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Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules
... NCR035, NCR055, NCR211, and NCR247, display some inhibitory effect on the proliferation of S. meliloti 2,9,15 . However, NCR peptides do not actually kill rhizobia during the symbiotic process; therefore, their real concentrations in planta must be lower than those in in vitro experimental conditions 16,17 . To date, it remains unclear how the antimicrobial activity of NCR peptides contributes to bacteroid differentiation 18 . ...
... It has been reported that physiologically active plant defensin with disulfide bonds can be expressed in E. coli as a fusion protein with thioredoxin 24 . Thus, we prepared recombinant M. truncatula NCR169 according to the protocol because NCR peptides have been classified as a "defensin-like" family 16 . After successful expression of the fusion protein and cleavage of the thioredoxin-tag, high-performance liquid chromatography (HPLC) purification unexpectedly resulted in two peaks (major and minor), both of which showed the mass of the NCR169 oxidized form (NCR169-ox, Fig. 1A,B). ...
... At present, the significance of the weak antimicrobial activity of NCR169 against S. meliloti in planta remains unclear. Since the concentration of NCR peptides in planta is believed to be very low compared to that in in vitro assays 16,17 , it is likely that NCR169 has a bacteriostatic action, rather than bactericidal, against rhizobia. The weak antimicrobial activity of NCR169 might be a factor that regulates the differentiation time of rhizobia. ...
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.
... Medicago truncatula with its small genome of 375 million base pairs (Mb) and relatively fast generation time is an intensively investigated model legume plant for functional genomics studies on plant development, abiotic stress, and plant-microbe interactions, in particular, rhizobial and mycorrhizal funguslegume symbiosis (Young et al. 2011). Its genome encodes 63 classical defensins each containing a tetradisulfide array and JWST955-c5-2-3-4 JWST955-de-Brujin June 19, 2019 11:14 Printer Name: Trim: 279mm × 216mm over 700 nodule-specific defensin-like peptides (Maróti et al. 2015). In this review, we describe the defensin gene family in this plant. ...
... Outside the eight conserved cysteines, considerable divergence was observed between defensins of each class. Since the completion of the M. truncatula genome sequence in 2011, a total of 63 genes with a potential to encode 83 unique defensins have now been identified (Maróti et al. 2015). A few of these genes are likely to be differentially spliced giving rise to more than one defensin peptide from a single gene. ...
... A few of these genes are likely to be differentially spliced giving rise to more than one defensin peptide from a single gene. A phylogenetic analysis of 27 defensins known to be expressed in various organs has been reported (Maróti et al. 2015). This analysis has revealed the presence of 8 anionic nodule-specific defensins present on a clade different from the ones containing the 19 cationic antifungal defensins (Maróti et al. 2015). ...
Abstract Plant defensins are cysteine‐rich peptides expressed ubiquitously in the plant kingdom. They are major players in the defense arsenal of plants against fungal and oomycete pathogens. A family of more than 50 defensin genes is present in the genome of the model legume Medicago truncatula indicating a rich diversity of variants. Defensin peptides encoded by this gene are structurally similar containing a conserved cysteine‐stabilized α/β motif, but differ in their amino acid sequences, net charge, and hydrophobicity. The antifungal activity of two members of the defensin family has been studied. These defensins exhibit potent antifungal activity at micromolar concentrations against several fungal pathogens tested. The sequence motifs governing their antifungal activity have been identified. The studies conducted during the past decade have revealed that they exhibit different modes of antifungal action. Their constitutive expression in transgenic plants provides resistance to fungal and oomycete pathogens. In this review, we describe recent advances and offer future perspectives on the utility of these peptides for engineering durable commercially useful disease resistance in transgenic crops.
... Protein sequences encoded by genes detected in nodules across all analyzed samples were inspected using a custom Python script searching conservative NCR-like structure according to Maróti et al. [98] and length < 150 bp. Sequences identified as NCR-like were merged with those detected as DE. ...
... Protein sequences of 37,415 genes detected in nodules across all samples were inspected for NCR-like structure [98]. This structure, with four or six conserved cysteines, was found in 33 sequences with a length of <150 bp. ...
Commonly studied in the context of legume–rhizobia symbiosis, biological nitrogen fixation (BNF) is a key component of the nitrogen cycle in nature. Despite its potential in plant breeding and many years of research, information is still lacking as to the regulation of hundreds of genes connected with plant–bacteria interaction, nodulation, and nitrogen fixation. Here, we compared root nodule transcriptomes of red clover (Trifolium pratense L.) genotypes with contrasting nitrogen fixation efficiency, and we found 491 differentially expressed genes (DEGs) between plants with high and low BNF efficiency. The annotation of genes expressed in nodules revealed more than 800 genes not yet experimentally confirmed. Among genes mediating nodule development, four nod-ule-specific cysteine-rich (NCR) peptides were confirmed in the nodule transcriptome. Gene duplication analyses revealed that genes originating from tandem and dispersed duplication are significantly over-represented among DEGs. Weighted correlation network analysis (WGCNA) organized expression profiles of the transcripts into 16 modules linked to the analyzed traits, such as nitrogen fixation efficiency or sample-specific modules. Overall, the results obtained broaden our knowledge about transcriptomic landscapes of red clover’s root nodules and shift the phenotypic description of BNF efficiency on the level of gene expression in situ.
... These proteins display little conservation in amino acid composition but possess four or six cysteine residues at conserved positions (Mergaert et al., 2003). The length of mature NCR peptides varies from about 20 to 50 amino acids and includes two or three disulfide bridges (Mar oti et al., 2015). NCR peptides can be classified as cationic (isoelectric point [pI] ≥ 8), neutral (6 ≤ pI < 8), or anionic (pI < 6) (Mar oti et al., 2015). ...
... The length of mature NCR peptides varies from about 20 to 50 amino acids and includes two or three disulfide bridges (Mar oti et al., 2015). NCR peptides can be classified as cationic (isoelectric point [pI] ≥ 8), neutral (6 ≤ pI < 8), or anionic (pI < 6) (Mar oti et al., 2015). Highly cationic peptides (pI ≥ 9.0) display antimicrobial activity in vitro, likely through disrupting microbial membranes, thereby leading to permeabilization and cell lysis (Mar oti et al., 2011;Tiricz et al., 2013). ...
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.
... S9) that is expressed in the zone of the nodule where bacterial infection occurs (12). The DNF1 complex has been implicated in processing the signal peptide from nodulespecific cysteine-rich (NCR) peptides (12,13), which are necessary for bacteroid development (14,15). dnf1 mutants produce small, white nodules (12) similar to two weak nin alleles ( fig. ...
... Thus, processing of NIN is only relevant for functions late in nodule development, not for activation of earlier genes such as CRE1 and NPL. Among the genes controlled by NIN at late stages of nodulation are leghemoglobins that buffer oxygen (24), NCR peptides that drive bacteroid differentiation (15), and thioredoxins that control the redox state of the nodule (25). NIN, as processed by the DNF1 complex, is both necessary and sufficient for the expression of these genes and the transition to the nitrogen-fixing state. ...
Nodulation regulation
Legumes convert atmospheric nitrogen into biologically useful ammonium with the help of symbiotic bacteria housed in root nodules. Much of nodule development is controlled by the transcription factor NODULE INCEPTION (NIN). Feng et al . show that NIN is proteolytically processed to release a fragment that regulates the later stages of nodulation when the nodules acquire nitrogen-fixing capability. In related work, Jiang et al . identified members of the NIN-like protein (NLP) transcription factor family as being regulators of leghemoglobin expression acting through an unusual promotor motif shared across legumes. —PJH
... NCRs are defensin-like peptides possessing antimicrobial activity and therefore are classified as AMPs ( Van de Velde et al., 2010;Maróti et al., 2015). NCRs are abundant and with more than 500 members in M. truncatula (Maróti et al., 2015), which are essential for bacteroid terminal differentiation Mergaert et al., 2006;Van de Velde et al., 2010;Farkas et al., 2014). ...
... NCRs are defensin-like peptides possessing antimicrobial activity and therefore are classified as AMPs ( Van de Velde et al., 2010;Maróti et al., 2015). NCRs are abundant and with more than 500 members in M. truncatula (Maróti et al., 2015), which are essential for bacteroid terminal differentiation Mergaert et al., 2006;Van de Velde et al., 2010;Farkas et al., 2014). During terminal differentiation of bacteria, NCRs were shown to suppress bacterial reproduction and increase bacterial membrane permeability, also indicating their positive roles during nodule senescence, when the fragilized bacteroid membranes can be efficiently degraded by the plant cell proteases (Vasse et al., 1990;Van de Velde et al., 2006;Van de Velde et al., 2010). ...
Leguminous plants are able to associate with rhizobium to develop de novo a root organ, the root nodule. Root nodule can reduce atmosphere nitrogen into available nitrogen to plant host. Thus, the nitrogen-fixing symbiosis plays important roles in agriculture with direct nitrogen inputs to crops. In the first part of the thesis, we intended to characterize the impact of the bacterial GSH deficiency on the bacteroid differentiation and the nodule functioning during the symbiotic interaction between Medicago truncatula and Sinorhizobium meliloti. Physiological, biochemical, cellular and genetic markers were used to describe the nodule functioning at ten and twenty days after plant inoculation. Our results showed that the bacterial GSH deficiency does not affect bacteroid differentiation. However, it induces an early nodule senescence process in M. truncatula. During nodule senescence, proteolytic activities are increased terminated with the final degradation of bacteroids and plant cells. Hence, proteases are found to be the hallmarks of nodule senescence. At the onset of nodule senescence, a papain family cysteine protease, MtCP6, is a good molecular marker for the initiation of nodules senescence. In the second part of the thesis, serial promoter deletion analysis of a cysteine protease gene MtCP6 was conducted to identify cis regulatory element involved in the transcriptional regulation of nodule senescence of M. truncatula. Thereafter, identified cis-regulating region (67bp, NS) was validated to function in transcription activation in nodule zone III-IV. We have shown that tetramers of NS can increase the transcription into nodule senescence zone. In order to determine the significance of NS to the expression compared to the full promoter (-1,710bp), a functional analysis was conducted with deletion of NS from the full promoter and the analysis of a minimal promoter (-254bp). In addition, the potential roles of CAAT, WRKY and Dof motifs localized at 5’ of NS sequence was validated by site-specific mutagenesis. Finally, yeast one hybrid experiment was performed to identify transcription factors interacting with NS DNA fragment. Preliminary resulrs are presented. Taken together, these data allow a better understanding of the regulation and the operation of the nodule senescence.
... This terminal differentiation process is orchestrated by ∼700 nodule-specific cysteine-rich (NCR) peptides expressed in successive spatiotemporal waves during nodule development (2)(3)(4). Genetic studies have revealed that the absence of specific NCR peptides results in the breakdown of the symbiotic relationship and the cessation of nitrogen fixation in M. truncatula (5)(6)(7). ...
... These two families have likely evolved in parallel in this legume to play different biological roles. While defensins have evolved for defense against harmful pathogens early in the evolution of land plants, NCRs have evolved relatively recently and act as effectors inducing differentiation of rhizobia into nitrogen-fixing bacteroids (5). Therefore, antifungal activity is a secondary attribute of NCR044. ...
Significance
Several nodule-specific cysteine-rich (NCR) peptides expressed in a model legume Medicago truncatula exhibit potent antimicrobial activity. However, their structure–activity relationships and mechanisms of action against fungal pathogens of plants are still largely unknown. A small highly cationic peptide NCR044 with potent antifungal activity has been identified. This peptide has a unique highly dynamic structure and exhibits multifaceted mechanisms of action against a fungal pathogen Botrytis cinerea . Exogenous application of this peptide confers resistance to a gray mold disease caused by B. cinerea in tobacco and tomato plants as well as postharvest products. Our work paves the way for future development of NCR peptides as spray-on antifungal agents.
... Помимо дефензинов у растений выявляется также несколько семейств дефензинподобных последовательностей, часть из которых обладает более специфичным характером экспрессии и более направленной функциональностью. Так, у части бобовых растений обнаружена группа клубенек-специфичных NCR-пептидов, играющих важную роль в дифференцировке бактероидов и таким образом контролирующих развитие симбиоза [34]. Целью данного исследования была идентификация и характеристика представителей семейств дефензинов и NCR-пептидов у гороха посевного. ...
... Сравнительный транскриптомный анализ корней и клубеньков, образуемых в ходе развития актиноризного симбиоза растением Datisca glomerata и представителями рода Frankia, позволил выявить отдельную группу дефензинов, специфично экспрессирующихся в клубеньках и отличающихся от других дефензинов наличием особого терминального домена [35]. Клубенек-специфичная экспрессия ряда генов, кодирующих дефензины, была продемонстрирована и в исследованиях M. truncatula [34]. Активация экспрессии ряда генов, кодирующих дефензинподобные пептиды, была выявлена и при взаимодействии лядвенца японского (Lotus japonicus (Regel.) ...
Background. The active and careless applying of antibiotics in medicine and agriculture leads to the emergence of resistance to the existing antimicrobial drugs, which reduces the effectiveness of their use. One of the ways to solve this problem is the development of new antibiotics based on plant peptides with antimicrobial activity, for example plant defensins (which identified in all plants) and NCR peptides that are specifically synthesized in nodules of some leguminous plants.
Materials and methods. In the present study, a meta-assembly of a transcriptome was constructed based on publicly available RNA-sequencing transcriptomes of pea nodules (Pisum sativum L.). This meta-assembly was used to search for sequences encoding antimicrobial peptides.
Results. As a result, 55 and 908 unique sequences encoding defensins and NCR peptides, respectively, were identified. The recognition site for the signal peptidase was predicted and sequences were divided into the signal and mature part of the peptide. Among mature defensins, 22 peptides possess in silico predicted antimicrobial activity, and for the NCR peptides family their number was 422.
Conclusion. Sequences encoding defensins and NCR peptides expressed in nitrogen-fixing pea nodules were identified. They are candidates for testing their antimicrobial activity in vitro.
... The main goals of traditional ABD therapy are to lessen inflammation and control the dysregulated immune response, which will lessen disease activity and stop more tissue damage. Here, we explore the underlying basic mechanics of these traditional treatments: [11,12,13.14] Corticosteroid: -For ABDs, corticosteroids continue to be the mainstay of treatment because of their strong antiinflammatory and immunosuppressive qualities. Corticosteroids work mechanistically by attaching to glucocorticoid receptors inside cells. ...
Autoimmune blistering diseases, such as Linear IgA Bullous Dermatosis (LABD) and Chronic Bullous Disease of Childhood (CBDC), provide considerable therapeutic treatment issues. Samana Aushadhis are regularly prescribed by Ayurvedic practitioners as part of therapy regimens for dermatological diseases including LABD and CBDC. Although anecdotal data and clinical observations point to the potential usefulness of traditional medicines in promoting long-term remission and alleviating symptoms, the precise mechanisms by which they exert their therapeutic benefits in LABD/CBD remain incompletely known in contemporary science. The potential advantages of traditional therapies in autoimmune blistering illnesses are being investigated through clinical trials, observational research, and mechanistic investigations. The therapeutic potential of these herbal remedies in LABD and CBDC is being investigated through clinical trials, observational research, and mechanistic investigations. Clinical trials, observational studies, and mechanistic investigations are being conducted in an effort to better understand the therapeutic potential of conventional medications in autoimmune blistering disorders. The effectiveness of these herbal treatments in causing remission and easing symptoms in LABD and CBDC is being closely examined. Overall, while traditional therapies hold promise in the management of autoimmune blistering diseases like LABD and CBDC, more research is necessary to fully understand their mechanisms of action and therapeutic potential. Through rigorous scientific inquiry, the integration of traditional and contemporary medicine may offer new avenues for managing these difficult conditions. Clinical trials, observational research, and mechanistic investigations are all contributing to the exploration of the therapeutic potential of these herbal remedies in LABD and CBDC.
... Many of them, when forming complexes with MtNCC1 1-78 can be detected in the nuclei. Some of them could be involved in epigenetic regulation (SAM synthases; Liu et al., 2020), in the response to plant-microbe interactions (pathogenesis-related proteins; Kaur et al., 2017), or in the coordination between host and bacteroid (nodule-specific cysteine-rich peptides, NCRs) (Maróti et al., 2015), but their specific roles in symbiotic nitrogen fixation still remain to be determined. Our metalloproteomic and pulldown assays have unveiled not only a range of known copper proteins, but also a number of new putative copper proteins that have to be further validated biochemically and placed in the context of symbiotic nitrogen fixation. ...
Cu⁺‐chaperones are a diverse group of proteins that allocate Cu⁺ ions to specific copper proteins, creating different copper pools targeted to specific physiological processes.
Symbiotic nitrogen fixation carried out in legume root nodules indirectly requires relatively large amounts of copper, for example for energy delivery via respiration, for which targeted copper deliver systems would be required.
MtNCC1 is a nodule‐specific Cu⁺‐chaperone encoded in the Medicago truncatula genome, with a N‐terminus Atx1‐like domain that can bind Cu⁺ with picomolar affinities. MtNCC1 is able to interact with nodule‐specific Cu⁺‐importer MtCOPT1. MtNCC1 is expressed primarily from the late infection zone to the early fixation zone and is located in the cytosol, associated with plasma and symbiosome membranes, and within nuclei. Consistent with its key role in nitrogen fixation, ncc1 mutants have a severe reduction in nitrogenase activity and a 50% reduction in copper‐dependent cytochrome c oxidase activity.
A subset of the copper proteome is also affected in the ncc1 mutant nodules. Many of these proteins can be pulled down when using a Cu⁺‐loaded N‐terminal MtNCC1 moiety as a bait, indicating a role in nodule copper homeostasis and in copper‐dependent physiological processes. Overall, these data suggest a pleiotropic role of MtNCC1 in copper delivery for symbiotic nitrogen fixation.
... In particular, EPS-I confers resistance to the antimicrobial activity of NCR247 [63,65], which belongs to a diverse family of small, nodule-specific cysteine-rich (NCR) peptides encoded by certain legumes [10,66,67]. Structurally similar to host defensins [68], different NCR peptides regulate bacterial load in nodules and influence distinct aspects of terminal bacteroid differentiation, including maintaining survival and preventing premature senescence [69][70][71][72][73][74]. In addition to EPS-I, other bacterial factors can modulate the effects of NCR peptides [63,[75][76][77]. ...
Sinorhizobium meliloti is a model alpha-proteobacterium for investigating microbe-host interactions, in particular nitrogen-fixing rhizobium-legume symbioses. Successful infection requires complex coordination between compatible host and endosymbiont, including bacterial production of succinoglycan, also known as exopolysaccharide-I (EPS-I). In S . meliloti EPS-I production is controlled by the conserved ExoS-ChvI two-component system. Periplasmic ExoR associates with the ExoS histidine kinase and negatively regulates ChvI-dependent expression of exo genes, necessary for EPS-I synthesis. We show that two extracytoplasmic proteins, LppA (a lipoprotein) and JspA (a lipoprotein and a metalloprotease), jointly influence EPS-I synthesis by modulating the ExoR-ExoS-ChvI pathway and expression of genes in the ChvI regulon. Deletions of jspA and lppA led to lower EPS-I production and competitive disadvantage during host colonization, for both S . meliloti with Medicago sativa and S . medicae with M . truncatula . Overexpression of jspA reduced steady-state levels of ExoR, suggesting that the JspA protease participates in ExoR degradation. This reduction in ExoR levels is dependent on LppA and can be replicated with ExoR, JspA, and LppA expressed exogenously in Caulobacter crescentus and Escherichia coli . Akin to signaling pathways that sense extracytoplasmic stress in other bacteria, JspA and LppA may monitor periplasmic conditions during interaction with the plant host to adjust accordingly expression of genes that contribute to efficient symbiosis. The molecular mechanisms underlying host colonization in our model system may have parallels in related alpha-proteobacteria.
... CRPs are predominantly copious during plant reproduction. Due to their role in various reproductive processes, CRPs occur in female and male gametophytes, unlike PTM peptides that are mainly present in vegetative tissues (Hara et al. 2007;Sugano et al. 2010;Maróti et al. 2015;Bircheneder and Dresselhaus 2016). CRPs do not undergo post-translational modifications. ...
... The defensins display a wide-spectrum toxicity against bacterial, fungal, and viral microorganisms [120,136]. Some of the NCR-peptides are involved in bacteroid differentiation and survival [137,138]. In M. truncatula, more than 700 NCR-peptide genes are present. ...
Plants often experience unfavorable conditions during their life cycle that impact their growth and sometimes their survival. A temporary phase of such stress, which can result from heavy metals, drought, salinity, or extremes of temperature or pH, can cause mild to enormous damage to the plant depending on its duration and intensity. Besides environmental stress, plants are the target of many microbial pathogens, causing diseases of varying severity. In plants that harbor mutualistic bacteria, stress can affect the symbiotic interaction and its outcome. To achieve the full potential of a symbiotic relationship between the host and rhizobia, it is important that the host plant maintains good growth characteristics and stay healthy under challenging environmental conditions. The host plant cannot provide good accommodation for the symbiont if it is infested with diseases and prone to other predators. Because the bacterium relies on metabolites for survival and multiplication, it is in its best interests to keep the host plant as stress-free as possible and to keep the supply stable. Although plants have developed many mitigation strategies to cope with stress, the symbiotic bacterium has developed the capability to augment the plant’s defense mechanisms against environmental stress. They also provide the host with protection against certain diseases. The protective features of rhizobial–host interaction along with nitrogen fixation appear to have played a significant role in legume diversification. When considering a legume–rhizobial symbiosis, extra benefits to the host are sometimes overlooked in favor of the symbionts’ nitrogen fixation efficiency. This review examines all of those additional considerations of a symbiotic interaction that enable the host to withstand a wide range of stresses, enabling plant survival under hostile regimes. In addition, the review focuses on the rhizosphere microbiome, which has emerged as a strong pillar of evolutionary reserve to equip the symbiotic interaction in the interests of both the rhizobia and host. The evaluation would draw the researchers’ attention to the symbiotic relationship as being advantageous to the host plant as a whole and the role it plays in the plant’s adaptation to unfavorable environmental conditions.
... NCR peptides (nodule-specific cysteine-rich peptides), a large group of defensin-like antimicrobial peptides, are produced in nodules of M. truncatula and control rhizobial development [43,44]. Interestingly, we found that whereas almost all NCR peptides were nodule-specific, a single NCR peptide gene (NCR150) was expressed at our earliest time point, before nodules had developed, and then was turned back off at nodule emergence. ...
Nodule number regulation in legumes is controlled by a feedback loop that integrates nutrient and rhizobia symbiont status signals to regulate nodule development. Signals from the roots are perceived by shoot receptors, including a CLV1-like receptor-like kinase known as SUNN in Medicago truncatula. In the absence of functional SUNN, the autoregulation feedback loop is disrupted, resulting in hypernodulation. To elucidate early autoregulation mechanisms disrupted in SUNN mutants, we searched for genes with altered expression in the loss-of-function sunn-4 mutant and included the rdn1-2 autoregulation mutant for comparison. We identified constitutively altered expression of small groups of genes in sunn-4 roots and in sunn-4 shoots. All genes with verified roles in nodulation that were induced in wild-type roots during the establishment of nodules were also induced in sunn-4, including autoregulation genes TML2 and TML1. Only an isoflavone-7-O-methyltransferase gene was induced in response to rhizobia in wild-type roots but not induced in sunn-4. In shoot tissues of wild-type, eight rhizobia-responsive genes were identified, including a MYB family transcription factor gene that remained at a baseline level in sunn-4; three genes were induced by rhizobia in shoots of sunn-4 but not wild-type. We cataloged the temporal induction profiles of many small secreted peptide (MtSSP) genes in nodulating root tissues, encompassing members of twenty-four peptide families, including the CLE and IRON MAN families. The discovery that expression of TML2 in roots, a key factor in inhibiting nodulation in response to autoregulation signals, is also triggered in sunn-4 in the section of roots analyzed, suggests that the mechanism of TML regulation of nodulation in M. truncatula may be more complex than published models.
... Previously, it was believed that Cys-rich peptides function in plants mainly as antimicrobial compounds upon infection by pathogenic microorganisms [11,29]. However, it was further discovered that these peptides may play an important role in stomatal structure and density, symbiosis, and a wide range of reproductive processes such as tube pollen germination, induction and rupture, gamete activation, and seed development [30][31][32][33]. Consistent with their role in reproduction, Cys-rich peptides are widely present in both female and male gametophytes, in contrast to post-translationally modified peptides, which are predominantly found in vegetative tissues. ...
Peptides perform many functions, participating in the regulation of cell differentiation, regulating plant growth and development, and also involved in the response to stress factors and in antimicrobial defense. Peptides are an important class biomolecules for intercellular communication and in the transmission of various signals. The intercellular communication system based on the ligand-receptor bond is one of the most important molecular bases for creating complex multicellular organisms. Peptide-mediated intercellular communication plays a critical role in the coordination and determination of cellular functions in plants. The intercellular communication system based on the receptor-ligand is one of the most important molecular foundations for creating complex multicellular organisms. Peptide-mediated intercellular communication plays a critical role in the coordination and determination of cellular functions in plants. The identification of peptide hormones, their interaction with receptors, and the molecular mechanisms of peptide functioning are important for understanding the mechanisms of both intercellular communications and for regulating plant development. In this review, we drew attention to some peptides involved in the regulation of root development, which implement this regulation by the mechanism of a negative feedback loop.
... Eight DAPs involved in cysteine and methionine metabolism were induced at inflorescence development stages, which suggested that cysteine and methionine metabolism play critical roles in the regulation of inflorescence development in castor. In general, many plant cysteine-rich peptides with potential antimicrobial properties have been predicted [23]. At the same time, plants can defend against tolerant environmental stresses and plant pathogens by some proteins having plant-specific cysteine-rich motifs [24]. ...
Castor (Ricinus communis L.) is one of ten oil crops in the world and has complex inflorescence styles. Generally, castor has three inflorescence types: single female inflorescence (SiFF), standard female inflorescence (StFF) and bisexual inflorescence (BF). StFF is realized as a restorer line and as a maintainer line, which was applied to castor hybrid breeding. However, the developmental mechanism of the three inflorescences is not clear. Therefore, we used proteomic techniques to analyze different inflorescence styles. A total of 72 diferentially abundant protein species (DAPs) were detected. These DAPs are primarily involved in carbon and energy metabolism and carbon fixation in the photosynthetic organism pathway. The results showed that DAPs are involved in photosynthesis to control the distribution of imported carbohydrates and exported photoassimilates and thus affect the inflorescence development of castor. In addition, these DAPs are also involved in cysteine and methionine metabolism. Quantitative real-time PCR (qRT-PCR) results demonstrated that the proteomics data collected in this study were reliable. Our findings indicate that the carbon cycle and amino acid metabolism influence the inflorescence development of castor.
... NCRs produced by legumes are diverse, they do not display any significant conservation of the amino acid sequence except for 4 or 6 conserved cysteine residues [146]. The mechanisms of NCRs action on bacterial cells also vary: some cationic NCRs effect the cell surface and form pores in the membranes causing cell lysis [147], while others are internalized and bind to different proteins in the cell provoking global transcriptional changes in symbiotic bacteria [148]. ...
People widely use antibiotics in healthcare, agriculture, and for preservation of food for almost a century. However, the spread of antimicrobial resistance across the populations of clinically and industrially important microorganisms reduces the arsenal of compounds we can rely on in the fight against undesirable bacteria. Furthermore, the rates of new compound discovery using classical screening approaches dropped dramatically in the past decades primarily due to the high levels of rediscovery of known molecules. The search for novel antibiotics starting from the analysis of accumulated genomic data (“genome mining”) is a viable alternative, which makes use of the improvements in DNA sequencing technologies.
In this work, using genome mining, we identified a biosynthetic gene cluster (BGC) of a putative new antibiotic in the genome of a symbiotic nitrogen-fixing bacterium Rhizobium sp. Pop5. Further, we purified and characterized the compound, which biosynthesis is guided by the BGC. The molecule, which we named phazolicin (PHZ) belongs to the growing class of ribosomally synthesized posttranslationally-modified peptide (RiPP) natural products. PHZ is a linear azol(in)e-containing peptide (LAP) exhibiting narrow-spectrum activity against the strains of rhizobia closely related to its producer. PHZ inhibits bacterial translation by the obstruction of the ribosomal nascent peptide exit tunnel and demonstrates the mode of interaction with the ribosome, which is distinct from that of previously described translation-targeting antibiotics. PHZ uses two different non-specific peptide import systems to enter susceptible cells. Such dual mode of
uptake dramatically decreases the levels of spontaneous PHZ resistance acquisition. PHZ production solely defines the ability of Rhizobium sp. Pop5 to eliminate closely related strains from the co-culture. However, our attempts to investigate the role of PHZ production in soil and upon the nodulation of plants did not reveal any competitive advantage of PHZ-producing strains. Finally, we perform a systematic search for BGCs of novel putative linear azol(in)e-containing peptides in the publicly available bacterial genomes and describe several previously overlooked groups, which are interesting leads for further experimental validation.
The work present is a comprehensive study of a single compound starting with its prediction by the methods of bioinformatics and finishing with the experiments aiming to validate the role of its production in the complex environment mimicking natural conditions. The application of diverse methodologies of biochemistry, genetic engineering, microbiology, and structural biology enabled us to characterize PHZ from both chemical and biological points of view.
... Hairpinins exert either antifungal or proteinase inhibitory activity [31]. NCR peptides comprise a large family of highly diverse peptides with a conserved 4-or 6-Cys motif which are specifically expressed in legume nodules [32]. The cysteine motif in 4-Cys-containing NCR peptides conforms to the following signature: C-X-{5}-C-X{n}-C-X{4}-C. Their 3D structure includes either one short C-terminal antiparallel β-sheet and a short α-helix as in NCR044 or only the β-sheet without α-helix as in NCR169 [33,34]. ...
Antimicrobial peptides (AMPs) constitute an essential part of the plant immune system. They are regarded as alternatives to conventional antibiotics and pesticides. In this study, we have identified the γ-core motifs, which are associated with antimicrobial activity, in 18 AMPs from grasses and assayed their antimicrobial properties against nine pathogens, including yeasts affecting humans, as well as plant pathogenic bacteria and fungi. All the tested peptides displayed antimicrobial properties. We discovered a number of short AMP-derived peptides with high antimicrobial activity both against human and plant pathogens. For the first time, antimicrobial activity was revealed in the peptides designed from the 4-Cys-containing defensin-like peptides, whose role in plant immunity has remained unknown, as well as the knottin-like peptide and the C-terminal prodomain of the thionin, which points to the direct involvement of these peptides in defense mechanisms. Studies of the mode of action of the eight most active γ-core motif peptides on yeast cells using staining with propidium iodide showed that all of them induced membrane permeabilization leading to cell lysis. In addition to identification of the antimicrobial determinants in plant AMPs, this work provides short candidate peptide molecules for the development of novel drugs effective against opportunistic fungal infections and biopesticides to control plant pathogens.
... broadly known as CRISPs, act as ion channel inhibitors and anticoagulants in reptile venoms [14]. CRISPs also play important roles in reproduction and immune systems in various organisms of the animal kingdom [15][16][17]. Despite being rich in cysteine residues, Ae. aegypti CRVP379 belongs to a broad group of proteins known as the cysteine-rich trypsin inhibitor-like proteins (TIL; PF01826.) ...
Cysteine-rich trypsin inhibitor-like domain (TIL)-harboring proteins are broadly distributed in nature but remain understudied in vector mosquitoes. Here we have explored the biology of a TIL domain-containing protein of the arbovirus vector Aedes aegypti, cysteine-rich venom protein 379 (CRVP379). CRVP379 was previously shown to be essential for dengue virus infection in Ae. aegypti mosquitoes. Gene expression analysis showed CRVP379 to be highly expressed in pupal stages, male testes, and female ovaries. CRVP379 expression is also increased in the ovaries at 48 h post-blood feeding. We used CRISPR-Cas9 genome editing to generate two mutant lines of CRVP379 with mutations inside or outside the TIL domain. Female mosquitoes from both mutant lines showed severe defects in their reproductive capability; mutant females also showed differences in their follicular cell morphology. However, the CRVP379 line with a mutation outside the TIL domain did not affect male reproductive performance, suggesting that some CRVP379 residues may have sexually dimorphic functions. In contrast to previous reports, we did not observe a noticeable difference in dengue virus infection between the wild-type and any of the mutant lines. The importance of CRVP379 in Ae. aegypti reproductive biology makes it an interesting candidate for the development of Ae. aegypti population control methods.
... Thus, the antimicrobial NCRs are collectively used by legumes as a tool rather than a weapon (Alunni and Gourion 2016). However, this does not exclude the possibility that some symbiotic NCRs have multiple functions and may participate in plant defense against bacterial infections (Maroti et al. 2015). The key point is the potential Fig. 5. Effects of Astragalus sinicus nodule-specific cysteine-rich peptides (AsNCRs) on rhizobial growth in vitro. ...
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 .
... Most known AMPs are of animal origin (Wang et al., 2016), however, plants have evolved an extremely rich source of AMPs that act as the most prominent defense barriers. The largest group of plant AMPs are cysteine-rich defensins characterized by four conserved disulfide bridges, positive net charge and amphipathic character and capability to interact with microbial membranes, and by entering the cells to inhibit translation and enzymatic activity (Maróti et al., 2015;Tam et al., 2015). ...
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.
... After successful invasion and nodule formation, it has been found that proliferation of disease causing microbes decreases but this does not help in efficient nitrogen fixation. Thus several adaptations have taken place in order to overcome these shortcomings such as: many plant genera have short peptide known as defensin that controls the behavior of the symbiotic microorganism; some plant clades have small, nodule-specific and cysteine-rich peptides that interferes with cell cycle of both plant and bacterial genomes, perturbs membrane stability, alters gene expression, and promotes terminal differentiation of the Rhizobium species (Maróti et al., 2015). ...
The productivity of crop plants gets limited by the access of fixed or available forms of nitrogen and also affects the production of food. Reduction in the dependence on nitrogenous fertilizers provides significant potential to the developing countries and developed world and hence a considerable amount of research is performed in this field. The mechanism of biological nitrogen fixation involves the conversion of N2 to NH3 that can be used efficiently by the plants. Microorganisms take active part in biogeochemical cycle of nitrogen, where gaseous nitrogen of air is converted into multiple chemical forms as it circulates among atmosphere, terrestrial, and aquatic ecosystems. Diverse microbial species play the key role in various stages of nitrogen cycle. Phototrophic diazotrophs are the group of photosynthetic purple and green bacteria containing chlorophyll that play important role in the mechanism of nitrogen fixation. ATP and reductant needed to support N2 fixation are produced by the process of photosynthesis but the two processes are indirectly related by the accumulation of carbon reserves. In this chapter we will focus on various bacterial species that are associated with the autotrophic fixation of nitrogen, highlighting the underlying mechanism of fixation with an emphasis on its genetic regulation.
... Indeed, in our root expression data, we observed 11 defense response genes, including three defensins (Medtr8g012810, Medtr8g012815, and Medtr8g012845) were strongly upregulated in Ri09-colonized roots, but not in Ga165-colonized roots (Supplemental Fig. S9b; Supplemental Table S5). Defensins are known to inhibit pathogen growth (Maróti et al. 2015), so it is tempting to speculate that Ga165 may employ a suite of effectors to inhibit the expression of defensins and thereby temper host defense responses to avoid detection and sanctioning. Not all defensins play a role in pathogen response; some may play a role in the establishment of the AM symbiosis. ...
Arbuscular mycorrhizal (AM) fungi form a root endosymbiosis with many agronomically important crop species. They enhance the ability of their host to obtain nutrients from the soil and increase the tolerance to biotic and abiotic stressors. However, AM fungal species can differ in the benefits they provide to their host plants. Here, we examined the putative molecular mechanisms involved in the regulation of the physiological response of Medicago truncatula to colonization by Rhizophagus irregularis or Glomus aggregatum, which have previously been characterized as high- and low-benefit AM fungal species, respectively. Colonization with R. irregularis led to greater growth and nutrient uptake than colonization with G. aggregatum. These benefits were linked to an elevated expression in the roots of strigolactone biosynthesis genes (NSP1, NSP2, CCD7, and MAX1a), mycorrhiza-induced phosphate (PT8), ammonium (AMT2;3), and nitrate (NPF4.12) transporters and the putative ammonium transporter NIP1;5. R. irregularis also stimulated the expression of photosynthesis-related genes in the shoot and the upregulation of the sugar transporters SWEET1.2, SWEET3.3, and SWEET 12 and the lipid biosynthesis gene RAM2 in the roots. In contrast, G. aggregatum induced the expression of biotic stress defense response genes in the shoots, and several genes associated with abiotic stress in the roots. This suggests that either the host perceives colonization by G. aggregatum as pathogen attack or that G. aggregatum can prime host defense responses. Our findings highlight molecular mechanisms that host plants may use to regulate their association with high- and low-benefit arbuscular mycorrhizal symbionts.
... In S. meliloti, BacA and YejABEF are involved in the transport of nodule-specific cysteine-rich peptides (NCRs) [19,25]. NCRs are a diverse family of ribosomally-synthesized defensin-like peptides encoded in the genomes of some plants (such as Medicago) with lengths varying usually from 24 to 65 amino acids [40,41]. Cationic NCRs exhibit antimicrobial activity by targeting cell membranes of both Gram-positive and Gram-negative bacteria [42]. ...
Phazolicin (PHZ) is a peptide antibiotic exhibiting narrow-spectrum activity against rhizobia closely related to its producer Rhizobium sp. Pop5. Using genetic and biochemical techniques, we here identified BacA and YejABEF as two importers of PHZ in a sensitive model strain Sinorhizobium meliloti Sm1021. BacA and YejABEF are members of SLiPT and ABC transporter families of non-specific peptide importers, respectively. The uptake of PHZ by two distinct families of transporters dramatically decreases the naturally occurring rate of resistance. Moreover, since both BacA and YejABEF are essential for the development of functional symbiosis of rhizobia with leguminous plants, the acquisition of PHZ resistance via the inactivation of transporters is further disfavoured since single bacA or yejABEF mutants are unable to propagate in root nodules. Crystal structures of the periplasmic subunit YejA from S. meliloti and Escherichia coli revealed fortuitous bound peptides, suggesting a non-specific peptide-binding mechanism that facilitates the uptake of PHZ and other antimicrobial peptides.
SIGNIFICANCE
Many bacteria produce antimicrobial peptides to eliminate competitors and create an exclusive niche. These peptides kill bacteria by either membrane disruption or inhibiting essential intracellular processes. The Achilles heel of the latter type of antimicrobials is their dependence on transporters to enter the susceptible bacteria since mutations in such transporters result in resistance. We describe here how the ribosome-targeting peptide phazolicin, produced by Rhizobium sp. Pop5, uses two different transporters, BacA and YejABEF, to get into the cells of the symbiotic bacterium Sinorhizobium meliloti . This dramatically reduces the probability of resistance acquisition. Both transporters need to be inactivated for phazolicin resistance acquisition. Since these transporters are also crucial in S. meliloti for its symbiotic association with host plants, their inactivation in biological settings is highly unlikely. This makes PHZ an attractive lead for the development of a biocontrol agent with potential for use in agriculture.
... For example, CRPs (∼5 kDa) which contain 2 to 16 Cys residues have been shown to form disulfide bridges and mainly function as antimicrobial peptides (van der Weerden et al., 2013;Tavormina et al., 2015;reviewed by De Coninck and De Smet, 2016). The CRs also regulate stomatal patterning and density, symbiosis and a wide range of reproductive processes such as pollen tube germination, guidance and burst, gamete activation, and seed development (Hara et al., 2007;Sugano et al., 2010;Maróti et al., 2015;Bircheneder and Dresselhaus, 2016;De Coninck and De Smet, 2016). ...
In this review, we describe and integrate the latest knowledge on the signaling role of proteins and peptides in the stress-induced microspore embryogenesis (ME) in some crop plants with agricultural importance (i.e., oilseed rape, tobacco, barley, wheat, rice, triticale, rye). Based on the results received from the most advanced omix analyses, we have selected some inconspicuous but possibly important players in microspores reprogramming toward embryogenic development. We provide an overview of the roles and downstream effect of stress-related proteins (e.g., β-1,3-glucanases, chitinases) and small signaling peptides, especially cysteine—(e.g., glutathione, γ-thionins, rapid alkalinization factor, lipid transfer, phytosulfokine) and glycine-rich peptides and other proteins (e.g., fasciclin-like arabinogalactan protein) on acclimation ability of microspores and the cell wall reconstruction in a context of ME induction and haploids/doubled haploids (DHs) production. Application of these molecules, stimulating the induction and proper development of embryo-like structures and green plant regeneration, brings significant improvement of the effectiveness of DHs procedures and could result in its wider incorporation on a commercial scale. Recent advances in the design and construction of synthetic peptides–mainly cysteine-rich peptides and their derivatives–have accelerated the development of new DNA-free genome-editing techniques. These new systems are evolving incredibly fast and soon will find application in many areas of plant science and breeding.
... Studies involving mutant legumes unable to nodulate or form mycorrhizal symbioses revealed significant effects of symbiont absence on bacteriome structure in the rhizosphere (Uroz et al., 2019). Nodulation induces unique metabolic changes within the legume host that may explain this effect, including the production of cysteine-rich peptides in some legumes that control bacteroid differentiation but also act as antimicrobials against non-symbionts (Maróti et al., 2015). However, the role of these peptides in modulating microbial community structure has not yet been thoroughly tested. ...
Legumes are of primary importance for agroecosystems because they provide protein-rich foods and enhance soil fertility through fixed atmospheric nitrogen. The legume-rhizobia symbiosis that makes this possible has been extensively studied, from basic research on biochemical signaling to practical applications in cropping systems. While rhizobia are the most-studied group of associated microorganisms, the functional benefit they confer to their legume hosts by fixing nitrogen is not performed in isolation. Indeed, non-rhizobia members of the rhizosphere and nodule microbiome are now understood to contribute in multiple ways to nodule formation, legume fitness, and other agroecosystem services. In this review, we summarize advances contributing to our understanding of the diversity and composition of bacterial members of the belowground legume microbiome. We also highlight applied work in legume food and forage crops that link microbial community composition with plant functional benefits. Ultimately, further research will assist in the development of multi-species microbial inoculants and cropping systems that maximize plant nutrient benefits, while reducing sources of agricultural pollution.
... Legumes in the inverted repeat-lacking clade (but not legumes in the related robinioid clade) produce hundreds of small, nodule-specific, and cysteine-rich peptides. These peptides perturb the cell cycle, leading to endoreduplication of both plant and bacterial genomes, disruption of membrane stability, alteration of gene ex expression, and promotion of terminal differentiation of the Rhizobium species (Maróti et al., (2015). More recently, sets of defensin-like peptides with similar properties have been found in dalbergioid legumes Czernic et al., (2015) and in three genera of actinorhizal hosts (Carro et al., 2013). ...
Nitrogen is the limiting nutrient element after carbon, hydrogen and oxygen for photosynthetic process, Phyto hormonal, proteomic changes and growth development of plants to complete its lifecycle. However excessive and inefficient use of N fertilizer results in enhanced crop production costs and atmospheric pollution. Atmospheric nitrogen (71%) in the molecular form is not available for the plants. For world's sustainable food production and atmospheric benefits, there is an urgent need to upgrade nitrogen use efficiency in agricultural farming system. Nitrogen use efficiency is the product of nitrogen uptake efficiency and nitrogen utilization efficiency; varies from 30.2 to 53.2%. Nitrogen losses are too high, due to excess amount, low plant population, poor application methods etc., which can go up to 70% of total available nitrogen. Adopting improved agronomic approaches, losses can be minimized up to 15-30%, such as optimal dosage of nitrogen, application of N by using canopy sensors, maintaining plant population, drip fertigation and legume based intercropping. Recent developments and future prospects of improving nitrogen use efficiency (NUE) in crops using various complementary approaches. Including conventional breeding and molecular genetics, in addition to alternative farming techniques based on no-till continuous cover cropping cultures and/or organic nitrogen (N) nutrition. Whatever the mode of N fertilization, an increased knowledge of the mechanisms controlling plant N economy is essential for improving (NUE) and for reducing excessive input of fertilizers, while maintaining an acceptable yield and sufficient profit margin for the farmers. Using plants grown under agronomic conditions, with different tillage conditions, in pure or associated cultures, at low and high N mineral fertilizer input, or using organic fertilization, it is now possible to develop further whole plant agronomic and physiological studies. These can be combined with gene, protein and metabolite profiling to build up a comprehensive picture depicting the different steps of N uptake, assimilation and recycling to produce either biomass in vegetative organs or proteins in storage organs. Providing a critical overview as to how our understanding of the agro-eco physiological and molecular controls of N assimilation in crops, under varying environmental conditions, has been improved. Long-term sustainability may require a gradual transition from synthetic N inputs to legume-based crop rotation, including continuous cover cropping systems, where these may be possible in certain areas of the world, depending on climatic conditions. Current knowledge and prospects for future agronomic development and application for breeding crops adapted to lower mineral fertilizer input and to alternative farming techniques are explored, whilst taking into account the constraints of both the current world economic situation and the environment. Nitrate reductase, nitrite reductase, glutamine synthetase, glutamine oxoglutarate aminotransferase and asparagine synthetase enzyme have a great role in nitrogen metabolism. However, further studies on carbon-nitrogen metabolism and molecular changes atomic levels are required by using "whole genome sequencing technology" to improve nitrogen use efficiency (NUE).
... In such defensins, cysteine crosslinks hold together α-helical and β sheet regions such that highly variable "interactive" domains can bind efficiently to proteins (Shafee et al., 2017). NCRs are similar to, but different from most cysteine-rich defensins, usually having 4 or 6 cysteine residues rather than 8 or 10 seen in true defensins (Maroti et al., 2015). The sequences of NCR peptides are highly diverse and fall into cationic, anionic, and neutral groupings. ...
In nitrogen-fixing nodules of legumes such as pea (Pisum) and Medicago spp. the plant induces terminal differentiation in the rhizobial endosymbionts by targeting nodule-specific cysteine-rich defensin-like peptides into the bacteria. However, in nodules of other legumes such as soybean and Lotus spp. terminal bacterial differentiation does not occur; these legumes lack genes encoding equivalent peptides controlling rhizobial development. Here, we review the effects of some of these peptides on rhizobia and address the question as to how and why such peptides may have evolved to enslave rhizobia and become essential for nitrogen fixation in some clades of legumes but not in others.
... In addition to playing a key role in symbiotic signalling with the host plant, which most probably suppresses plant defence responses [142], a number of functional studies indicate that both S. meliloti EPS play a protective role against not only different abiotic stresses, such as detergents, salt, acidic pH, and heat [143] but also certain responses from the host plant, such as ROS that are present within IT [144] and, maybe, against antimicrobial peptides that are produced inside nodules of certain host legumes belonging to the IRLC (inverting-repeat lacking clade) such as Medicago [145]. S. meliloti EPS, especially EPS II, are also important for biofilm formation [146]. ...
Rhizobia are soil proteobacteria able to engage in a nitrogen-fixing symbiotic interaction with legumes that involves the rhizobial infection of roots and the bacterial invasion of new organs formed by the plant in response to the presence of appropriate bacterial partners. This interaction relies on a complex molecular dialogue between both symbionts. Bacterial N-acetyl-glucosamine oligomers called Nod factors are indispensable in most cases for early steps of the symbiotic interaction. In addition, different rhizobial surface polysaccharides, such as exopolysaccharides (EPS), may also be symbiotically relevant. EPS are acidic polysaccharides located out of the cell with little or no cell association that carry out important roles both in free-life and in symbiosis. EPS production is very complexly modulated and, frequently, co-regulated with Nod factors, but the type of co-regulation varies depending on the rhizobial strain. Many studies point out a signalling role for EPS-derived oligosaccharides in root infection and nodule invasion but, in certain symbiotic couples, EPS can be dispensable for a successful interaction. In summary, the complex regulation of the production of rhizobial EPS varies in different rhizobia, and the relevance of this polysaccharide in symbiosis with legumes depends on the specific interacting couple.
... The first evidence of a new class of late-stage symbiotic signals emerged from a transcriptomic analysis in M. truncatula, where a large assortment of hundreds of host-derived nodule-specific cysteine-rich (NCR) peptides was observed 21 . Subsequent studies of NCR peptides revealed that they possess structural and bactericidal properties similar to the defensin class of antimicrobial peptides 22,23 . At non-lethal doses, NCR peptides have several effects on rhizobial cells: they permeabilize membranes, promote genome endoreduplication, and drive cell-enlargement and branching, all of which presumably facilitate effective nitrogen fixation, nutrient exchange, and suppression of rhizobial proliferation [24][25][26][27][28][29] . ...
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.
... The cysteine-rich peptides, defensins and defensin-like peptides are the most abundant in plants, e.g., in legume-rhizobial symbiosis which can inhibit pathogen growth and control rhizobial differentiation in legume nodules (Maróti et al. 2015). ...
... The cysteine-rich peptides, defensins and defensin-like peptides are the most abundant in plants, e.g., in legume-rhizobial symbiosis which can inhibit pathogen growth and control rhizobial differentiation in legume nodules (Maróti et al. 2015). ...
... The side chain of cysteine contains sulfhydryl group, which can make a covalent coupling with an amino group of the protein via a cross-linker molecule such as sulfo-SMCC (sulfosuccinimidyl 4-(N-maleimidomethyl) cyclohexane-1-carboxylate). The cysteine-rich AMPs were isolated from leguminous plants and the granular hemocytes of mangrove crabs (Sivakamavalli, Nirosha & Vaseeharan, 2015;Maróti, Downie & Kondorosi, 2015). The functionalized textiles and nasal prongs modified with L-cysteine exhibited antimicrobial activity against Staphylococcus aureus and Klebsiella pneumoniae (Gouveia, Sá & Henriques, 2012;Caldeira et al., 2013;Xu et al., 2017;Odeberg et al., 2018). ...
Background
Antimicrobial peptides have a broad spectrum of antimicrobial activities and are attracting attention as promising next-generation antibiotics against multidrug-resistant (MDR) bacteria. The all-d-enantiomer [ D (KLAKLAK) 2 ] has been reported to have antimicrobial activity against Escherichia coli and Pseudomonas aeruginosa , and to be resistant to protein degradation in bacteria because it is composed of D-enantiomer compounds. In this study, we demonstrated that modification of [ D (KLAKLAK) 2 ] by the addition of an L-cysteine residue to its N- or C- terminus markedly enhanced its antimicrobial activities against Gram-negative bacteria such as MDR Acinetobacter baumannii , E. coli , and P. aeruginosa .
Methods
The peptides [ D (KLAKLAK) 2 ] (DP), DP to which L-cysteine was added at the N-terminus C-DP, and DP to which L-cysteine was added at the C-terminus DP-C, were synthesized at >95% purity. The minimum inhibitory concentrations of peptides and antibiotics were determined by the broth microdilution method. The synergistic effects of the peptides and the antibiotics against MDR P. aeruginosa were evaluated using the checkerboard dilution method. In order to assess how these peptides affect the survival of human cells, cell viability was determined using a Cell Counting Kit-8.
Results
C-DP and DP-C enhanced the antimicrobial activities of the peptide against MDR Gram-negative bacteria, including A. baumannii , E. coli, and P. aeruginosa . The antimicrobial activity of DP-C was greater than that of C-DP, with these peptides also having antimicrobial activity against drug-susceptible P. aeruginosa and drug-resistant P. aeruginosa overexpressing the efflux pump components. C-DP and DP-C also showed antimicrobial activity against colistin-resistant E. coli harboring mcr-1 , which encodes a lipid A modifying enzyme. DP-C showed synergistic antimicrobial activity against MDR P. aeruginosa when combined with colistin. The LD 50 of DP-C against a human cell line HepG2 was six times higher than the MIC of DP-C against MDR P. aeruginosa. The LD 50 of DP-C was not altered by incubation with low-dose colistin.
Conclusion
Attachment of an L-cysteine residue to the N- or C-terminus of [ D (KLAKLAK) 2 ] enhanced its antimicrobial activity against A. baumannii , E. coli , and P. aeruginosa . The combination of C-DP or DP-C and colistin had synergistic effects against MDR P. aeruginosa . In addition, DP-C and C-DP showed much stronger antimicrobial activity against MDR A. baumannii and E. coli than against P. aeruginosa .
... Nodules belonging to the inverted-repeat lacking clade (IRLC) of legumes are marked by more extreme bacteroid differentiation, and this is mediated, at least in part, by antimicrobial peptides belonging to the nodule-specific cysteine-rich (NCR) family (Van de Velde et al., 2010;Roy et al., 2020). The role of NCR peptides is best understood in M. truncatula, which has over 700 inferred NCRs to date (Maróti et al., 2015), although it is unclear if all NCR family members are involved in regulating symbiosis since their expression levels and pattern can vary [reviewed in Roy et al., 2020]. Patterns of NCR peptide expression vary greatly between nodules of M. truncatula accessions but show little variation within accessions in response to different strains of rhizobia (Nallu et al., 2014). ...
Leguminous plants possess the almost unique ability to enter symbiosis with soil-resident, nitrogen fixing bacteria called rhizobia. During this symbiosis, the bacteria physically colonize specialized organs on the roots of the host plant called nodules, where they reduce atmospheric nitrogen into forms that can be assimilated by the host plant and receive photosynthates in return. In order for nodule development to occur, there is extensive chemical cross-talk between both parties during the formative stages of the symbiosis. The vast majority of the legume family are capable of forming root nodules and typically rhizobia are only able to fix nitrogen within the context of this symbiotic association. However, many legume species only enter productive symbiosis with a few, or even single rhizobial species or strains, and vice-versa. Permitting symbiosis with only rhizobial strains that will be able to fix nitrogen with high efficiency is a crucial strategy for the host plant to prevent cheating by rhizobia. This selectivity is enforced at all stages of the symbiosis, with partner choice beginning during the initial communication between the plant and rhizobia. However, it can also be influenced even once nitrogen-fixing nodules have developed on the root. This review sets out current knowledge about the molecular mechanisms employed by both parties to influence host range during legume-rhizobia symbiosis.
... Exopolysaccharide-deficient mutants of rhizobia that infect plants producing indeterminate-type nodules (e.g., Rhizobium leguminosarum and Sinorhizobium meliloti) were compromised in their ability to induce root hair curling and to form infection-threads (Leigh et al., 1985;Cheng and Walker, 1998;Niehaus and Becker, 1998;Pellock et al., 2000). In addition, exopolysaccharides represent the major component of the biofilm matrix and are thus important for bacterial attachment to surfaces, protection against environmental stresses and antimicrobial compounds, and evasion of host defense responses (Santos et al., 2001;Downie, 2010;Ferreira et al., 2010Ferreira et al., , 2011Geddes et al., 2014;Maroti et al., 2015;Arnold et al., 2018). Some polysaccharides have also been shown to suppress plant defense responses and to be involved in plant signaling by binding to specific membrane-spanning receptorlike proteins with significant similarity to Nod factor receptor 1 (NFR1) (Kawaharada et al., 2015). ...
Paraburkholderia phymatum is a rhizobial strain that belongs to the beta-proteobacteria, a group known to form efficient nitrogen-fixing symbioses within root nodules of several legumes, including the agriculturally important common bean. The establishment of the symbiosis requires the exchange of rhizobial and plant signals such as lipochitooligosaccharides (Nod factors), polysaccharides, and flavonoids. Inspection of the genome of the competitive rhizobium P. phymatum revealed the presence of several polysaccharide biosynthetic gene clusters. In this study, we demonstrate that bceN, a gene encoding a GDP-D-mannose 4,6-dehydratase, which is involved in the production of the exopolysaccharide cepacian, an important component of biofilms produced by closely related opportunistic pathogens of the Burkholderia cepacia complex (Bcc), is required for efficient plant colonization. Wild-type P. phymatum was shown to produce cepacian while a bceN mutant did not. Additionally, the bceN mutant produced a significantly lower amount of biofilm and formed less root nodules compared to the wild-type strain with Phaseolus vulgaris as host plant. Finally, expression of the operon containing bceN was induced by the presence of germinated P. vulgaris seeds under nitrogen limiting conditions suggesting a role of this polysaccharide in the establishment of this ecologically important symbiosis.
... Small peptides play critical roles in cell proliferation, differentiation and mediate biotic and abiotic stress stimulation with metabolic intermediates (Mandal et al., 2008). Small signalling cysteine rich peptides have also been reported to play essential roles in stomatal patterning and density, symbiosis and a wide range of reproductive events viz., pollen tube formation, guidance and burst, activation of gametes and seed development (Hara et al., 2007;Sugano et al., 2010;Maróti et al., 2015;Bircheneder & Dresselhaus, 2016). Few of these have been isolated and characterized in details. ...
Plants confront an array of environmental stresses by tightly regulating their signalling pathways involving low molecular weight peptide synthesis. In the conducted study, initially growth parameters and ion contents were measured from salt stressed and haloprimed seedlings of blackgram (Vigna mungo L.) and pigeonpea (Cajanus cajan L.). Seedlings raised from nonprimed and haloprimed seeds were grown in hydroponic solution supplemented with desired concentration of NaCl for 3 weeks under controlled physiological conditions. Attempts were made to isolate low molecular weight peptide(s) from non-stressed, NaCl stressed and haloprimed seedlings of blackgram and pigeonpea, to quantify their variations and detect their molecular weight using HPLC and MALDI-TOF analysis respectively. Free radical scavenging activities of these peptides were studied under NaCl exposure. Subsequent bioassays were performed to determine the effect of these peptides on their respective physiological parameters. Peptide abundance was maximum in control seedlings of both the cultivars, which under NaCl stress became scanty. CD spectroscopic analysis confirmed reduced secondary conformations and more unordered peptides under NaCl stress. Haloprimed seedlings recovered such adversities to variable extents, resulting in improved germination and growth of test seedlings.
... Defensin-like peptides called nodule-specific cysteine-rich peptides (NCR) can possess antimicrobial functions but also control rhizobial differentiation to increase efficiency of nitrogen fixation in root nodules of legumes (Maroti et al., 2015). Typical motifs for NCRs were found for Os04g0381500 ( Figure S2), which was 1.5 fold upregulated by the endophyte but not modulated by Xoo (Table S3) and could have a potential role in Azoarcus-rice mutualism. ...
Research on the interaction between the non-nodule-forming bacterial endophytes and their host plants is still in its infancy. Especially the understanding of plant control mechanisms which govern endophytic colonization is very limited. The current study sets out to determine which hormonal signaling pathway controls endophytic colonization in rice, and whether the mechanisms deviate for a pathogen. The endophyte Azoarcus olearius BH72—rice model was used to investigate root responses to endophytes in comparison to the recently established pathosystem of rice blight Xanthomonas oryzae pv. oryzae PXO99 (Xoo) in flooded roots. In the rice root transcriptome, 523 or 664 genes were found to be differentially expressed in response to Azoarcus or Xoo colonization, respectively; however, the response was drastically different, with only 6% of the differentially expressed genes (DEGs) overlapping. Overall, Xoo infection induced a much stronger defense reaction than Azoarcus colonization, with the latter leading to down-regulation of many defense related DEGs. Endophyte-induced DEGs encoded several enzymes involved in phytoalexin biosynthesis, ROS (reactive oxygen species) production, or pathogenesis-related (PR) proteins. Among putative plant markers related to signal transduction pathways modulated exclusively during Azoarcus colonization, none overlapped with previously published DEGs identified for another rice endophyte, Azospirillum sp. B510. This suggests a large variation in responses of individual genotypic combinations. Interestingly, the DEGs related to jasmonate (JA) signaling pathway were found to be consistently activated by both beneficial endophytes. In contrast, the salicylate (SA) pathway was activated only in roots infected by the pathogen. To determine the impact of SA and JA production on root colonization by the endophyte and the pathogen, rice mutants with altered hormonal responses were employed: mutant cpm2 deficient in jasmonate synthesis, and RNA interference (RNAi) knockdown lines of NPR1 decreased in salicylic acid-mediated defense responses (NPR1-kd). Only in cpm2, endophytic colonization of Azoarcus was significantly increased, while Xoo colonization was not affected. Surprisingly, NPR1-kd lines showed slightly decreased colonization by Xoo, contrary to published results for leaves. These outcomes suggest that JA but not SA signaling is involved in controlling the Azoarcus endophyte density in roots and can restrict internal root colonization, thereby shaping the beneficial root microbiome.
... In this regards , various thaumatin-like proteins have been reported to generate tolerance to fungal pathogens Arabidopsis (41), potato (42) and grape (43). Moreover, cysteine-rich proteins have been informed to confer resistance for fungal diseases such as maize fungal pathogens (44) and legume modules (45)(46)(47). ...
For the majority of world populations, wheat (Triticum aestivum L.) would be the first essential and economic cereal grain crop. Pests and pathogens in both rich and developing countries are constantly threatening wheat production and sustainable development. Multiple gene pathways were recorded to share an association with fungal pathogens with wheat biological resistance. Our aim to use such tools in order to detect and classify fungal resistance genes in wheat through sequence alignment, protein domain identification and phylogenetic analysis. In addition the introduction for restriction fragment length polymorphism (RFLP) for such genes in the new primer database. Approximately 138 sequences of DNA were recovered from the wheat genome by aligning 3845 anti-fungal amino acids through tblastn tool. The NCBI blastn online tool used to detect sequences with functional genes, where 92 genes have been detected. The total number of nucleotides was 48385, where the smallest DNA sequence have 302 bp and the longest contains 977 bp with an average length of 525.9 bp per sequence. The wheat chromosomes 3D, and 4B have the highest number of sequences (9) followed by chromosomes 3B (7) and 3A(6), where wheat genomes A, B and D have 30, 35 and 27 genes, respectively. Five different amino acids motifs have been revealed among studied wheat amino acid sequences. The gene annotation tools used to infer studied amino acid gene annotation. Amino acid sequences belongs to lectin, kinase, tyrosine-protein kinase (STK), thaumatin, and cysteine-rich repeats representing 2, 9, 8, 19, 23 genes respectively, in addition to 31 hypothetical genes. The proteins chemical content have been assessed through 16 different amino acid chemical and physical characteristics.
Adaptive mechanisms for unfavorable environments have evolved in plants for thousands of generations, primarily in the form of endogenous chemical signals and the coordination of physiological processes. Signaling peptides (SPs) are diverse molecular messengers in various stress responses which have been identified in different plant families. SPs are recognized by the membrane-localized receptors and co-receptors, leading to downstream signaling for various plant responses. Progress in in silico analysis, along with other factors, has increased our understanding of the signaling peptide-mediated regulatory mechanisms underlying the entire plant life cycle. SPs mediate both long-distance (root-to-shoot-to-root) and local cell–cell communication via vascular system to communicate and coordinate with plant organs at distant locations. During abiotic stress, SPs inside plant cells perceive stress signals and transfer information at short and long physiological ranges through the signal transduction pathway, causing stress-responsive gene expression. SPs interact with pathogens and mediate cell-to-cell communication via signaling pathways. There are intriguing relationships between phytohormones and the secondary signaling cascades which are mediated by SPs. During biotic or abiotic stress, different peptides trigger jasmonic acid, ethylene, and ABA signaling, involving several secondary messengers. These messengers mediate the stress response via shared signaling components of ROS, Ca²⁺, and MAPKs, and they modify the gene expression for different phytohormones. In this review, we highlight current knowledge on the role of signaling peptides in plant adaptation, growth, and development. We aim to analyze the SP-receptor interactions and the significance of crosstalk between a few sample SPs and phytohormones. Potential directions on how scientists can use this information for crop improvement are also suggested.
The legume species Astragalus sinicus (Chinese milk vetch, CMV) has been widely cultivated for centuries in southern China as one of the most important green manures/cover crops to improve rice productivity and prevent soil degeneration. In this study, we generated the first chromosome-scale reference genome of CMV using a combination of PacBio, Illumina sequencing, and high-throughput chromatin conformation capture(Hi-C) technology. The CMV genome was 595.52 Mb, with a contig N50 size of 1.50 Mb. Long terminal repeats (LTR) were amplified and contributed to the genome size expansion in CMV. CMV underwent two whole-genome duplication (WGD) events, and the retained genes following WGD, which were shared by Papilionoideae species, shaped the hormonal regulation of nodulation and the symbiosis with rhizobia. The chalcone synthase (CHS) gene family was expanded and expressed primarily in the root of CMV. Intriguingly, we found that R genes were highly expressed in root than in the nodules of legume species, suggesting that they may be hired to increased plant immunity in root to cope with pathogen infection in legume species. Our work sheds light on the genetic basis of nodulation and symbiosis in CMV and provides a benchmark for accelerating genetic research and molecular breeding in the future.
The alphaproteobacterium Sinorhizobium meliloti secretes two acidic exopolysaccharides (EPS), succinoglycan (EPSI) and galactoglucan (EPSII), which differentially enable it to adapt to a changing environment. Succinoglycan is essential for invasion of plant hosts, and thus for formation of nitrogen-fixing root nodules. Galactoglucan is critical for population-based behaviors such as swarming and biofilm formation, and can facilitate invasion in the absence of succinoglycan on some host plants. Biosynthesis of galactoglucan is not as completely understood as that of succinoglycan. We devised a pipeline to: identify putative pyruvyltransferase and acetyltransferase genes; construct genomic deletions in strains engineered to produce either succinoglycan or galactoglucan; and analyze EPS from mutant bacterial strains. EPS samples were examined by ¹³ C cross-polarization magic-angle spinning (CPMAS) solid-state nuclear magnetic resonance (NMR). CPMAS NMR is uniquely suited to defining chemical composition in complex samples and enable detection and quantification of distinct EPS functional groups. Galactoglucan was isolated from mutant strains, with deletions in five candidate acyl/acetyltransferase genes ( exoZ , exoH , SMb20810 , SMb21188 , SMa1016 ) and a putative pyruvyltransferase ( wgaE or SMb21322). Most samples were similar in composition to wild-type EPSII by CPMAS NMR analysis. However, galactoglucan produced from a strain lacking wgaE exhibited a significant reduction in pyruvylation. Pyruvylation was restored through ectopic expression of plasmid-encoded wgaE . Our work has thus identified WgaE as a galactoglucan pyruvyltransferase. This exemplifies how the systematic combination of genetic analyses and solid-state NMR detection is a rapid means to identify genes responsible for modification of rhizobial exopolysaccharides.
IMPORTANCE
Nitrogen-fixing bacteria are crucial for geochemical cycles and global nitrogen nutrition. Symbioses between legumes and rhizobial bacteria establish root nodules, where bacteria convert dinitrogen to ammonia for plant utilization. Secreted exopolysaccharides (EPS) produced by Sinorhizobium meliloti (succinoglycan and galactoglucan) play important roles in soil and plant environments. Biosynthesis of galactoglucan is not as well characterized as succinoglycan. We employed solid-state nuclear magnetic resonance (NMR) to examine intact EPS from wild type and mutant S. meliloti strains. NMR analysis of EPS isolated from a wgaE gene mutant revealed a novel pyruvyltransferase that modifies galactoglucan. Few EPS pyruvyltransferases have been characterized. Our work provides insight into biosynthesis of an important S. meliloti EPS and expands knowledge of enzymes that modify polysaccharides.
Legumes form two types of root organs in response to signals from microbes, namely, nodules and root galls. In the field, these interactions occur concurrently and often interact with each other. The outcomes of these interactions vary and can depend on natural variation in rhizobia and nematode populations in the soil as well as abiotic conditions. While rhizobia are symbionts that contribute fixed nitrogen to their hosts, parasitic root-knot nematodes (RKN) cause galls as feeding structures that consume plant resources without a contribution to the plant. Yet, the two interactions share similarities, including rhizosphere signaling, repression of host defense responses, activation of host cell division, and differentiation, nutrient exchange, and alteration of root architecture. Rhizobia activate changes in defense and development through Nod factor signaling, with additional functions of effector proteins and exopolysaccharides. RKN inject large numbers of protein effectors into plant cells that directly suppress immune signaling and manipulate developmental pathways. This review examines the molecular control of legume interactions with rhizobia and RKN to elucidate shared and distinct mechanisms of these root-microbe interactions. Many of the molecular pathways targeted by both organisms overlap, yet recent discoveries have singled out differences in the spatial control of expression of developmental regulators that may have enabled activation of cortical cell division during nodulation in legumes. The interaction of legumes with symbionts and parasites highlights the importance of a comprehensive view of root-microbe interactions for future crop management and breeding strategies.
[Formula: see text] Copyright © 2021 The Author(s). This is an open access article distributed under the CC BY-NC-ND 4.0 International license .
Plants are continuously exposed to various biotic and abiotic factors that may trigger cascade reactions aimed at maintaining homeostasis. One of the most important components of plant protection from biotic factors is the synthesis of antimicrobial peptides (AMPs). AMPs are a large group of peptides present in insects, animals and plants. Plant innate immunity is provided by AMPs from different families that are categorized according to sequence similarity, the number and order of amino acid residues, and the tertiary structure of the mature peptide. AMPs may also participate in plant response to abiotic stresses such as high salinity, drought, high or low temperature, and heavy metals. In nitrogen-fixing nodules of some members of the Fabaceae family, AMP-like molecules named NCR peptides promote the differentiation of the symbiotic bacteria into bacteroids. Thus, AMPs are used by plants for fine tuning their responses to biotic and abiotic factors alike.
Food spoilage resulting from the presence of yeast is a common problem in the food industry. The development of natural food preservatives is a growing area of interest for the food industry. The application of antimicrobial peptides derived from plants can be a simple and natural method of preserving food. This study looked at the antiyeast activity of two chemically synthesised radish antimicrobial peptides, Rs-AFP1 and Rs-AFP2, for their inhibitory effect against different yeast species. The minimum inhibitory concentration (MIC) of both peptides was generated. Two mechanisms of action were studied (membrane permeabilisation and the overproduction of reactive oxygen species (ROS)) and both were found to occur with Rs-AFP2, while only the overproduction of ROS was detected for Rs-AFP1. The effect of the peptides on the yeast cells was also visualised by scanning electron microscopy. Their safety in terms of human consumption was studied and no adverse effects were found. Lastly, the stability of the peptides in different conditions, such as high salt, heat and a range of pH were studied in addition to their antiyeast activity in different food matrices such as soft drink, fruit juices and salad dressing, further supporting the peptides’ potential for use in food preservation.
Lipid transfer proteins (LTPs) are secreted cysteine‐rich proteins highly distributed among the plant kingdom, characterized by lipid‐binding capacity and possessing antimicrobial activity. Several biological roles have been proposed for LTPs in plants, including response to biotic stress. In Medicago truncatula, the N5 LTP has been proved to be necessary for the establishment of a successful symbiosis with Sinorhizobium meliloti by limiting bacterial spread at the epidermal level and promoting nodule primordia invasion. We identified two novel MtLTPs, named LTP3 and LTP7 highly similar to MtN5 for the amino acid sequence and the cysteine pattern. Their putative promoter regulatory regions contain response elements related to rhizobial symbiosis. We demonstrated that the expression of these LTP genes is induced after infection, particularly in nodule primordia and mature nodules. LTP7‐silencing impaired nodule formation: the number of nodules in MtLTP7‐silenced roots was reduced by ∼60% as compared with control roots. When roots were inoculated with mycorrhiza, we did not observe any change in LTP3 and LTP7 expression, suggesting that these LTPs specifically respond to the rhizobial symbiont. These data suggest that LTPs might represent a novel group of proteins involved in the regulation of the symbiosis. In this chapter, we propose several hypotheses about the mechanisms of action of these LTPs.
In this chapter, the authors show that in the symbiosis‐defective dnf1 of Medicago truncatula, bacteroid, and symbiosome development are blocked. The dnf1 gene was identified as encoding a subunit of a signal peptidase complex that is highly expressed in nodules. By analyzing data from whole‐genome expression, the authors propose that correct symbiosome development in M. truncatula requires the orderly secretion of protein constituents through coordinated upregulation of a nodule‐specific pathway exemplified by DNF1. Cell‐wall‐bound infection threads (ITs) in these cells grow toward and penetrate cells that are newly added to the central tissue by the meristem. Unwalled infection droplets extrude from the ITs, after which the bacteria are endocytosed into the cytoplasm; The rhizobia thus become surrounded by a plant‐derived membrane and form organelle‐like symbiosomes. The authors also show that the formation of the organelle‐like N2‐fixing symbiosomes is controlled by DMI2.
Actinorhizal plant growth in pioneer ecosystems depends on the symbiosis with the nitrogen-fixing actinobacterium Frankia cells that are housed in special root organs called nodules. Nitrogen fixation occurs in differentiated Frankia cells known as vesicles. Vesicles lack a pathway for assimilating ammonia beyond the glutamine stage and are supposed to transfer reduced nitrogen to the plant host cells. However, a mechanism for the transfer of nitrogen-fixation products to the plant cells remains elusive. Here, new elements for this metabolic exchange are described. We show that Alnus glutinosa nodules express defensin-like peptides, and one of these, Ag5, was found to target Frankia vesicles. In vitro and in vivo analyses showed that Ag5 induces drastic physiological changes in Frankia, including an increased permeability of vesicle membranes. A significant release of nitrogen-containing metabolites, mainly glutamine and glutamate, was found in N2-fixing cultures treated with Ag5. This work demonstrates that the Ag5 peptide is central for Frankia physiology in nodules and uncovers a novel cellular function for this large and widespread defensin peptide family.The ISME Journal advance online publication, 20 January 2015; doi:10.1038/ismej.2014.257.
The increasing number of multidrug-resistant microbes now emerging necessitates the identification of novel antimicrobial agents. Plants produce a great variety of antimicrobial peptides including hundreds of small, nodule-specific cysteine-rich NCR peptides that, in the legume Medicago truncatula, govern the differentiation of endosymbiotic nitrogen fixing bacteria and, in vitro, can display potent antibacterial activities. In this study, the potential candidacidal activity of 19 NCR peptides was investigated. Cationic NCR peptides having an isoelectric point above 9 were efficient in killing Candida albicans, one of the most common fungal pathogens of humans. None of the tested NCR peptides were toxic for immortalized human epithelial cells at concentrations that effectively killed the fungus; however, at higher concentrations, some of them inhibited the division of the cells. Furthermore, the cationic peptides successfully inhibited C. albicans induced human epithelial cell death in an in vitro coculture model. These results highlight the therapeutic potential of cationic NCR peptides in the treatment of candidiasis.
Background
Legumes form root nodules to house nitrogen fixing bacteria of the rhizobium family. The rhizobia are located intracellularly in the symbiotic nodule cells. In the legume Medicago truncatula these cells produce high amounts of Nodule-specific Cysteine-Rich (NCR) peptides which induce differentiation of the rhizobia into enlarged, polyploid and non-cultivable bacterial cells. NCRs are similar to innate immunity antimicrobial peptides. The NCR gene family is extremely large in Medicago with about 600 genes.
Results
Here we used the Medicago truncatula Gene Expression Atlas (MtGEA) and other published microarray data to analyze the expression of 334 NCR genes in 267 different experimental conditions. We find that all but five of these genes are expressed in nodules but in no other plant organ or in response to any other biotic interaction or abiotic stress tested. During symbiosis, none of the genes are induced by Nod factors. The NCR genes are activated in successive waves during nodule organogenesis, correlated with bacterial infection of the nodule cells and with a specific spatial localization of their transcripts from the apical to the proximal nodule zones. However, NCR expression is not associated with nodule senescence. According to their Shannon entropy, a measure expressing tissue specificity of gene expression, the NCR genes are among the most specifically expressed genes in M. truncatula. Moreover, when activated in nodules, their expression level is among the highest of all genes.
Conclusions
Together, these data show that the NCR gene expression is subject to an extreme tight regulation and is only activated during nodule organogenesis in the polyploid symbiotic cells.
Electronic supplementary material
The online version of this article (doi:10.1186/1471-2164-15-712) contains supplementary material, which is available to authorized users.
Since the beginning of the 90s lots of cationic plant, cysteine-rich antimicrobial peptides (AMP) have been studied. However, Broekaert et al. (1995) only coined the term "plant defensin," after comparison of a new class of plant antifungal peptides with known insect defensins. From there, many plant defensins have been reported and studies on this class of peptides encompass its activity toward microorganisms and molecular features of the mechanism of action against bacteria and fungi. Plant defensins also have been tested as biotechnological tools to improve crop production through fungi resistance generation in organisms genetically modified (OGM). Its low effective concentration towards fungi, ranging from 0.1 to 10 μM and its safety to mammals and birds makes them a better choice, in place of chemicals, to control fungi infection on crop fields. Herein, is a review of the history of plant defensins since their discovery at the beginning of 90s, following the advances on its structure conformation and mechanism of action towards microorganisms is reported. This review also points out some important topics, including: (i) the most studied plant defensins and their fungal targets; (ii) the molecular features of plant defensins and their relation with antifungal activity; (iii) the possibility of using plant defensin(s) genes to generate fungi resistant GM crops and biofungicides; and (iv) a brief discussion about the absence of products in the market containing plant antifungal defensins.
Significance
Intracellular endosymbiotic bacteria in diverse symbiotic systems are under the control of host-derived symbiosis-specific peptides. These peptides have mostly unknown activities. In the facultative rhizobium-legume symbiosis, the bacteria differentiate in many legumes to large polyploid noncultivable bacteroids. This terminal differentiation is achieved by concerted actions of hundreds of nodule-specific cysteine-rich (NCR) peptides. Although in vitro antimicrobial activities were demonstrated for a few NCR peptides, their mode of action in planta remained unexplored. Our work reveals a complex interaction network of NCR247, which affects bacterial cell division machinery, translation, and protein folding. These findings give an insight how the host can modulate the physiology of the endosymbionts and may serve as a paradigm for other symbiotic systems.
The nodule cysteine-rich (NCR) groups of Defensin-Like (DEFL) genes are one of the largest gene families expressed in the nodules of some legume plants. They have only been observed in the Inverted Repeat Loss Clade (IRLC) of legumes which includes the model legume Medicago truncatula. NCRs are reported to play an important role in the plant-microbe interactions. To understand their diversity we analyzed their expression and sequence polymorphisms among four accessions of M. truncatula. A significant expression and nucleotide variation was observed among the genes. We then used twenty-six accessions to estimate the selection pressures shaping evolution among the accessions by calculating the nucleotide diversity at non-synonymous and synonymous sites in the coding region. The mature peptides of the orthologous NCRs had signatures of both purifying and diversifying selection pressures, unlike the seed DEFLs, which predominantly exhibited purifying selection. The expression, sequence variation, and apparent diversifying selection in NCRs within the Medicago species indicates rapid and recent evolution and suggests that this family of genes is actively evolving to adapt to different environments and acquiring new functions.This article is protected by copyright. All rights reserved.
Significance
Sinorhizobium meliloti and its legume hosts establish a symbiosis in which bacterial fixed nitrogen is exchanged for plant carbon compounds. We study this symbiosis because it is agriculturally and ecologically important and to identify mechanisms used in host–microbe interactions. S. meliloti is internalized in specialized host nodule cells that then use small, cysteine-rich peptides to drive their differentiation into polyploid cells that fix nitrogen. We found that a representative host peptide blocks cell division, in part by eliciting significant changes in the expression of genes that regulate the cell cycle and mediate cell division. We also found that the peptide activated pathways conserved in related pathogens. Our study provides insights into how host peptides cause differentiation of S. meliloti during symbiosis.
Rhizobium-induced root nodules are organs specialized for symbiotic nitrogen fixation. Indeterminate-type nodules are formed from an apical meristem and exhibit a spatial zonation which corresponds to successive developmental stages. To get a dynamic and integrated view of plant and bacterial gene expression associated with nodule development, we set up a sensitive and comprehensive approach based upon oriented high depth RNA-seq coupled to laser micro-dissection of nodule regions. This study, focused on the association between the model legume Medicago truncatula and its symbiont Sinorhizobium meliloti, led to the production of 942 million sequencing read pairs that were unambiguously mapped on plant and bacterial genomes. Bioinformatic and statistical analyses enabled in-depth comparison, at a whole genome level, of gene expression in specific nodule zones. Previously characterized symbiotic genes displayed the expected spatial pattern of expression, thus validating the robustness of the approach. We illustrate the interest of this resource by examining gene expression associated with three essential elements of nodule development, namely meristem activity, cell differentiation and selected signaling processes related to bacterial Nod factors and to the redox status. We found that transcription factor genes essential for the control of the root apical meristem were also expressed in the nodule meristem, while plant mRNAs most enriched in nodules compared to roots were mostly associated with zones comprising both plant and bacterial partners. The data, accessible on a dedicated web site, represent a rich resource for microbiologists and plant biologists to address a variety of questions of both fundamental and applied interest. This article is protected by copyright. All rights reserved.
MtDef4 is a 47-amino acid cysteine-rich evolutionary conserved defensin from a model legume Medicago truncatula. It is an apoplast-localized plant defense protein that inhibits the growth of the ascomycetous fungal pathogen Fusarium graminearum in vitro at micromolar concentrations. Little is known about the mechanisms by which MtDef4 mediates its antifungal activity. In this study, we show that MtDef4 rapidly permeabilizes fungal plasma membrane and is internalized by the fungal cells where it accumulates in the cytoplasm. Furthermore, analysis of the structure of MtDef4 reveals the presence of a positively charged γ-core motif composed of β2 and β3 strands connected by a positively charged RGFRRR loop. Replacement of the RGFRRR sequence with AAAARR or RGFRAA abolishes the ability of MtDef4 to enter fungal cells, suggesting that the RGFRRR loop is a translocation signal required for the internalization of the protein. MtDef4 binds to phosphatidic acid (PA), a precursor for the biosynthesis of membrane phospholipids and a signaling lipid known to recruit cytosolic proteins to membranes. Amino acid substitutions in the RGFRRR sequence which abolish the ability of MtDef4 to enter fungal cells also impair its ability to bind PA. These findings suggest that MtDef4 is a novel antifungal plant defensin capable of entering into fungal cells and affecting intracellular targets and that these processes are mediated by the highly conserved cationic RGFRRR loop via its interaction with PA.
Transcription factors (TFs) are thought to regulate many aspects of nodule and symbiosis development in legumes, although few TFs have been characterized functionally. Here, we describe REGULATOR OF SYMBIOSOME DIFFERENTIATION (RSD) of Medicago truncatula, a member of the Cysteine-2/Histidine-2 (C2H2) family of plant TFs that is required for normal symbiosome differentiation during nodule development. RSD is expressed in a nodule-specific manner, with maximal transcript levels in the bacterial invasion zone. A tobacco (Nicotiana tabacum) retrotransposon (Tnt1) insertion rsd mutant produced nodules that were unable to fix nitrogen and that contained incompletely differentiated symbiosomes and bacteroids. RSD protein was localized to the nucleus, consistent with a role of the protein in transcriptional regulation. RSD acted as a transcriptional repressor in a heterologous yeast assay. Transcriptome analysis of an rsd mutant identified 11 genes as potential targets of RSD repression. RSD interacted physically with the promoter of one of these genes, VAMP721a, which encodes vesicle-associated membrane protein 721a. Thus, RSD may influence symbiosome development in part by repressing transcription of VAMP721a and modifying vesicle trafficking in nodule cells. This establishes RSD as a TF implicated directly in symbiosome and bacteroid differentiation and a transcriptional regulator of secretory pathway genes in plants.
Although evidence has accumulated on the role of plant peptides in the response to external conditions, the number of peptide-encoding
genes in the genome is still underestimated. Using tiling arrays, we identified 176 unannotated transcriptionally active regions
(TARs) in Arabidopsis thaliana that were induced upon oxidative stress generated by the herbicide paraquat (PQ). These 176 TARs could be translated into
575 putative oxidative stress-induced peptides (OSIPs). A high-throughput functional assay was used in the eukaryotic model
organism Saccharomyces cerevisiae allowing us to test for bioactive peptides that increase oxidative stress tolerance. In this way, we identified three OSIPs
that, upon overexpression in yeast, resulted in a significant rise in tolerance to hydrogen peroxide (H2O2). For one of these peptides, the decapeptide OSIP108, exogenous application to H2O2-treated yeast also resulted in significantly increased survival. OSIP108 is contained within a pseudogene and is induced in A. thaliana leaves by both the reactive oxygen species-inducer PQ and the necrotrophic fungal pathogen Botrytis cinerea. Moreover, infiltration and overexpression of OSIP108 in A. thaliana leaves resulted in increased tolerance to treatment with PQ. In conclusion, the identification and characterization of OSIP108
confirms the validity of our high-throughput approach, based on tiling array analysis in A. thaliana and functional screening in yeast, to identify bioactive peptides.
Symbiosis between Rhizobium bacteria and legumes leads to the formation of the root nodule. The endosymbiotic bacteria reside in polyploid host cells as membrane-surrounded vesicles where they reduce atmospheric nitrogen to support plant growth by supplying ammonia in exchange for carbon sources and energy. The morphology and physiology of endosymbionts, despite their common function, are highly divergent in different hosts. In galegoid plants, the endosymbionts are terminally differentiated, uncultivable polyploid cells, with remarkably elongated and even branched Y-shaped cells. Bacteroid differentiation is controlled by host peptides, many of which have antibacterial activity and require the bacterial function of BacA. Although the precise and combined action of several hundred host peptides and BacA has yet to be discovered, similarities, especially to certain insect-bacterium symbioses involving likewise host peptides for manipulation of endosymbionts, suggest convergent evolution. Rhizobium-legume symbiosis provides a rich source of information for understanding host-controlled endosymbiotic life in eukaryotic cells.
Leguminous plants establish symbiosis with nitrogen-fixing α- and β-Proteobacteria, collectively called rhizobia, which provide combined nitrogen to support plant growth. Members of the Inverted Repeat-Lacking Clade of legumes impose terminal differentiation on their endosymbiotic bacterium partners with the help of the nodule-specific cysteine-rich (NCR) peptide family composed of close to 600 members. Among the few tested NCR peptides, cationic ones had anti-rhizobial activity measured by reduction or elimination of the colony forming units and uptake of the membrane impermeable dye, propidium iodide. Here, the antimicrobial spectrum of two of these peptides, NCR247 and NCR335, were investigated as well as their effect on the transcriptome of the natural target Sinorhizobium meliloti was characterized. Both peptides were able to kill quickly a wide range of Gram-negative and Gram-positive bacteria, however their spectra were only partially overlapping and differencies were found also in their efficacy on given strains indicating that the action of NCR247 and NCR335 might be similar though not identical. Treatment of S. meliloti cultures with either peptide resulted in a quick down-regulation of genes involved in basic cellular functions such as transcription-translation and energy production as well as up-regulation of genes involved in stress and oxidative stress responses and membrane transport. Similar changes provoked mainly in Gram-positive bacteria by antimicrobial agents were coupled with the destruction of membrane potential indicating that it might also be a common step in bactericidal action of NCR247 and NCR335.
Plant genomes contain several hundred defensin-like (DEFL) genes that encode short cysteine-rich proteins resembling defensins, which are well known antimicrobial polypeptides. Little is known about the expression patterns or functions of many DEFLs because most were discovered recently and hence are not well represented on standard microarrays. We designed a custom Affymetrix chip consisting of probe sets for 317 and 684 DEFLs from Arabidopsis thaliana and Medicago truncatula, respectively for cataloging DEFL expression in a variety of plant organs at different developmental stages and during symbiotic and pathogenic associations. The microarray analysis provided evidence for the transcription of 71% and 90% of the DEFLs identified in Arabidopsis and Medicago, respectively, including many of the recently annotated DEFL genes that previously lacked expression information. Both model plants contain a subset of DEFLs specifically expressed in seeds or fruits. A few DEFLs, including some plant defensins, were significantly up-regulated in Arabidopsis leaves inoculated with Alternaria brassicicola or Pseudomonas syringae pathogens. Among these, some were dependent on jasmonic acid signaling or were associated with specific types of immune responses. There were notable differences in DEFL gene expression patterns between Arabidopsis and Medicago, as the majority of Arabidopsis DEFLs were expressed in inflorescences, while only a few exhibited root-enhanced expression. By contrast, Medicago DEFLs were most prominently expressed in nitrogen-fixing root nodules. Thus, our data document salient differences in DEFL temporal and spatial expression between Arabidopsis and Medicago, suggesting distinct signaling routes and distinct roles for these proteins in the two plant species.
Antimicrobial peptides are a vital component of the innate immune system of all eukaryotic organisms and many of these peptides have potent antifungal activity. They have potential application in the control of fungal pathogens that are a serious threat to both human health and food security. Development of antifungal peptides as therapeutics requires an understanding of their mechanism of action on fungal cells. To date, most research on antimicrobial peptides has focused on their activity against bacteria. Several antimicrobial peptides specifically target fungal cells and are not active against bacteria. Others with broader specificity often have different mechanisms of action against bacteria and fungi. This review focuses on the mechanism of action of naturally occurring antifungal peptides from a diverse range of sources including plants, mammals, amphibians, insects, crabs, spiders, and fungi. While antimicrobial peptides were originally proposed to act via membrane permeabilization, the mechanism of antifungal activity for these peptides is generally more complex and often involves entry of the peptide into the cell.
Defensins are a class of small and diverse cysteine-rich proteins found in plants, insects, and vertebrates, which share a common tertiary structure and usually exert broad-spectrum antimicrobial activities. We used a bioinformatic approach to scan the Vitis vinifera genome and identified 79 defensin-like sequences (DEFL) corresponding to 46 genes and allelic variants, plus 33 pseudogenes and gene fragments. Expansion and diversification of grapevine DEFL has occurred after the split from the last common ancestor with the genera Medicago and Arabidopsis. Grapevine DEFL localization on the 'Pinot Noir' genome revealed the presence of several clusters likely evolved through local duplications. By sequencing reverse-transcription polymerase chain reaction products, we could demonstrate the expression of grapevine DEFL with no previously reported record of expression. Many of these genes are predominantly or exclusively expressed in tissues linked to plant reproduction, consistent with findings in other plant species, and some of them accumulated at fruit ripening. The transcripts of five DEFL were also significantly upregulated in tissues infected with Botrytis cinerea, a necrotrophic mold, suggesting a role of these genes in defense against this pathogen. Finally, three novel defensins were discovered among the identified DEFL. They inhibit B. cinerea conidia germination when expressed as recombinant proteins.
The root nodules of certain legumes including Medicago truncatula produce >300 different nodule-specific cysteine-rich (NCR) peptides. Medicago NCR antimicrobial peptides (AMPs) mediate the differentiation of the bacterium, Sinorhizobium meliloti into a nitrogen-fixing bacteroid within the legume root nodules. In vitro, NCR AMPs such as NCR247 induced bacteroid features and exhibited antimicrobial activity against S. meliloti. The bacterial BacA protein is critical to prevent S. meliloti from being hypersensitive toward NCR AMPs. NCR AMPs are cationic and have conserved cysteine residues, which form disulfide
(S–S) bridges. However, the natural configuration of NCR AMP S–S bridges and the role of these in the activity of the peptide
are unknown. In this study, we found that either cysteine replacements or S–S bond modifications influenced the activity of
NCR247 against S. meliloti. Specifically, either substitution of cysteines for serines, changing the S–S bridges from cysteines 1–2, 3–4 to 1–3, 2–4
or oxidation of NCR247 lowered its activity against S. meliloti. We also determined that BacA specifically protected S. meliloti against oxidized NCR247. Due to the large number of different NCRs synthesized by legume root nodules and the importance
of bacterial BacA proteins for prolonged host infections, these findings have important implications for analyzing the function
of these novel peptides and the protective role of BacA in the bacterial response toward these peptides.
Legumes (Fabaceae or Leguminosae) are unique among cultivated plants for their ability to carry out endosymbiotic nitrogen fixation with rhizobial bacteria, a process that takes place in a specialized structure known as the nodule. Legumes belong to one of the two main groups of eurosids, the Fabidae, which includes most species capable of endosymbiotic nitrogen fixation. Legumes comprise several evolutionary lineages derived from a common ancestor 60 million years ago (Myr ago). Papilionoids are the largest clade, dating nearly to the origin of legumes and containing most cultivated species. Medicago truncatula is a long-established model for the study of legume biology. Here we describe the draft sequence of the M. truncatula euchromatin based on a recently completed BAC assembly supplemented with Illumina shotgun sequence, together capturing ∼94% of all M. truncatula genes. A whole-genome duplication (WGD) approximately 58 Myr ago had a major role in shaping the M. truncatula genome and thereby contributed to the evolution of endosymbiotic nitrogen fixation. Subsequent to the WGD, the M. truncatula genome experienced higher levels of rearrangement than two other sequenced legumes, Glycine max and Lotus japonicus. M. truncatula is a close relative of alfalfa (Medicago sativa), a widely cultivated crop with limited genomics tools and complex autotetraploid genetics. As such, the M. truncatula genome sequence provides significant opportunities to expand alfalfa's genomic toolbox.
Plant defensins are small cysteine-rich antimicrobial proteins. Their three-dimensional structures are similar in that they consist of an α-helix and three anti-parallel β-strands stabilized by four disulfide bonds. Plant defensins MsDef1 and MtDef4 are potent inhibitors of the growth of several filamentous fungi including Fusarium graminearum. However, they differ markedly in their antifungal properties as well as modes of antifungal action. MsDef1 induces prolific hyperbranching of fungal hyphae, whereas MtDef4 does not. Both defensins contain a highly conserved γ-core motif (GXCX(3-9)C), a hallmark signature present in the disulfide-stabilized antimicrobial peptides, composed of β2 and β3 strands and the interposed loop. The γ-core motifs of these two defensins differ significantly in their primary amino acid sequences and in their net charge. In this study, we have found that the major determinants of the antifungal activity and morphogenicity of these defensins reside in their γ-core motifs. The MsDef1-γ4 variant in which the γ-core motif of MsDef1 was replaced by that of MtDef4 was almost as potent as MtDef4 and also failed to induce hyperbranching of fungal hyphae. Importantly, the γ-core motif of MtDef4 alone was capable of inhibiting fungal growth, but that of MsDef1 was not. The analysis of synthetic γ-core variants of MtDef4 indicated that the cationic and hydrophobic amino acids were important for antifungal activity. Both MsDef1 and MtDef4 induced plasma membrane permeabilization; however, kinetic studies revealed that MtDef4 was more efficient in permeabilizing fungal plasma membrane than MsDef1. Furthermore, the in vitro antifungal activity of MsDef1, MsDef1-γ4, MtDef4 and peptides derived from the γ-core motif of each defensin was not solely dependent on their ability to permeabilize the fungal plasma membrane. The data reported here indicate that the γ-core motif defines the unique antifungal properties of each defensin and may facilitate de novo design of more potent antifungal peptides.
The legume plant Medicago truncatula establishes a symbiosis with the nitrogen-fixing bacterium Sinorhizobium meliloti which takes place in root nodules. The formation of nodules employs a complex developmental program involving organogenesis, specific cellular differentiation of the host cells and the endosymbiotic bacteria, called bacteroids, as well as the specific activation of a large number of plant genes. By using a collection of plant and bacterial mutants inducing non-functional, Fix(-) nodules, we studied the differentiation processes of the symbiotic partners together with the nodule transcriptome, with the aim of unravelling links between cell differentiation and transcriptome activation. Two waves of transcriptional reprogramming involving the repression and the massive induction of hundreds of genes were observed during wild-type nodule formation. The dominant features of this "nodule-specific transcriptome" were the repression of plant defense-related genes, the transient activation of cell cycle and protein synthesis genes at the early stage of nodule development and the activation of the secretory pathway along with a large number of transmembrane and secretory proteins or peptides throughout organogenesis. The fifteen plant and bacterial mutants that were analyzed fell into four major categories. Members of the first category of mutants formed non-functional nodules although they had differentiated nodule cells and bacteroids. This group passed the two transcriptome switch-points similarly to the wild type. The second category, which formed nodules in which the plant cells were differentiated and infected but the bacteroids did not differentiate, passed the first transcriptome switch but not the second one. Nodules in the third category contained infection threads but were devoid of differentiated symbiotic cells and displayed a root-like transcriptome. Nodules in the fourth category were free of bacteria, devoid of differentiated symbiotic cells and also displayed a root-like transcriptome. A correlation thus exists between the differentiation of symbiotic nodule cells and the first wave of nodule specific gene activation and between differentiation of rhizobia to bacteroids and the second transcriptome wave in nodules. The differentiation of symbiotic cells and of bacteroids may therefore constitute signals for the execution of these transcriptome-switches.
The nitrogen-fixing symbiosis between Sinorhizobium meliloti and its leguminous host plant Medicago truncatula occurs in a specialized root organ called the nodule. Bacteria that are released into plant cells are surrounded by a unique
plant membrane compartment termed a symbiosome. We found that in the symbiosis-defective dnf1 mutant of M. truncatula, bacteroid and symbiosome development are blocked. We identified the DNF1 gene as encoding a subunit of a signal peptidase complex that is highly expressed in nodules. By analyzing data from whole-genome
expression analysis, we propose that correct symbiosome development in M. truncatula requires the orderly secretion of protein constituents through coordinated up-regulation of a nodule-specific pathway exemplified
by DNF1.
Legume Symbiosome
Leguminous plants (peas and beans) are major players in global nitrogen cycling by virtue of their symbioses with nitrogen-fixing bacteria that are harbored in specialized structures, called nodules, on the plant's roots. Van de Velde et al. (p. 1122 ) show that the host plant, Medicago truncatula produces nodule-specific cysteine-rich peptides, resembling natural plant defense peptides. The peptides enter the bacterial cells and promote its development into the mature symbiont. In a complementary study, D. Wang et al. (p. 1126 ), have identified the signal peptidase, also encoded by the plant, that is required for processing these specialized peptides into their active form.
Defensins are small cysteine-rich peptides with antimicrobial activity.
We demonstrate that the alfalfa antifungal peptide (alfAFP) defensin isolated
from seeds of Medicago sativa displays strong activity against the
agronomically important fungal pathogen Verticillium dahliae. Expression
of the alfAFP peptide in transgenic potato plants provides robust resistance
in the greenhouse. Importantly, this resistance is maintained under field
conditions. There have been no previous demonstrations of a single transgene
imparting a disease resistance phenotype that is at least equivalent to those
achieved through current practices using fumigants.
We isolated a stamen-specific cDNA, BSD1 (Brassica stamen specific plant defensin 1) that encodes a novel plant defensin peptide in Chinese cabbage (Brassica campestris L. ssp. pekinensis). Plant defensins are antimicrobial peptides containing eight highly conserved cysteine residues linked by disulfide bridges. In BSD1, the eight cysteine residues and a glutamate residue at position 29 are conserved whereas other amino acid residues of the plant defensins consensus sequence are substituted. BSD1 transcripts accumulate specifically in the stamen of developing flowers and its level drops as the flowers mature. The recombinant BSD1 produced in Escherichia coli showed antifungal activity against several phytopathogenic fungi. Furthermore, constitutive over-expression of the BSD1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter conferred enhanced tolerance against the Phytophthora parasitica in the transgenic tobacco plants.
A novel cDNA clone, Tad1, was isolated from crown tissue of winter wheat after differential screening of cold acclimation-induced genes. The Tad1 cDNA encoded a 23kDa polypeptide with a potential N-terminal signal sequence. The putative mature sequence showed striking similarity to plant defensins or gamma-thionins, representing low molecular size antipathogenic polypeptides. High levels of Tad1 mRNA accumulation occurred within one day of cold acclimation in crown tissue and the level was maintained throughout 14 days of cold acclimation. Similar rapid induction was observed in young seedlings treated with low temperature but not with exogenous abscisic acid. In contrast to defensins from other plant species, neither salicylic acid nor methyl jasmonate induced expression of Tad1. The recombinant mature form of TAD1 polypeptide inhibited the growth of the phytopathogenic bacteria, Pseudomonas cichorii; however, no antifreeze activity was detected. Collectively, these data suggested that Tad1 is induced in cold-acclimated winter wheat independent of major defense signaling(s) and is involved in low temperature-induced resistance to pathogens during winter hardening.
Plant defensins are small, basic peptides that have a characteristic three-dimensional folding pattern that is stabilized by eight disulfide-linked cysteines. They are termed plant defensins because they are structurally related to defensins found in other types of organism, including humans. To date, sequences of more than 80 different plant defensin genes from different plant species are available. In Arabidopsis thaliana, at least 13 putative plant defensin genes (PDF) are present, encoding 11 different plant defensins. Two additional genes appear to encode plant defensin fusions. Plant defensins inhibit the growth of a broad range of fungi but seem nontoxic to either mammalian or plant cells. Antifungal activity of defensins appears to require specific binding to membrane targets. This review focuses on the classification of plant defensins in general and in Arabidopsis specifically, and on the mode of action of plant defensins against fungal pathogens.
Transgenic rice ( Oryza sativa cv. Sasanishiki) overexpressing the wasabi defensin gene, a plant defensin effective against the rice blast fungus, was generated by Agrobacterium tumefaciens-mediated transformation. Twenty-two T2 homozygous lines harboring the wasabi defensin gene were challenged by the blast fungus. Transformants exhibited resistance to rice blast at various levels. The inheritance of the resistance over generations was investigated. T3 plants derived from two highly blast-resistant T2 lines (WT14-5 and WT43-5) were challenged with the blast fungus using the press-injured spots method. The average size of disease lesions of the transgenic line WT43-5 was reduced to about half of that of non-transgenic plants. The 5-kDa peptide, corresponding to the processed form of the wasabi defensin, was detected in the total protein fraction extracted from the T3 progeny. Transgenic rice plants overproducing wasabi defensin are expected to possess a durable and wide-spectrum resistance (i.e. field resistance) against various rice blast races.
Transcriptome analysis of Medicago truncatula nodules has led to the discovery of a gene family named NCR (nodule-specific cysteine rich) with more than 300 members. The encoded polypeptides were short (60-90 amino acids), carried a conserved signal peptide, and, except for a conserved cysteine motif, displayed otherwise extensive sequence divergence. Family members were found in pea (Pisum sativum), broad bean (Vicia faba), white clover (Trifolium repens), and Galega orientalis but not in other plants, including other legumes, suggesting that the family might be specific for galegoid legumes forming indeterminate nodules. Gene expression of all family members was restricted to nodules except for two, also expressed in mycorrhizal roots. NCR genes exhibited distinct temporal and spatial expression patterns in nodules and, thus, were coupled to different stages of development. The signal peptide targeted the polypeptides in the secretory pathway, as shown by green fluorescent protein fusions expressed in onion (Allium cepa) epidermal cells. Coregulation of certain NCR genes with genes coding for a potentially secreted calmodulin-like protein and for a signal peptide peptidase suggests a concerted action in nodule development. Potential functions of the NCR polypeptides in cell-to-cell signaling and creation of a defense system are discussed.
The Fabaceae, the third largest family of plants and the source of many crops, has been the target of many genomic studies. Currently, only the grasses surpass the legumes for the number of publicly available expressed sequence tags (ESTs). The quantity of sequences from diverse plants enables the use of computational approaches to identify novel genes in specific taxa. We used BLAST algorithms to compare unigene sets from Medicago truncatula, Lotus japonicus, and soybean (Glycine max and Glycine soja) to nonlegume unigene sets, to GenBank's nonredundant and EST databases, and to the genomic sequences of rice (Oryza sativa) and Arabidopsis. As a working definition, putatively legume-specific genes had no sequence homology, below a specified threshold, to publicly available sequences of nonlegumes. Using this approach, 2,525 legume-specific EST contigs were identified, of which less than three percent had clear homology to previously characterized legume genes. As a first step toward predicting function, related sequences were clustered to build motifs that could be searched against protein databases. Three families of interest were more deeply characterized: F-box related proteins, Pro-rich proteins, and Cys cluster proteins (CCPs). Of particular interest were the >300 CCPs, primarily from nodules or seeds, with predicted similarity to defensins. Motif searching also identified several previously unknown CCP-like open reading frames in Arabidopsis. Evolutionary analyses of the genomic sequences of several CCPs in M. truncatula suggest that this family has evolved by local duplications and divergent selection.
Plant defensins are a family of small Cys-rich antifungal proteins that play important roles in plant defense against invading fungi. Structures of several plant defensins share a Cys-stabilized alpha/beta-motif. Structural determinants in plant defensins that govern their antifungal activity and the mechanisms by which they inhibit fungal growth remain unclear. Alfalfa (Medicago sativa) seed defensin, MsDef1, strongly inhibits the growth of Fusarium graminearum in vitro, and its antifungal activity is markedly reduced in the presence of Ca(2+). By contrast, MtDef2 from Medicago truncatula, which shares 65% amino acid sequence identity with MsDef1, lacks antifungal activity against F. graminearum. Characterization of the in vitro antifungal activity of the chimeras containing portions of the MsDef1 and MtDef2 proteins shows that the major determinants of antifungal activity reside in the carboxy-terminal region (amino acids 31-45) of MsDef1. We further define the active site by demonstrating that the Arg at position 38 of MsDef1 is critical for its antifungal activity. Furthermore, we have found for the first time, to our knowledge, that MsDef1 blocks the mammalian L-type Ca(2+) channel in a manner akin to a virally encoded and structurally unrelated antifungal toxin KP4 from Ustilago maydis, whereas structurally similar MtDef2 and the radish (Raphanus sativus) seed defensin Rs-AFP2 fail to block the L-type Ca(2+) channel. From these results, we speculate that the two unrelated antifungal proteins, KP4 and MsDef1, have evolutionarily converged upon the same molecular target, whereas the two structurally related antifungal plant defensins, MtDef2 and Rs-AFP2, have diverged to attack different targets in fungi.
Plant defensins are small (c.a. 5 kDa), basic, cysteine-rich proteins with antimicrobial activities. They are ubiquitous in plants and form part of the innate immunity arsenal. Plant defensins are encoded by small multigene families and are expressed in various plant tissues, but are best characterized in seeds. They are typically produced as preproteins, however, a small subset are produced as larger precursors with C-terminal prodomains. To date, the three-dimensional solution structures of seven seed- and two floral-derived defensins have been elucidated by (1)H-NMR spectroscopy. Despite limited amino acid sequence identities, these defensins have comparable global folds with features that are characteristic of the cysteine-stabilized alphabeta (CSalphabeta) motif. Interestingly, their structures are remarkably similar to those of insect defensins and scorpion toxins. Functionally, these proteins exhibit a diverse array of biological activities, although they all serve a common function as defenders of their hosts. This review describes the distribution, biosynthesis, structure, function and mode of action of plant defensins and reflects on their potential in agribiotechnological applications.
Defensins represent an ancient and diverse set of small, cysteine-rich, antimicrobial peptides in mammals, insects, and plants. According to published accounts, most species' genomes contain 15 to 50 defensins. Starting with a set of largely nodule-specific defensin-like sequences (DEFLs) from the model legume Medicago truncatula, we built motif models to search the near-complete Arabidopsis (Arabidopsis thaliana) genome. We identified 317 DEFLs, yet 80% were unannotated at The Arabidopsis Information Resource and had no prior evidence of expression. We demonstrate that many of these DEFL genes are clustered in the Arabidopsis genome and that individual clusters have evolved from successive rounds of gene duplication and divergent or purifying selection. Sequencing reverse transcription-PCR products from five DEFL clusters confirmed our gene predictions and verified expression. For four of the largest clusters of DEFLs, we present the first evidence of expression, most frequently in floral tissues. To determine the abundance of DEFLs in other plant families, we used our motif models to search The Institute for Genomic Research's gene indices and identified approximately 1,100 DEFLs. These expressed DEFLs were found mostly in reproductive tissues, consistent with our reverse transcription-PCR results. Sequence-based clustering of all identified DEFLs revealed separate tissue- or taxon-specific subgroups. Previously, we and others showed that more than 300 DEFL genes were expressed in M. truncatula nodules, organs not present in most plants. We have used this information to annotate the Arabidopsis genome and now provide evidence of a large DEFL superfamily present in expressed tissues of all sequenced plants.
A large gene family encoding the putative cysteine-rich defensins was discovered in Medicago truncatula. Sixteen members of the family were identified by screening a cloned seed defensin from M. sativa (Gao et al. 2000) against the Institute for Genomic Research's (TIGR) M. truncatula gene index (MtGI version 7). Based on the comparison of their amino acid sequences, M. truncatula defensins fell arbitrarily into three classes displaying extensive sequence divergence outside of the eight canonical cysteine residues. The presence of Class II defensins is reported for the first time in a legume plant. In silico as well as Northern blot and RT-PCR analyses indicated these genes were expressed in a variety of tissues including leaves, flowers, developing pods, mature seed and roots. The expression of these genes was differentially induced in response to a variety of biotic and abiotic stimuli. For the first time, a defensin gene (TC77480) was shown to be induced in roots in response to infection by the mycorrhizal fungus, Glomus versiforme. Northern blot analysis indicated that the tissue-specific expression patterns of the cloned Def1 and Def2 genes differed substantially between M. truncatula and M. sativa. Furthermore, the induction profiles of the Def1 and Def2 genes in response to the signaling molecules methyl jasmonate, ethylene and salicylic acid differed markedly between these two legumes.
Symbiosis between legumes and Rhizobium bacteria leads to the formation of root nodules where bacteria in the infected plant cells are converted into nitrogen-fixing bacteroids. Nodules with a persistent meristem are indeterminate, whereas nodules without meristem are determinate. The symbiotic plant cells in both nodule types are polyploid because of several cycles of endoreduplication (genome replication without mitosis and cytokinesis) and grow consequently to extreme sizes. Here we demonstrate that differentiation of bacteroids in indeterminate nodules of Medicago and related legumes from the galegoid clade shows remarkable similarity to host cell differentiation. During bacteroid maturation, repeated DNA replication without cytokinesis results in extensive amplification of the entire bacterial genome and elongation of bacteria. This finding reveals a positive correlation in prokaryotes between DNA content and cell size, similar to that in eukaryotes. These polyploid bacteroids are metabolically functional but display increased membrane permeability and are nonviable, because they lose their ability to resume growth. In contrast, bacteroids in determinate nodules of the nongalegoid legumes lotus and bean are comparable to free-living bacteria in their genomic DNA content, cell size, and viability. Using recombinant Rhizobium strains nodulating both legume types, we show that bacteroid differentiation is controlled by the host plant. Plant factors present in nodules of galegoid legumes but absent from nodules of nongalegoid legumes block bacterial cell division and trigger endoreduplication cycles, thereby forcing the endosymbionts toward a terminally differentiated state. Hence, Medicago and related legumes have evolved a mechanism to dominate the symbiosis.
• antimicrobial activity
• bacteroid
• endoreduplication
• Medicago
• nitrogen fixation
We isolated a stamen-specific cDNA, BSD1 (Brassica stamen specific plant defensin 1) that encodes a novel plant defensin peptide in Chinese cabbage (Brassica campestris L. ssp. pekinensis). Plant defensins are antimicrobial peptides containing eight highly conserved cysteine residues linked by disulfide bridges. In BSD1, the eight cysteine residues and a glutamate residue at position 29 are conserved whereas other amino acid residues of the plant defensins consensus sequence are substituted. BSD1 transcripts accumulate specifically in the stamen of developing flowers and its level drops as the flowers mature. The recombinant BSD1 produced in Escherichia coli showed antifungal activity against several phytopathogenic fungi. Furthermore, constitutive over-expression of the BSD1 gene under the control of the cauliflower mosaic virus (CaMV) 35S promoter conferred enhanced tolerance against the Phytophthora parasitica in the transgenic tobacco plants.
A cDNA encoding a small cysteine-rich protein designated VrCRP was isolated from a bruchid-resistant mungbean. VrCRP encodes a protein of 73 amino acids containing a 27 amino acid signal peptide and 8 cysteines. On the basis of the amino acid sequence similarity and conserved residues, it is suggested that VrCRP is a member of the plant defensin family. VrCRP protein was obtained by overexpression of VrCRP with a truncated signal peptide in an IMPACT system. Artificial seeds containing 0.2% (w/w) of the purified VrCRP-TSP were lethal to larvae of the bruchid Callosobruchus chinensis. VrCRP is apparently the first reported plant defensin exhibiting in vitro insecticidal activity against C, chinensis.
The symbiosis of Medicago truncatula with Sinorhizobium meliloti or S. medicae soil bacteria results in the formation of root nodules where bacteria inside the plant cells are irreversibly converted to polyploid, non-dividing 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 8 NCR peptides has been demonstrated so far. The predicted NCRs are secreted peptides targeted to the endosymbionts. Correspondingly, all the 8 detected peptides were present in the bacteroids. Here we report on large scale detection of NCR peptides from nodules and from isolated, semi-purified 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. This article is protected by copyright. All rights reserved.
This article is protected by copyright. All rights reserved.
Crop losses due to pathogens are a major threat to global food security. Plants employ a multilayer defense against a pathogen including the use of physical barriers (cell wall), induction of hypersensitive defense response (HR), resistance (R) proteins, and synthesis of antimicrobial peptides (AMPs). Unlike a complex R gene-mediated immunity, AMPs directly target diverse microbial pathogens. Many a times, R-mediated immunity breaks down and plant defense is compromised. Although R-gene dependent pathogen resistance has been well studied, comparatively little is known about the interactions of AMPs with host defense and physiology. AMPs are ubiquitous, low molecular weight peptides that display broad spectrum resistance against bacteria, fungi and viruses. In plants, AMPs are mainly classified into cyclotides, defensins, thionins, lipid transfer proteins, snakins, and hevein-like vicilin-like and knottins. Genetic distance lineages suggest their conservation with minimal effect of speciation events during evolution. AMPs provide durable resistance in plants through a combination of membrane lysis and cellular toxicity of the pathogen. Plant hormones - gibberellins, ethylene, jasmonates, and salicylic acid, are among the physiological regulators that regulate the expression of AMPs. Transgenically produced AMP-plants have become a means showing that AMPs are able to mitigate host defense responses while providing durable resistance against pathogens.
MsDef1 and MtDef4 from Medicago spp. are small cysteine-rich defensins with potent antifungal activity against a broad range of filamentous fungi. Each defensin has a hallmark γ-core motif (GXCX3-9C), which contains major determinants of its antifungal activity. In this study, the antifungal activities of MsDef1, MtDef4, and peptides derived from their γ-core motifs, were characterized during colony initiation in the fungal model, Neurospora crassa. These defensins and their cognate peptides inhibited conidial germination and accompanying cell fusion with different potencies. The inhibitory effects of MsDef1 were strongly mediated by the plasma membrane localized sphingolipid glucosylceramide. Cell fusion was selectively inhibited by the hexapeptide RGFRRR derived from the γ-core motif of MtDef4. Fluorescent labelling of this hexapeptide showed that it strongly bound to the germ tube plasma membrane / cell wall. Using N. crassa expressing the Ca2+ reporter aequorin, MsDef1, MtDef4 and their cognate peptides were each shown to perturb Ca2+ homeostasis in specific and distinct ways, and the disruptive effects of MsDef1 on Ca2+ were mediated by glucosylceramide. Together, our results demonstrate that MsDef1 and MtDef4 differ markedly in their antifungal properties and specific domains within their γ-core motifs play important roles in their different modes of antifungal action.
The Medicago truncatula gene encoding an evolutionarily conserved antifungal defensin MtDef4.2 was cloned and characterized. In silico expression analysis indicated that MtDef4.2 is expressed in many tissues during the normal growth and development of M. truncatula. MtDef4.2 exhibits potent broad-spectrum antifungal activity against various Fusarium spp. Transgenic Arabidopsis thaliana lines in which MtDef4.2 was targeted to three different subcellular compartments were generated. These lines were tested for resistance to the obligate biotrophic oomycete Hyaloperonospora arabidopsidis Noco2 and the hemibiotrophic fungal pathogen Fusarium graminearum PH-1. MtDef4.2 directed to the extracellular space, but not to the vacuole or retained in the endoplasmic reticulum, conferred robust resistance to H. arabidopsidis. Siliques of transgenic Arabidopsis lines expressing either extracellularly or intracellularly targeted MtDef4.2 displayed low levels of resistance to F. graminearum, but accumulated substantially reduced levels of the mycotoxin deoxynivalenol. The data presented here suggest that extracellularly targeted MtDef4.2 is sufficient to provide strong resistance to the biotrophic oomycete, consistent with the extracellular lifestyle of this pathogen. However, the co-expression of extracellular and intracellular MtDef4.2 is probably required to achieve strong resistance to the hemibiotrophic pathogen F. graminearum which grows extracellularly and intracellularly.
Rhizobium-legume symbiosis has been considered as a mutually favorable relationship for both partners. However, in certain phylogenetic groups of legumes, the plant directs the bacterial symbiont into an irreversible terminal differentiation. This is mediated by the actions of hundreds of symbiosis-specific plant peptides resembling antimicrobial peptides, the effectors of innate immunity. The bacterial BacA protein, associated in animal pathogenic bacteria with the maintenance of chronic intracellular infections, is also required for terminal differentiation of rhizobia. Thus, a virulence factor of pathogenesis and effectors of the innate immunity were adapted in symbiosis for the benefit of the plant partner.
Antimicrobial peptides (So-D1-7) were isolated from a crude cell wall preparation from spinach leaves (Spinacia oleracea cv. Matador) and, judged from their amino acid sequences, six of them (So-D2-7) represented a novel structural subfamily of plant defensins (group IV). Group-IV defensins were also functionally distinct from those of groups I-III. They were active at concentrations < 20 microM against Gram-positive (Clavibacter michiganensis) and Gram-negative (Ralstonia solanacearum) bacterial pathogens, as well as against fungi, such as Fusarium culmorum, F. solani, Bipolaris maydis, and Colletotrichum lagenarium. Fungal inhibition occurred without hyphal branching. Group-IV defensins were preferentially distributed in the epidermal cell layer of leaves and in the subepidermal region of stems.
Conventional analyses distinguish between antimicrobial peptides by differences in amino acid sequence. Yet structural paradigms common to broader classes of these molecules have not been established. The current analyses examined the potential conservation of structural themes in antimicrobial peptides from evolutionarily diverse organisms. Using proteomics, an antimicrobial peptide signature was discovered to integrate stereospecific sequence patterns and a hallmark three-dimensional motif. This striking multidimensional signature is conserved among disulfide-containing antimicrobial peptides spanning biological kingdoms, and it transcends motifs previously limited to defined peptide subclasses. Experimental data validating this model enabled the identification of previously unrecognized antimicrobial activity in peptides of known identity. The multidimensional signature model provides a unifying structural theme in broad classes of antimicrobial peptides, will facilitate discovery of antimicrobial peptides as yet unknown, and offers insights into the evolution of molecular determinants in these and related host defense effector molecules.
A cDNA encoding a small cysteine-rich protein designated VrCRP was isolated from a bruchid-resistant mungbean. VrCRP encodes a protein of 73 amino acids containing a 27 amino acid signal peptide and 8 cysteines. On the basis of the amino acid sequence similarity and conserved residues, it is suggested that VrCRP is a member of the plant defensin family. VrCRP protein was obtained by overexpression of VrCRP with a truncated signal peptide in an IMPACT system. Artificial seeds containing 0.2% (w/w) of the purified VrCRP-TSP were lethal to larvae of the bruchid Callosobruchus chinensis. VrCRP is apparently the first reported plant defensin exhibiting in vitro insecticidal activity against C. chinensis.
Multicellular organisms produce small cysteine-rich antimicrobial peptides as an innate defense against pathogens. While defensins, a well-known class of such peptides, are common among eukaryotes, there are other classes restricted to the plant kingdom. These include thionins, lipid transfer proteins and snakins. In earlier work, we identified several divergent classes of small putatively secreted cysteine-rich peptides (CRPs) in legumes [Graham et al. (2004)Plant Physiol. 135, 1179-97]. Here, we built sequence motif models for each of these classes of peptides, and iteratively searched for related sequences within the comprehensive UniProt protein dataset, the Institute for Genomic Research's 33 plant gene indices, and the entire genomes of the model dicot, Arabidopsis thaliana, and the model monocot and crop species, Oryza sativa (rice). Using this search strategy, we identified approximately 13,000 plant genes encoding peptides with common features: (i) an N-terminal signal peptide, (ii) a small divergent charged or polar mature peptide with conserved cysteines, (iii) a similar intron/exon structure, (iv) spatial clustering in the genomes studied, and (v) overrepresentation in expressed sequences from reproductive structures of specific taxa. The identified genes include classes of defensins, thionins, lipid transfer proteins, and snakins, plus other protease inhibitors, pollen allergens, and uncharacterized gene families. We estimate that these classes of genes account for approximately 2-3% of the gene repertoire of each model species. Although 24% of the genes identified were not annotated in the latest Arabidopsis genome releases (TIGR5, TAIR6), we confirmed expression via RT-PCR for 59% of the sequences attempted. These findings highlight limitations in current annotation procedures for small divergent peptide classes.
RsAFP2 (Raphanus sativus antifungal peptide 2), an antifungal plant defensin isolated from seed of R. sativus, interacts with glucosylceramides (GlcCer) in membranes of susceptible yeast and fungi and induces membrane permeabilization and fungal cell death. However, using carboxyfluorescein-containing small unilamellar vesicles containing purified GlcCer, we could not observe permeabilization as a consequence of insertion of RsAFP2 in such vesicles. Therefore, we focused on a putative RsAFP2-induced signaling cascade downstream of RsAFP2-binding to GlcCer in fungal membranes. We show that RsAFP2 induces reactive oxygen species (ROS) in Candida albicans wild type in a dose-dependent manner, but not at all in an RsAFP2-resistant DeltagcsC. albicans mutant that lacks the RsAFP2-binding site in its membranes. These findings indicate that upstream binding of RsAFP2 to GlcCer is needed for ROS production leading to yeast cell death. Moreover, the antioxidant ascorbic acid blocks RsAFP2-induced ROS generation, as well as RsAFP2 antifungal activity. These data point to the presence of an intracellular plant defensin-induced signaling cascade, which involves ROS generation and leads to fungal cell growth arrest.
To defend themselves against invading fungal pathogens, plants and insects largely depend on the production of a wide array of antifungal molecules, including antimicrobial peptides such as defensins. Interestingly, plant and insect defensins display antimicrobial activity not only against plant and insect pathogens but also against human fungal pathogens, including Candida spp. and Aspergillus spp. This review focuses on these defensins as novel leads for antifungal therapeutics. Their mode of action, involving interaction with fungus-specific sphingolipids, and heterologous expression, required for cost-effective production, are major assets for development of plant and insect defensins as antifungal leads. Studies evaluating their in vivo antifungal efficacy demonstrate their therapeutic potential.
Defensins are small (~5 kDa), basic, cysteine-rich antimicrobial peptides that fulfill an important role in the innate immunity of their host by combating pathogenic invading micro-organisms. Defensins can inhibit the growth or virulence of microorganisms directly or can do so indirectly by enhancing the host's immune system. Because of their wide distribution in nature, defensins are believed to be ancient molecules with a common ancestor that arose more than a billion years ago. This review summarizes current knowledge concerning the mode of antifungal action of plant, insect and human defensins.
- Bp Thomma
- Bp Cammue
- K Thevissen
Thomma BP, Cammue BP, Thevissen K: Plant defensins. Planta
2002, 216:193-202.