural mutualist populations. In some species of legumes that form root nodule symbioses with nitrogen‐fixing rhizobial bacteria, hosts secrete nodule‐specific cysteine‐rich (NCR) peptides that cause rhizobia to differentiate in the nodule environment. However, rhizobia can cleave NCR peptides through the expression of genes like the plasmid‐borne Host range restriction peptidase (hrrP), whose product degrades specific NCR peptides. Although hrrP activity can confer host exploitation by depressing host fitness and enhancing symbiont fitness, the effects of hrrP on symbiosis phenotypes depend strongly on the genotypes of the interacting partners. However, the effects of hrrP have yet to be characterised in a natural population context, so its contribution to variation in wild mutualist populations is unknown. To understand the distribution of effects of hrrP in wild rhizobia, we measured mutualism phenotypes conferred by hrrP in 12 wild Ensifer medicae strains. To evaluate context dependency of hrrP effects, we compared hrrP effects across two Medicago polymorpha host genotypes and across two experimental years for five E. medicae strains. We show for the first time in a natural population context that hrrP has a wide distribution of effect sizes for many mutualism traits, ranging from strongly positive to strongly negative. Furthermore, we show that hrrP effect size varies across host genotypes and experiment years, suggesting that researchers should be cautious about extrapolating the role of genes in natural populations from controlled laboratory studies of single genetic variants.

The Genome of the Acid Soil-Adapted Strain Rhizobium favelukesii OR191 Encodes Determinants for Effective Symbiotic Interaction With Both an Inverted Repeat Lacking Clade and a Phaseoloid Legume Host.

Although Medicago sativa forms highly effective symbioses with the comparatively acid-sensitive genus Ensifer, its introduction into acid soils appears to have selected for symbiotic interactions with acid-tolerant R. favelukesii strains. Rhizobium favelukesii has the unusual ability of being able to nodulate and fix nitrogen, albeit sub-optimally, not only with M. sativa but also with the promiscuous host Phaseolus vulgaris. Here we describe the genome of R. favelukesii OR191 and genomic features important for the symbiotic interaction with both of these hosts. The OR191 draft genome contained acid adaptation loci, including the highly acid-inducible lpiA/acvB operon and olsC, required for production of lysine- and ornithine-containing membrane lipids, respectively. The olsC gene was also present in other acid-tolerant Rhizobium strains but absent from the more acid-sensitive Ensifer microsymbionts. The OR191 symbiotic genes were in general more closely related to those found in Medicago microsymbionts. OR191 contained the nodA, nodEF, nodHPQ, and nodL genes for synthesis of polyunsaturated, sulfated and acetylated Nod factors that are important for symbiosis with Medicago, but contained a truncated nodG, which may decrease nodulation efficiency with M. sativa. OR191 contained an E. meliloti type BacA, which has been shown to specifically protect Ensifer microsymbionts from Medicago nodule-specific cysteine-rich peptides. The nitrogen fixation genes nifQWZS were present in OR191 and P. vulgaris microsymbionts but absent from E. meliloti-Medicago microsymbionts. The ability of OR191 to nodulate and fix nitrogen symbiotically with P. vulgaris indicates that this host has less stringent requirements for nodulation than M. sativa but may need rhizobial strains that possess nifQWZS for N2-fixation to occur. OR191 possessed the exo genes required for the biosynthesis of succinoglycan, which is required for the Ensifer-Medicago symbiosis. However, 1H-NMR spectra revealed that, in the conditions tested, OR191 exopolysaccharide did not contain a succinyl substituent but instead contained a 3-hydroxybutyrate moiety, which may affect its symbiotic performance with Medicago hosts. These findings provide a foundation for the genetic basis of nodulation requirements and symbiotic effectiveness with different hosts.

Microscopic and Transcriptomic Analyses of Dalbergoid Legume Peanut Reveal a Divergent Evolution Leading to Nod-Factor-Dependent Epidermal Crack-Entry and Terminal Bacteroid Differentiation.

Root nodule symbiosis (RNS) is the pillar behind sustainable agriculture and plays a pivotal role in the environmental nitrogen cycle. Most of the genetic, molecular, and cell-biological knowledge on RNS comes from model legumes that exhibit a root-hair mode of bacterial infection, in contrast to the Dalbergoid legumes exhibiting crack-entry of rhizobia. As a step toward understanding this important group of legumes, we have combined microscopic analysis and temporal transcriptome to obtain a dynamic view of plant gene expression during Arachis hypogaea (peanut) nodule development. We generated comprehensive transcriptome data by mapping the reads to A. hypogaea, and two diploid progenitor genomes. Additionally, we performed BLAST searches to identify nodule-induced yet-to-be annotated peanut genes. Comparison between peanut, Medicago truncatula, Lotus japonicus, and Glycine max showed upregulation of 61 peanut orthologs among 111 tested known RNS-related genes, indicating conservation in mechanisms of nodule development among members of the Papilionoid family. Unlike model legumes, recruitment of class 1 phytoglobin-derived symbiotic hemoglobin (SymH) in peanut indicates diversification of oxygen-scavenging mechanisms in the Papilionoid family. Finally, the absence of cysteine-rich motif-1-containing nodule-specific cysteine-rich peptide (NCR) genes but the recruitment of defensin-like NCRs suggest a diverse molecular mechanism of terminal bacteroid differentiation. In summary, our work describes genetic conservation and diversification in legume-rhizobia symbiosis in the Papilionoid family, as well as among members of the Dalbergoid legumes.[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.

Plant circadian clock control of Medicago truncatula nodulation via regulation of nodule cysteine-rich peptides.

Legumes house nitrogen-fixing endosymbiotic rhizobia in specialized polyploid cells within root nodules, which undergo tightly regulated metabolic activity. By carrying out expression analysis of transcripts over time in Medicago truncatula nodules, we found that the circadian clock enables coordinated control of metabolic and regulatory processes linked to nitrogen fixation. This involves the circadian clock-associated transcription factor LATE ELONGATED HYPOCOTYL (LHY), with lhy mutants being affected in nodulation. Rhythmic transcripts in root nodules include a subset of nodule-specific cysteine-rich peptides (NCRs) that have the LHY-bound conserved evening element in their promoters. Until now, studies have suggested that NCRs act to regulate bacteroid differentiation and keep the rhizobial population in check. However, these conclusions came from the study of a few members of this very large gene family that has complex diversified spatio-temporal expression. We suggest that rhythmic expression of NCRs may be important for temporal coordination of bacterial activity with the rhythms of the plant host, in order to ensure optimal symbiosis.

A conserved rhizobial peptidase that interacts with host-derived symbiotic peptides

In the Medicago truncatula-Sinorhizobium meliloti symbiosis, chemical signaling initiates rhizobial infection of root nodule tissue, where a large portion of the bacteria are endocytosed into root nodule cells to function in nitrogen-fixing organelles. These intracellular bacteria are subjected to an arsenal of plant-derived nodule-specific cysteine-rich (NCR) peptides, which induce the physiological changes that accompany nitrogen fixation. NCR peptides drive these intracellular bacteria toward terminal differentiation. The bacterial peptidase HrrP was previously shown to degrade host-derived NCR peptides and give the bacterial symbionts greater fitness at the expense of host fitness. The hrrP gene is found in roughly 10% of Sinorhizobium isolates, as it is carried on an accessory plasmid. The objective of the present study is to identify peptidase genes in the core genome of S. meliloti that modulate symbiotic outcome in a manner similar to the accessory hrrP gene. In an overexpression screen of annotated peptidase genes, we identified one such symbiosis-associated peptidase (sap) gene, sapA (SMc00451). When overexpressed, sapA leads to a significant decrease in plant fitness. Its promoter is active in root nodules, with only weak expression evident under free-living conditions. The SapA enzyme can degrade a broad range of NCR peptides in vitro.

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

A 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.

Symbiotic NCR Peptide Fragments Affect the Viability, Morphology and Biofilm Formation of Candida Species.

The increasing rate of fungal infections causes global problems not only in human healthcare but agriculture as well. To combat fungal pathogens limited numbers of antifungal agents are available therefore alternative drugs are needed. Antimicrobial peptides are potent candidates because of their broad activity spectrum and their diverse mode of actions. The model legume Medicago truncatula produces >700 nodule specific cysteine-rich (NCR) peptides in symbiosis and many of them have in vitro antimicrobial activities without considerable toxicity on human cells. In this work we demonstrate the anticandidal activity of the NCR335 and NCR169 peptide derivatives against five Candida species by using the micro-dilution method, measuring inhibition of biofilm formation with the XTT (2,3-Bis-(2-Methoxy-4-Nitro-5-Sulfophenyl)-2H-Tetrazolium-5-Carboxanilide) assay, and assessing the morphological change of dimorphic Candida species by microscopy. We show that both the N- and C-terminal regions of NCR335 possess anticandidal activity as well as the C-terminal sequence of NCR169. The active peptides inhibit biofilm formation and the yeast-hypha transformation. Combined treatment of C. auris with peptides and fluconazole revealed synergistic interactions and reduced 2-8-fold the minimal inhibitory concentrations. Our results demonstrate that shortening NCR peptides can even enhance and broaden their anticandidal activity and therapeutic potential.

Investigating the Involvement of Cytoskeletal Proteins MreB and FtsZ in the Origin of Legume-Rhizobial Symbiosis

Rhizobia are rod-shaped bacteria that form nitrogen-fixing root nodules on leguminous plants, but they don't carry MreB, a key determinant of rod-like cell shape. Here, we introduced an actin-like mreB homologue from pseudomonad into Mesorhizobium huakuii 7653R (a microsymbiont of Astragalus sinicus L.) and examined the molecular, cellular and symbiotic phenotypes of the resultant mutant. Exogenous mreB caused an enlarged cell size and slower growth in laboratory medium. However, the mutant formed small ineffective nodules on A. sinicus (Nod+ Fix-), and rhizobial cells in the infection zone were unable to differentiate into bacteroids. RNA-seq analysis also revealed minor effects of mreB on global gene expression in free-living cells but larger effects for cells grown in planta. Differentially expressed nodule-specific genes include cell cycle regulators such as the tubulin-like ftsZ1 and ftsZ2. Unlike the ubiquitous FtsZ1, FtsZ2 was commonly found in Rhizobium, Sinorhizobium and Mesorhizobium, but not in closely related non-symbiotic species. Bacterial Two-Hybrid analysis revealed that MreB interacts with FtsZ1 and FtsZ2, which are targeted by the host-derived NCR peptides. Significantly, MreB mutation D283A disrupted the protein-protein interactions and restored the aforementioned phenotypic defects caused by MreB in M. huakuii. Together, our data indicate that MreB is detrimental for modern rhizobia and its interaction with FtsZ1/FtsZ2 cause the symbiotic process to cease at the late stage of bacteroid differentiation. These findings led to a hypothesis that loss of mreB in the common ancestor of Rhizobiales and subsequent acquisition of ftsZ2 are critical evolutionary steps leading to legume-rhizobial symbiosis.

Proteome Analysis Reveals a Significant Host-Specific Response in Rhizobium leguminosarum bv. viciae Endosymbiotic Cells

AbstractThe 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.

Antifungal symbiotic peptide NCR044 exhibits unique structure and multifaceted mechanisms of action that confer plant protection

In the indeterminate nodules of a model legume Medicago truncatula, ∼700 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 β-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.

Symbiotic Outcome Modified by the Diversification from 7 to over 700 Nodule-Specific Cysteine-Rich Peptides.

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.