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Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules
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ScienceDirect Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules Gergely Maro´ti1, J Allan Downie2 and E´va Kondorosi1 Plants must co-exist with both pathogenic and beneficial microbes. Antimicrobial peptides with broad antimicrobial activities represent one of the first lines of defense against pathogens. Many plant cysteine-rich peptides with potential antimicrobial properties have been predicted. Amongst them, defensins and defensin-like peptides are the most abundant and plants can express several hundreds of them. In some rhizobial–legume symbioses special defensin-like peptides, the nodule-specific cysteine-rich (NCR) peptides have evolved in those legumes whose symbiotic partner terminally differentiates. In Medicago truncatula, >700 NCRs exist and collectively act as plant effectors inducing irreversible differentiation of rhizobia to nitrogen-fixing bacteroids. Cationic NCR peptides have a broad range of potent antimicrobial activities but do not kill the endosymbionts. Addresses 1 Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Temesva´ri krt. 62., Szeged 6726, Hungary 2 John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK Corresponding author: Kondorosi, E´va ([email protected], [email protected])
Current Opinion in Plant Biology 2015, 26:57–63 This review comes from a themed issue on Biotic interactions Edited by Uta Paszkowski and D Barry Scott
http://dx.doi.org/10.1016/j.pbi.2015.05.031 1369-5266/# 2015 Elsevier Ltd. All rights reserved.
Introduction Although plants are frequently attacked by micro-organisms, relatively few of these attacks lead to systemic infection and the development of full-blown disease. This is because plants have highly developed mechanisms of defense against pathogenic micro-organisms. These can be divided into two broad categories: induced resistance and innate immunity, that provides broad-spectrum resistance against fungi, bacteria and viruses [1–3]. However, as plants also interact with beneficial micro-organisms, a key aspect of plant defense mechanisms is that they should be able to distinguish between pathogens and microorganisms that promote plant growth [4]. Such beneficial www.sciencedirect.com
micro-organisms include growth-promoting rhizosphere and endophytic micro-organisms and intracellular symbionts including mycorrhizal fungi, and the rhizobia and Frankia spp. of bacteria that produce nitrogen-fixing nodules on legumes and related genera [5]. In this article we focus on one aspect of the multifaceted mechanisms of innate immunity, namely the short cysteine-rich antimicrobial peptides called defensins and defensin-like peptides. They are one of the largest families of antimicrobial peptides in plants and their constitutive, regulated or induced expression confers durable resistance to pathogens. They can inhibit fungal [6,7], bacterial [8,9] and insect [10] growth and can confer broad-spectrum resistance to pathogens in crops [6,11]. The signal peptides of plant defensins direct them to the secretory pathway resulting in their specific destination, mostly secretion from the cells but also vacuolar localization. Typical plant defensins are 45–70 amino acids in length (without the signal peptide), contain four conserved disulphide bonds and share a backbone structure in which an a-helix is stabilized through disulphide bridging to three antiparallel b-strands [12–15]. These antimicrobial effectors have primary sequence differences that determine specificity and different mechanisms of action [15–20]. In Arabidopsis thaliana, 317 defensin-like genes have been identified; in M. truncatula 63 classical defensin genes have been found with the potential (allowing for splicing variants) of encoding over 80 peptides [21,22,23]. Given the apparently diverse effects of defensins, it might be predicted that, to allow the growth of rhizobia in legume nodules (which can host up to 108 bacteria in a nodule, the size of a match-head), legumes would switch off the production of defensins and defensin-like proteins in nodules. However in some legumes the reality is strikingly different, because some legumes have massively increased numbers of defensin-like genes that are specifically expressed in nodules. Over 700 genes have been identified as encoding these defensin-like proteins [24– 26]. Nodule-specific Cysteine-Rich peptides (NCRs) are small secreted peptides, structurally related to defensins but contain only 2 or 3 disulfide bridges. Instead of primarily acting in defense they have symbiotic functions in root nodules, guiding differentiation of the endosymbionts to nitrogen-fixing bacteroids, while limiting their cell division [27]. Paradoxically, some legumes produce nitrogen-fixing nodules with rhizobia in the absence of Current Opinion in Plant Biology 2015, 26:57–63
58 Biotic interactions
these large numbers of NCR peptides and we address the question why this may occur. The main objective of this review is to use M. truncatula as a model to analyse the relatedness of true defensins to the nodule-expressed cysteine-rich peptides with the aim of trying to gain an insight into the evolution and the functional roles of both groups of proteins.
M. truncatula defensins The 63 M. truncatula classical defensin genes are scattered along the eight chromosomes; most contain a large intron between the coding sequences of the mature peptides and the hydrophobic signal peptides. Sixteen of the mature ‘MtDef’ peptides were classified earlier based on sequence similarity, but only limited structural and functional work has been done on them [28]. A threedimensional structure was determined for the M. truncatula defensin MtDef4 (locus tag: MTR_8g070770): it has a positively-charged g-core motif composed of b2 and b3 strands connected by a positively-charged RGFRRR loop [29]. Analyses of gene expression with Affymetrix arrays containing probes for 27 of the standard MtDef genes showed that most of these genes had tissue or organspecific expression, whereas MtDef4 (MTR_8g070770) was highly expressed in all organs investigated (Medicago Gene Atlas: http://mtgea.noble.org/v3/). MtDef1 (MTR_ 2g079440) and MtDef2 (MTR_2g079430) are specifically expressed in seeds whereas eight MtDefs are specifically expressed in nodules, hereafter we refer to these defensins as MtDefNS1-8 (Nodule-Specific MtDefs). Basic data including locus tag, Affymetrix probe IDs, name, expression data as well as sequences of the 27 MtDefs are shown in Supplementary Files 1 and 2. Surprisingly seven of these eight nodule-specific defensins are encoded on chromosome 8 suggesting some specialisation. Most of these nodule-specific defensins (six out of eight) have lower isoelectric points compared to defensins expressed in other organs: MtDefNS1, 2, 4, 5, 7 and 8 have a pI value below 5, while none of the further 19 MtDefs have a pI value below 6 (Supplementary File 1). Most of the nodule-expressed defensins are phylogenetically clustered and are related to, but in a different clade from, other classical defensins (Figure 1, Supplementary File 2). The different mechanisms of action of defensins can be explained by the variable primary sequences; the major determinants of the antifungal and morphogen activities of defensins reside in their g-core motifs [18,30]. MsDef1 (defensin 1.3 of M. sativa) and MtDef4 each contain a conserved g-core structural motif (GXCX3–9C, where X is any amino acid), which is a hallmark signature of disulfide-stabilized antimicrobial peptides [31]. The g-core motifs of MsDef1 and MtDef4 differ in primary sequences and net charge (MsDef1: GRCRDDFRC, MtDef4: GRCRGFRRRC); although they share only 41% amino acid sequence identity, both inhibit the growth of several filamentous fungi at micromolar Current Opinion in Plant Biology 2015, 26:57–63
concentrations. MsDef1 inhibits Fusarium graminearum growth by inducing hyperbranching of hyphae, whereas MtDef4 inhibits polar growth of hyphae but does not induce hyperbranching. MsDef1 blocks a Ca2+-channel in mammalian cells and the plasma-membrane-associated sphingolipid glucosylceramide (GlcCer) was identified as a MsDef1 receptor in F. graminearum; a mutant lacking GlcCer is resistant to MsDef1, but retains sensitivity to MtDef4 demonstrating they act on different targets [32]. It was shown in transgenic A. thaliana lines that extracellularly targeted MtDef4 is sufficient to provide strong resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis, while the co-expression of extracellular and intracellular MtDef4 is required to achieve resistance to the hemibiotrophic pathogen F. graminearum [33].
NCRs: host effectors of endosymbionts In addition to the typical defensins, M. truncatula has more than 700 genes encoding defensin-like polypeptides with conserved cysteine residues, but varying in the primary amino acid sequence of the mature proteins [24–26]. These were identified from a combination of transcriptional profiling of M. truncatula root nodules and genome sequencing [23,34]. Most of these M. truncatula peptides are in the NCR (nodule-specific cysteine rich peptides) family and play various — so far mostly unknown — roles in the development of rhizobial–legume symbioses [35]. The NCR-encoding genes are spread along the eight chromosomes and have 2 or 3 exons. Similar to defensins, the coding sequences are short and the first exons encode the NCR signal peptides. The mature peptides contain 4 or 6 cysteines, are usually 30–55 amino acids long, but differ greatly in their amino acid sequence, composition and charge: there is an almost equal distribution of cationic, anionic, and neutral NCRs [24]. Conservation of 4 or 6 cysteine residues with specific pattern is a hallmark of the NCR peptides and this distinguishes them from typical defensins and other cysteine cluster peptides (Figure 2). It is nearly impossible to generate an accurate and reliable multiple sequence alignment using several hundred NCR peptides strongly varying in their primary sequence. However, clustering analysis on 413 unique NCR peptides and 27 standard defensin peptides revealed that the NCRs with 4 and 6 cysteines (forming presumably 2 or 3 disulfide bridges) represent different NCR subgroups which are separated from each other as well as from the typical defensins (Supplementary File 3). Studies in vitro with synthetic NCR247 (containing 4 cysteines) indicate that disulfide bridges form between the 1st and 2nd and the 3rd and 4th cysteine residues [36]; this remains to be confirmed with plant-made NCR247. The importance of disulfide bridges for the in vitro and in planta activities of NCRs may differ. The C-terminal half of NCR247 containing only the 3rd and 4th cysteine residues, and mutant forms of NCR247 with individual cysteines replaced with www.sciencedirect.com
Standard defensins and defensin-like peptides Maro´ti, Downie and Kondorosi 59
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Phylogenetic tree of M. truncatula defensins. The phylogenetic tree is generated on the basis of the amino acid sequences of 27 M. truncatula standard defensins expressed in various organs. For clarity reason, locus tags were used for the identification of the individual sequences instead of using tentative consensus sequences (TC), although the tree is based on the deduced amino acid sequences. The nodule-specific defensins (red) are clustered and are positioned in a different clade from the described antifungal defensins (blue: MtDef4, green: MtDef1 and MtDef2). The Geneious software (version 8.0.5) was used for generating consensus sequence and phylogenetic tree, Blosum50 cost matrix was applied for the global alignment.
serines retained some antimicrobial activity. However with NCR169, replacing any cysteine with alanine abolished the in planta function of the peptides (Horva´th B, et al. unpublished). Cationic NCR peptides (pI > 9) show a broad range of bactericidal and fungicidal activities, killing various Gram positive and Gram negative bacteria and fungi [37,38]. The in vitro antimicrobial activities of NCRs may depend primarily on the positive charge of the peptides and killing is probably caused by permeabilization of the microbial membranes leading to cell lysis. www.sciencedirect.com
Within root nodules, NCRs do not kill the S. meliloti or S. medicae endosymbionts, but control their differentiation converting them into elongated non-cultivable polyploid nitrogen-fixing bacteroids. The bacteria enter the roots via plant-made infection threads which eventually release the bacteria, endocytosed into a membrane-bound compartment known as a symbiosome [39]. The endosymbionts then undergo progressive and irreversible differentiation that adapts their physiology to an intracellular life-style, arresting their division but permitting cell enlargement, extensive amplification of their genome and Current Opinion in Plant Biology 2015, 26:57–63
60 Biotic interactions
Figure 2
Medicago truncatula defensins
X10-C1-X 8-G-X1-C2-X 5-8-C3-X 3-C4-X2-3-E-X4-5-G-X1-C5-X6-8-C6 -F/Y/M/W/I -C 7 -X3-C8-X n SIGNAL PEPTIDE CLEAVAGE SITE
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MATURE PEPTIDE (30-55 aa)
Current Opinion in Plant Biology
Comparison of the defensin and NCR peptide backbones in M. truncatula. Cysteine motifs conserved in defensins and in the two major types of NCR peptides (containing 4 and 6 cysteines, respectively). Conserved cysteines are in bold and numbered, the length of conserved spacings between cysteines are indicated (Xn). The structure of MtDef4 is shown illustrating in yellow the RGFRRR g-core motif, the eight conserved cysteines (C: one of which is C-terminal) and the N-terminus (N). The b sheets are shown highlighted as rectangular bocks and the a helix as a cylinder.
enabling them to fix nitrogen. This terminal bacteroid differentiation is plant controlled and NCR peptides are the major plant effectors [27,40]. The NCR genes are only expressed in nodules and are induced in successive waves as coordinated nodule development and rhizobial infection of nodules occurs [41,42]. Laser micro-dissection and RNA-Seq of nodule zones revealed which and how many NCRs may participate in a given developmental stage [43]. The NCR peptides are translocated across the endoplasmic reticulum membrane where a nodule-induced signal peptidase cleaves the signal peptide releasing the mature peptide. This results in the NCR peptides being present in the developing symbiosomes. Depending on the timings of gene expression, different sets of NCR peptides are delivered to the endosymbionts and so can mediate subsequent differentiation events. The essential role of appropriate NCR targeting was shown with the M. truncatula dnf1 mutant that lacks a nodule-specific subunit of the signal peptidase complex and forms non-functional nodules [44]. In this mutant, the unprocessed NCRs are blocked in the endoplasmic reticulum and so do not reach the bacteroids, resulting in the formation of ineffective Current Opinion in Plant Biology 2015, 26:57–63
nodules in which the bacteria remain undifferentiated [27]. A role for NCRs in rhizobial differentiation is supported by their accumulation in bacteroids within the symbiosomes. Proteomics of bacteroids from M. truncatula nodules identified 140 different NCR peptides in their cytosol, supporting the idea that NCR peptides can interact with intracellular bacterial targets [45]. It is unclear how NCR peptides enter the bacteria: addition of FITC-labeled NCR peptides to S. meliloti in vitro showed that certain cationic peptides could enter without causing membrane permeabilization [46]. Neutral and anionic peptides did not enter free-living bacteria even though most NCRs identified in bacteroids were anionic. The membranes of bacteroids seem to be different from those of cultured bacteria since the membrane-impermeable dye propidium-iodide can diffuse slowly into isolated bacteroids but not bacteria [40]. Altered membrane structure or permeability may facilitate uptake of peptides. Alternatively, the uptake of anioic NCRs may be enhanced in the presence of cationic peptides that could act to facilitate translocation of anionic or neutral NCRs. However in planta-expressed membrane receptors www.sciencedirect.com
Standard defensins and defensin-like peptides Maro´ti, Downie and Kondorosi 61
and/or oligopeptide transporters could also facilitate uptake that is not observed in vitro.
Key questions for future research The origins, activities and combined action of NCRs in the symbiotic cells remain to be discovered. NCR peptides are probably evolutionarily derived from one or more of the defensins. So far only a few NCR cationic peptides have been studied and mostly in vitro and so their roles remain enigmatic. Addition of NCR247 (which inhibits growth of different bacteria in vitro) to a growing culture of S. meliloti resulted in down-regulation of ribosomal protein genes and inhibition of protein synthesis via its interaction with various ribosomal proteins. It also bound to FtsZ, an essential bacterial cell division protein whose polymerization and mid-cell localization are required for septum assembly and cell division; presumably this binding contributed to the observed absence of septum formation and cell elongation. NCR247 also perturbed the expression of several critical cell-cycle regulator genes (ctrA, gcrA, dnaA) and thus could play an important role in arresting proliferation of bacteria in the host cells, contributing to their elongation [47]. NCR247 also bound to the GroEL chaperon, which has an important role in all phases of nodule development [46]. Why is it that the NCR peptides are found only in some clades of legumes? One possibility is that the nodule structures are critical. Those legumes in which they are found always have persistent infection threads and the rhizobia in those infection threads would be protected from the NCR peptides and so have a good chance of escaping to the soil following nodule senescence. In contrast, in nodules with a determinate meristem (e.g. as in Lotus spp. or soybean), there are very few persistent infection threads; if all the bacteroids in such nodules were driven to terminal differentiation by NCR peptides, there would be very few bacteria that could survive nodule senescence and so there would be a huge selective disadvantage. A final question is why there are so many NCR peptides and how many are essential for development of the symbiosis. Even though the genes are relatively small, there are many genes and yet, thus far, only mutations affecting two genes have been identified (Kim M, et al. unpublished, Horva´th B, et al. unpublished). One can expect that more essential NCRs will be identified, but the low frequency of mutant identification implies either genetic redundancy or that different subsets of NCRs may act on different rhizobial strains. Perhaps thinking about how NCR peptides developed in evolution could give an insight into the diversity of the peptides. If one makes the reasonable assumption (based on symbioses with no NCR peptides) that NCR peptides are not essential for nitrogen fixation per se, why should the www.sciencedirect.com
loss of one NCR peptide block the symbiosis? Perhaps this observation is telling us that, in the absence of one NCR peptide, other NCR peptides cause lethality. If this is the case then it is possible that, in order to arrest bacteroid growth and induce differentiation without causing lethality, different NCR peptides may have to target different components on a single pathway. Identifying the functions and targets of the NCR peptides will help answer such questions.
Conclusions Only a few of the very many defensin proteins identified in diverse plants have been characterized. This has revealed antimicrobial and primarily antifungal defensins with partially elucidated modes of action. It is now evident that some defensin-like peptides modulate rhizobial growth to the benefit of some legumes. In those legumes there has been huge diversification of a subset of defensin-like genes and at least some of these control rhizobial differentiation. However we do not know the individual and collective functions of NCRs and how they induce terminal differentiation; presumably it improves the efficiency of nitrogen fixation in some legumes. Could classical defensins similarly control and sustain the survival of some endophytic micro-organisms that promote plant growth? More work on defensins and defensin-like peptides will be required to understand their functions and roles in plant–microbe interactions.
Acknowledgements Our work is supported by the ‘SYM-BIOTICS’ Advanced Grant of the European Research Council to EK (grant number 269067) and by TA´MOP4.2.2.A-11/1/KONV-2012-0035 supported by the European Union and co˝cs at the financed by the European Social Fund. We thank Attila Szu Bioinformatics Platform for his help.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. pbi.2015.05.031.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Fan J, Doerner P: Genetic and molecular basis of nonhost disease resistance: complex, yes; silver bullet, no. Curr Opin Plant Biol 2012, 15:400-406.
2.
Lacerda AF, Vasconcelos EA, Pelegrini PB, Grossi de Sa MF: Antifungal defensins and their role in plant defense. Front Microbiol 2014, 5:116.
3.
Goyal RK, Mattoo AK: Multitasking antimicrobial peptides in plant development and host defense against biotic/abiotic stress. Plant Sci 2014, 228:135-149.
4.
Kereszt A, Mergaert P, Maro´ti G, Kondorosi E: Innate immunity effectors and virulence factors in symbiosis. Curr Opin Microbiol 2011, 14:76-81. Current Opinion in Plant Biology 2015, 26:57–63
62 Biotic interactions
5.
Carro L, Pujic P, Alloisio N, Fournier P, Boubakri H, Hay AE, Poly F, Franc¸ois P, Hocher V, Mergaert P et al.: Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia. ISME J 2015 http:// dx.doi.org/10.1038/ismej.2014257. This study demonstrates that Alnus glutinosa produces nodule-specific defensin-like peptides, and one of these peptides (Ag5) was shown to target the symbiotic bacterium partner Frankia. The peptide increased bacterial membrane permeability resulting in a significant release of nitrogen-containing metabolites, mainly glutamine and glutamate.
Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 2011, 480:520-524. 23. Tesfaye M, Silverstein KA, Nallu S, Wang L, Botanga CJ, Gomez SK, Costa LM, Harrison MJ, Samac DA, Glazebrook J et al.: Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes. PLoS One 2013, 8:e58992. The authors of this article described notable differences in DEFL gene expression patterns between Arabidopsis and Medicago. The study revealed, that most of the Arabidopsis DEFLs are expressed in inflorescences, while only a few exhibited root-enhanced expression. However, Medicago DEFLs were most prominently expressed in nitrogenfixing root nodules.
6.
Gao AG, Hakimi SM, Mittanck CA, Wu Y, Woerner BM, Stark DM, Shah DM, Liang J, Rommens CM: Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat Biotechnol 2000, 18:1307-1310.
7.
Park HC, Kang YH, Chun HJ, Koo JC, Cheong YH, Kim CY, Kim MC, Chung WS, Kim JC, Yoo JH et al.: Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage. Plant Mol Biol 2002, 50:59-69.
24. Mergaert P, Nikovics K, Kelemen Z, Maunoury N, Vaubert D, Kondorosi A, Kondorosi E: A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs. Plant Physiol 2003, 132:161-173.
8.
Koike M, Okamoto T, Tsuda S, Imai R: A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation. Biochem Biophys Res Commun 2002, 298:46-53.
25. Graham MA, Silverstein KAT, Cannon SB, VandenBosch KA: Computational identification and characterization of novel genes from legumes. Plant Physiol 2004, 135:1179-1197.
9.
Segura A, Moreno M, Molina A, Garcia-Olmedo F: Novel defensin subfamily from spinach (Spinacia oleracea). FEBS Lett 1998, 435:159-162.
26. Silverstein KAT, Moskal WA, Wu HC, Underwood BA, Graham MA, Town CD, VandenBosch KA: Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants. Plant J 2007, 51:262-280.
10. Chen KC, Lin CY, Kuan CC, Sung HY, Chen CS: A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. J Agric Food Chem 2002, 50:7258-7263. 11. Kanzaki H, Nirasawa S, Saitoh H, Ito M, Nishihara M, Terauchi R, Nakamura I: Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor Appl Genet 2002, 105:809-814. 12. Thomma BP, Cammue BP, Thevissen K: Plant defensins. Planta 2002, 216:193-202. 13. Lay FT, Anderson MA: Defensins, components of the innate immune system in plants. Curr Protein Pept Sci 2005, 6:85-101. 14. Aerts AM, Franc¸ois IE, Cammue BP, Thevissen K: The mode of antifungal action of plant, insect and human defensins. Cell Mol Life Sci 2008, 65:2069-2079. 15. van der Weerden NL, Bleackley MR, Anderson MA: Properties and mechanisms of action of naturally occurring antifungal peptides. Cell Mol Life Sci 2013, 70:3545-3570. 16. Thevissen K, Kristensen HH, Thomma BP, Cammue BP, Franc¸ois IE: Therapeutic potential of antifungal plant and insect defensins. Drug Discov Today 2007, 12:966-971. 17. Aerts AM, Franc¸ois IE, Meert EM, Li QT, Cammue BP, Thevissen K: The antifungal activity of RsAFP2, a plant defensin from Raphanus sativus, involves the induction of reactive oxygen species in Candida albicans. J Mol Microbiol Biotechnol 2007, 13:243-247. 18. Sagaram US, Pandurangi R, Kaur J, Smith TJ, Shah DM: Structure–activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum. PLoS One 2011, 6:e18550. 19. De Coninck B, Carron D, Tavormina P, Willem L, Craik DJ, Vos C, Thevissen K, Mathys J, Cammue BP: Mining the genome of Arabidopsis thaliana as a basis for the identification of novel bioactive peptides involved in oxidative stress tolerance. J Exp Bot 2013, 64:5297-5307. 20. Giacomelli L, Nanni V, Lenzi L, Zhuang J, Dalla Serra M, Banfield MJ, Town CD, Silverstein KA, Baraldi E, Moser C: Identification and characterization of the defensin-like gene family of grapevine. Mol Plant Microbe Interact 2012, 25: 1118-1131. 21. Silverstein KAT, Graham MA, Paape TD, VandenBosch KA: Genome organization of more than 300 defensin-like genes in Arabidopsis. Plant Physiol 2005, 138:600-610. 22. Young ND, Debelle´ F, Oldroyd GE, Geurts R, Cannon SB, Udvardi MK, Benedito VA, Mayer KF, Gouzy J, Schoof H et al.: The Current Opinion in Plant Biology 2015, 26:57–63
27. Van de Velde W, Zehirov G, Szatmari A, Debreczeny M, Ishihara H, Kevei Z, Farkas A, Mikulass K, Nagy A, Tiricz H et al.: Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 2010, 327:1122-1126. 28. Hanks JN, Snyder AK, Graham MA, Shah RK, Blaylock LA, Harrison MJ, Shah DM: Defensin gene family in Medicago truncatula: structure, expression and induction by signal molecules. Plant Mol Biol 2005, 58:385-399. 29. Sagaram US, El-Mounadi K, Buchko GW, Berg HR, Kaur J, Pandurangi RS, Smith TJ, Shah DM: Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin mtdef4: identification of an rgfrrr motif governing fungal cell entry. PLoS One 2013, 8:e82485. This study provides detailed analysis of the structure of MtDef4. Based on the results of mutational analyses the authors suggest that the RGFRRR loop is a translocation signal required for the internalization of the protein. Amino acid substitutions in the RGFRRR sequence abolished the ability of MtDef4 to enter fungal cells. 30. Munoz A, Chu M, Marris PI, Sagaram US, Kaur J, Shah DM, Read ND: Specific domains of plant defensins differentially disrupt colony initiation, cell fusion and calcium homeostasis in Neurospora crassa. Mol Microbiol 2014, 92:1357-1374. 31. Yount NY, Yeaman MR: Multidimensional signatures in antimicrobial peptides. Proc Natl Acad Sci USA 2004, 101:73637368. 32. Spelbrink RG, Dilmac N, Allen A, Smith TJ, Shah DM, Hockerman GH: Differential antifungal and calcium channelblocking activity among structurally related plant defensins. Plant Physiol 2004, 135:2055-2067. 33. Kaur J, Thokala M, Robert-Seilaniantz A, Zhao P, Peyret H, Berg H, Pandey S, Jones J, Shah D: Subcellular targeting of an evolutionarily conserved plant defensin MtDef4.2 determines the outcome of plant–pathogen interaction in transgenic Arabidopsis. Mol Plant Pathol 2012, 13:1032-1046. 34. Nallu S, Silverstein KA, Zhou P, Young ND, Vandenbosch KA: Patterns of divergence of a large family of nodule cysteine-rich peptides in accessions of Medicago truncatula. Plant J 2014, 78:697-705. 35. Kondorosi E, Mergaert P, Kereszt A: A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors. Annu Rev Microbiol 2013, 67:611-628. 36. Haag AF, Kerscher B, Dall’Angelo S, Sani M, Longhi R, Baloban M, Wilson HM, Mergaert P, Zanda M, Ferguson GP: Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide. J Biol Chem 2012, 287:10791-10798. www.sciencedirect.com
Standard defensins and defensin-like peptides Maro´ti, Downie and Kondorosi 63
In this study, the authors showed the significance of the proper formation of disulfide bridges in NCR247. They found that either cysteine replacements or S–S bond modifications influenced the activity of Medicago truncatula NCR247 against Sinorhizobium meliloti. Substitution of cysteines for serines, changing the S–S bridges from cysteines 1–2, 3– 4 to 1–3, 2–4 and oxidation of NCR247 lowered the peptide’s activity against S. meliloti.
Medicago truncatula nodulation are coupled to two transcriptome-switches. PLoS One 2010, 5:e9519. 43. Roux B, Rodde N, Jardinaud MF, Timmers T, Sauviac L, Cottret L, Carre`re S, Sallet E, Courcelle E, Moreau S et al.: An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing. Plant J 2014, 77:817-837.
37. Tiricz H, Szu˝cs A, Farkas A, Pap B, Lima RM, Maro´ti G, Kondorosi E´, Kereszt A: Antimicrobial nodule-specific cysteinerich peptides induce membrane depolarization-associated changes in the transcriptome of Sinorhizobium meliloti. Appl Environ Microbiol 2013, 79:6737-6746.
44. Wang D, Griffitts J, Starker C, Fedorova E, Limpens E, Ivanov S, Bisseling T, Long S: A nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis. Science 2010, 327:1126-1129.
38. O¨rdo¨gh L, Vo¨ro¨s A, Nagy I, Kondorosi E, Kereszt A: Symbiotic plant peptides eliminate Candida albicans both in vitro and in an epithelial infection model and inhibit the proliferation of immortalized human cells. Biomed Res Int 2014, 2014:320796 http://dx.doi.org/10.1155/2014/320796.
45. Du¨rgo˝ H, Klement E´, Hunyadi-Gulya´s E´, Szu˝cs A, Kereszt A, Medzihradszky KF, Kondorosi E´: Identification of nodulespecific cysteine-rich plant peptides in endosymbiotic bacteria. Proteomics 2015 http://dx.doi.org/10.1002/ pmic.201400385.
39. Sinharoy S, Torres-Jerez I, Bandyopadhyay K, Kereszt A, Pislariu CI, Nakashima J, Benedito VA, Kondorosi E, Udvardi MK: The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula. Plant Cell 2013, 25: 3584-3601.
46. Farkas A, Maro´ti G, Du¨rgo˝ H, Gyo¨rgypa´l Z, Lima RM, Medzihradszky KF, Kereszt A, Mergaert P, Kondorosi E´: The Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms. Proc Natl Acad Sci USA 2014, 111:5183-5188. This study shows the detailed in vivo and in vitro characterization of a Medicago truncatula NCR peptide (NCR247). It was shown that NCR247 penetrates the bacteria and forms complexes with many bacterial proteins including ribosomal proteins, FtsZ and GroEL. Interaction with bacterial FtsZ required for septum formation is one of the plant host interventions for inhibiting bacterial cell division.
40. Mergaert P, Uchiumi T, Alunni B, Evanno G, Cheron A, Catrice O, Mausset AE, Barloy-Hubler F, Galibert F, Kondorosi A et al.: Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium–legume symbiosis. Proc Natl Acad Sci USA 2006, 103:5230-5235. 41. Guefrachi I, Nagymihaly M, Pislariu CI, Van de Velde W, Ratet P, Mars M, Udvardi MK, Kondorosi E, Mergaert P, Alunni B: Extreme specificity of NCR gene expression in Medicago truncatula. BMC Genomics 2014, 15:712 http://dx.doi.org/10.1186/14712164-15-712. 42. Maunoury N, Redondo-Nieto M, Bourcy M, Van de Velde W, Alunni B, Laporte P, Durand P, Agier N, Marisa L, Vaubert D et al.: Differentiation of symbiotic cells and endosymbionts in
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47. Penterman J, Abo RP, De Nisco NJ, Arnold MF, Longhi R, Zanda M, Walker GC: Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis. Proc Natl Acad Sci USA 2014, 111:3561-3566. This study demonstrates that the Medicago truncatula NCR247 peptide specifically blocks bacterial cell division and antagonizes Z-ring function. Additionally, expression of critical cell-cycle regulators, including ctrA, and cell division genes were greatly attenuated in NCR-treated cells. NCR247 treatment was also shown to affect the expression of genes and functions important in symbiosis.
Current Opinion in Plant Biology 2015, 26:57–63
ScienceDirect Plant cysteine-rich peptides that inhibit pathogen growth and control rhizobial differentiation in legume nodules Gergely Maro´ti1, J Allan Downie2 and E´va Kondorosi1 Plants must co-exist with both pathogenic and beneficial microbes. Antimicrobial peptides with broad antimicrobial activities represent one of the first lines of defense against pathogens. Many plant cysteine-rich peptides with potential antimicrobial properties have been predicted. Amongst them, defensins and defensin-like peptides are the most abundant and plants can express several hundreds of them. In some rhizobial–legume symbioses special defensin-like peptides, the nodule-specific cysteine-rich (NCR) peptides have evolved in those legumes whose symbiotic partner terminally differentiates. In Medicago truncatula, >700 NCRs exist and collectively act as plant effectors inducing irreversible differentiation of rhizobia to nitrogen-fixing bacteroids. Cationic NCR peptides have a broad range of potent antimicrobial activities but do not kill the endosymbionts. Addresses 1 Institute of Biochemistry, Biological Research Center of the Hungarian Academy of Sciences, Temesva´ri krt. 62., Szeged 6726, Hungary 2 John Innes Centre, Norwich Research Park, Norwich NR4 7UH, UK Corresponding author: Kondorosi, E´va ([email protected], [email protected])
Current Opinion in Plant Biology 2015, 26:57–63 This review comes from a themed issue on Biotic interactions Edited by Uta Paszkowski and D Barry Scott
http://dx.doi.org/10.1016/j.pbi.2015.05.031 1369-5266/# 2015 Elsevier Ltd. All rights reserved.
Introduction Although plants are frequently attacked by micro-organisms, relatively few of these attacks lead to systemic infection and the development of full-blown disease. This is because plants have highly developed mechanisms of defense against pathogenic micro-organisms. These can be divided into two broad categories: induced resistance and innate immunity, that provides broad-spectrum resistance against fungi, bacteria and viruses [1–3]. However, as plants also interact with beneficial micro-organisms, a key aspect of plant defense mechanisms is that they should be able to distinguish between pathogens and microorganisms that promote plant growth [4]. Such beneficial www.sciencedirect.com
micro-organisms include growth-promoting rhizosphere and endophytic micro-organisms and intracellular symbionts including mycorrhizal fungi, and the rhizobia and Frankia spp. of bacteria that produce nitrogen-fixing nodules on legumes and related genera [5]. In this article we focus on one aspect of the multifaceted mechanisms of innate immunity, namely the short cysteine-rich antimicrobial peptides called defensins and defensin-like peptides. They are one of the largest families of antimicrobial peptides in plants and their constitutive, regulated or induced expression confers durable resistance to pathogens. They can inhibit fungal [6,7], bacterial [8,9] and insect [10] growth and can confer broad-spectrum resistance to pathogens in crops [6,11]. The signal peptides of plant defensins direct them to the secretory pathway resulting in their specific destination, mostly secretion from the cells but also vacuolar localization. Typical plant defensins are 45–70 amino acids in length (without the signal peptide), contain four conserved disulphide bonds and share a backbone structure in which an a-helix is stabilized through disulphide bridging to three antiparallel b-strands [12–15]. These antimicrobial effectors have primary sequence differences that determine specificity and different mechanisms of action [15–20]. In Arabidopsis thaliana, 317 defensin-like genes have been identified; in M. truncatula 63 classical defensin genes have been found with the potential (allowing for splicing variants) of encoding over 80 peptides [21,22,23]. Given the apparently diverse effects of defensins, it might be predicted that, to allow the growth of rhizobia in legume nodules (which can host up to 108 bacteria in a nodule, the size of a match-head), legumes would switch off the production of defensins and defensin-like proteins in nodules. However in some legumes the reality is strikingly different, because some legumes have massively increased numbers of defensin-like genes that are specifically expressed in nodules. Over 700 genes have been identified as encoding these defensin-like proteins [24– 26]. Nodule-specific Cysteine-Rich peptides (NCRs) are small secreted peptides, structurally related to defensins but contain only 2 or 3 disulfide bridges. Instead of primarily acting in defense they have symbiotic functions in root nodules, guiding differentiation of the endosymbionts to nitrogen-fixing bacteroids, while limiting their cell division [27]. Paradoxically, some legumes produce nitrogen-fixing nodules with rhizobia in the absence of Current Opinion in Plant Biology 2015, 26:57–63
58 Biotic interactions
these large numbers of NCR peptides and we address the question why this may occur. The main objective of this review is to use M. truncatula as a model to analyse the relatedness of true defensins to the nodule-expressed cysteine-rich peptides with the aim of trying to gain an insight into the evolution and the functional roles of both groups of proteins.
M. truncatula defensins The 63 M. truncatula classical defensin genes are scattered along the eight chromosomes; most contain a large intron between the coding sequences of the mature peptides and the hydrophobic signal peptides. Sixteen of the mature ‘MtDef’ peptides were classified earlier based on sequence similarity, but only limited structural and functional work has been done on them [28]. A threedimensional structure was determined for the M. truncatula defensin MtDef4 (locus tag: MTR_8g070770): it has a positively-charged g-core motif composed of b2 and b3 strands connected by a positively-charged RGFRRR loop [29]. Analyses of gene expression with Affymetrix arrays containing probes for 27 of the standard MtDef genes showed that most of these genes had tissue or organspecific expression, whereas MtDef4 (MTR_8g070770) was highly expressed in all organs investigated (Medicago Gene Atlas: http://mtgea.noble.org/v3/). MtDef1 (MTR_ 2g079440) and MtDef2 (MTR_2g079430) are specifically expressed in seeds whereas eight MtDefs are specifically expressed in nodules, hereafter we refer to these defensins as MtDefNS1-8 (Nodule-Specific MtDefs). Basic data including locus tag, Affymetrix probe IDs, name, expression data as well as sequences of the 27 MtDefs are shown in Supplementary Files 1 and 2. Surprisingly seven of these eight nodule-specific defensins are encoded on chromosome 8 suggesting some specialisation. Most of these nodule-specific defensins (six out of eight) have lower isoelectric points compared to defensins expressed in other organs: MtDefNS1, 2, 4, 5, 7 and 8 have a pI value below 5, while none of the further 19 MtDefs have a pI value below 6 (Supplementary File 1). Most of the nodule-expressed defensins are phylogenetically clustered and are related to, but in a different clade from, other classical defensins (Figure 1, Supplementary File 2). The different mechanisms of action of defensins can be explained by the variable primary sequences; the major determinants of the antifungal and morphogen activities of defensins reside in their g-core motifs [18,30]. MsDef1 (defensin 1.3 of M. sativa) and MtDef4 each contain a conserved g-core structural motif (GXCX3–9C, where X is any amino acid), which is a hallmark signature of disulfide-stabilized antimicrobial peptides [31]. The g-core motifs of MsDef1 and MtDef4 differ in primary sequences and net charge (MsDef1: GRCRDDFRC, MtDef4: GRCRGFRRRC); although they share only 41% amino acid sequence identity, both inhibit the growth of several filamentous fungi at micromolar Current Opinion in Plant Biology 2015, 26:57–63
concentrations. MsDef1 inhibits Fusarium graminearum growth by inducing hyperbranching of hyphae, whereas MtDef4 inhibits polar growth of hyphae but does not induce hyperbranching. MsDef1 blocks a Ca2+-channel in mammalian cells and the plasma-membrane-associated sphingolipid glucosylceramide (GlcCer) was identified as a MsDef1 receptor in F. graminearum; a mutant lacking GlcCer is resistant to MsDef1, but retains sensitivity to MtDef4 demonstrating they act on different targets [32]. It was shown in transgenic A. thaliana lines that extracellularly targeted MtDef4 is sufficient to provide strong resistance to the biotrophic oomycete Hyaloperonospora arabidopsidis, while the co-expression of extracellular and intracellular MtDef4 is required to achieve resistance to the hemibiotrophic pathogen F. graminearum [33].
NCRs: host effectors of endosymbionts In addition to the typical defensins, M. truncatula has more than 700 genes encoding defensin-like polypeptides with conserved cysteine residues, but varying in the primary amino acid sequence of the mature proteins [24–26]. These were identified from a combination of transcriptional profiling of M. truncatula root nodules and genome sequencing [23,34]. Most of these M. truncatula peptides are in the NCR (nodule-specific cysteine rich peptides) family and play various — so far mostly unknown — roles in the development of rhizobial–legume symbioses [35]. The NCR-encoding genes are spread along the eight chromosomes and have 2 or 3 exons. Similar to defensins, the coding sequences are short and the first exons encode the NCR signal peptides. The mature peptides contain 4 or 6 cysteines, are usually 30–55 amino acids long, but differ greatly in their amino acid sequence, composition and charge: there is an almost equal distribution of cationic, anionic, and neutral NCRs [24]. Conservation of 4 or 6 cysteine residues with specific pattern is a hallmark of the NCR peptides and this distinguishes them from typical defensins and other cysteine cluster peptides (Figure 2). It is nearly impossible to generate an accurate and reliable multiple sequence alignment using several hundred NCR peptides strongly varying in their primary sequence. However, clustering analysis on 413 unique NCR peptides and 27 standard defensin peptides revealed that the NCRs with 4 and 6 cysteines (forming presumably 2 or 3 disulfide bridges) represent different NCR subgroups which are separated from each other as well as from the typical defensins (Supplementary File 3). Studies in vitro with synthetic NCR247 (containing 4 cysteines) indicate that disulfide bridges form between the 1st and 2nd and the 3rd and 4th cysteine residues [36]; this remains to be confirmed with plant-made NCR247. The importance of disulfide bridges for the in vitro and in planta activities of NCRs may differ. The C-terminal half of NCR247 containing only the 3rd and 4th cysteine residues, and mutant forms of NCR247 with individual cysteines replaced with www.sciencedirect.com
Standard defensins and defensin-like peptides Maro´ti, Downie and Kondorosi 59
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Phylogenetic tree of M. truncatula defensins. The phylogenetic tree is generated on the basis of the amino acid sequences of 27 M. truncatula standard defensins expressed in various organs. For clarity reason, locus tags were used for the identification of the individual sequences instead of using tentative consensus sequences (TC), although the tree is based on the deduced amino acid sequences. The nodule-specific defensins (red) are clustered and are positioned in a different clade from the described antifungal defensins (blue: MtDef4, green: MtDef1 and MtDef2). The Geneious software (version 8.0.5) was used for generating consensus sequence and phylogenetic tree, Blosum50 cost matrix was applied for the global alignment.
serines retained some antimicrobial activity. However with NCR169, replacing any cysteine with alanine abolished the in planta function of the peptides (Horva´th B, et al. unpublished). Cationic NCR peptides (pI > 9) show a broad range of bactericidal and fungicidal activities, killing various Gram positive and Gram negative bacteria and fungi [37,38]. The in vitro antimicrobial activities of NCRs may depend primarily on the positive charge of the peptides and killing is probably caused by permeabilization of the microbial membranes leading to cell lysis. www.sciencedirect.com
Within root nodules, NCRs do not kill the S. meliloti or S. medicae endosymbionts, but control their differentiation converting them into elongated non-cultivable polyploid nitrogen-fixing bacteroids. The bacteria enter the roots via plant-made infection threads which eventually release the bacteria, endocytosed into a membrane-bound compartment known as a symbiosome [39]. The endosymbionts then undergo progressive and irreversible differentiation that adapts their physiology to an intracellular life-style, arresting their division but permitting cell enlargement, extensive amplification of their genome and Current Opinion in Plant Biology 2015, 26:57–63
60 Biotic interactions
Figure 2
Medicago truncatula defensins
X10-C1-X 8-G-X1-C2-X 5-8-C3-X 3-C4-X2-3-E-X4-5-G-X1-C5-X6-8-C6 -F/Y/M/W/I -C 7 -X3-C8-X n SIGNAL PEPTIDE CLEAVAGE SITE
MATURE DEFENSIN (50-70 aa)
Nodule-specific Cysteine-Rich peptides (6 Cys)
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X4-12-C1-X5-C2-X1-8-C3-X5-8-C4-X4-16-C5-X1-C6-Xn SIGNAL PEPTIDE CLEAVAGE SITE
MATURE PEPTIDE (30-55 aa)
γ
C C
C C
C
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Nodule-specific Cysteine-Rich peptides (4 Cys) X2-7-C1-X5-6-C2-X9-14-C3-X4-C4-Xn SIGNAL PEPTIDE CLEAVAGE SITE
C
MtDef4
MATURE PEPTIDE (30-55 aa)
Current Opinion in Plant Biology
Comparison of the defensin and NCR peptide backbones in M. truncatula. Cysteine motifs conserved in defensins and in the two major types of NCR peptides (containing 4 and 6 cysteines, respectively). Conserved cysteines are in bold and numbered, the length of conserved spacings between cysteines are indicated (Xn). The structure of MtDef4 is shown illustrating in yellow the RGFRRR g-core motif, the eight conserved cysteines (C: one of which is C-terminal) and the N-terminus (N). The b sheets are shown highlighted as rectangular bocks and the a helix as a cylinder.
enabling them to fix nitrogen. This terminal bacteroid differentiation is plant controlled and NCR peptides are the major plant effectors [27,40]. The NCR genes are only expressed in nodules and are induced in successive waves as coordinated nodule development and rhizobial infection of nodules occurs [41,42]. Laser micro-dissection and RNA-Seq of nodule zones revealed which and how many NCRs may participate in a given developmental stage [43]. The NCR peptides are translocated across the endoplasmic reticulum membrane where a nodule-induced signal peptidase cleaves the signal peptide releasing the mature peptide. This results in the NCR peptides being present in the developing symbiosomes. Depending on the timings of gene expression, different sets of NCR peptides are delivered to the endosymbionts and so can mediate subsequent differentiation events. The essential role of appropriate NCR targeting was shown with the M. truncatula dnf1 mutant that lacks a nodule-specific subunit of the signal peptidase complex and forms non-functional nodules [44]. In this mutant, the unprocessed NCRs are blocked in the endoplasmic reticulum and so do not reach the bacteroids, resulting in the formation of ineffective Current Opinion in Plant Biology 2015, 26:57–63
nodules in which the bacteria remain undifferentiated [27]. A role for NCRs in rhizobial differentiation is supported by their accumulation in bacteroids within the symbiosomes. Proteomics of bacteroids from M. truncatula nodules identified 140 different NCR peptides in their cytosol, supporting the idea that NCR peptides can interact with intracellular bacterial targets [45]. It is unclear how NCR peptides enter the bacteria: addition of FITC-labeled NCR peptides to S. meliloti in vitro showed that certain cationic peptides could enter without causing membrane permeabilization [46]. Neutral and anionic peptides did not enter free-living bacteria even though most NCRs identified in bacteroids were anionic. The membranes of bacteroids seem to be different from those of cultured bacteria since the membrane-impermeable dye propidium-iodide can diffuse slowly into isolated bacteroids but not bacteria [40]. Altered membrane structure or permeability may facilitate uptake of peptides. Alternatively, the uptake of anioic NCRs may be enhanced in the presence of cationic peptides that could act to facilitate translocation of anionic or neutral NCRs. However in planta-expressed membrane receptors www.sciencedirect.com
Standard defensins and defensin-like peptides Maro´ti, Downie and Kondorosi 61
and/or oligopeptide transporters could also facilitate uptake that is not observed in vitro.
Key questions for future research The origins, activities and combined action of NCRs in the symbiotic cells remain to be discovered. NCR peptides are probably evolutionarily derived from one or more of the defensins. So far only a few NCR cationic peptides have been studied and mostly in vitro and so their roles remain enigmatic. Addition of NCR247 (which inhibits growth of different bacteria in vitro) to a growing culture of S. meliloti resulted in down-regulation of ribosomal protein genes and inhibition of protein synthesis via its interaction with various ribosomal proteins. It also bound to FtsZ, an essential bacterial cell division protein whose polymerization and mid-cell localization are required for septum assembly and cell division; presumably this binding contributed to the observed absence of septum formation and cell elongation. NCR247 also perturbed the expression of several critical cell-cycle regulator genes (ctrA, gcrA, dnaA) and thus could play an important role in arresting proliferation of bacteria in the host cells, contributing to their elongation [47]. NCR247 also bound to the GroEL chaperon, which has an important role in all phases of nodule development [46]. Why is it that the NCR peptides are found only in some clades of legumes? One possibility is that the nodule structures are critical. Those legumes in which they are found always have persistent infection threads and the rhizobia in those infection threads would be protected from the NCR peptides and so have a good chance of escaping to the soil following nodule senescence. In contrast, in nodules with a determinate meristem (e.g. as in Lotus spp. or soybean), there are very few persistent infection threads; if all the bacteroids in such nodules were driven to terminal differentiation by NCR peptides, there would be very few bacteria that could survive nodule senescence and so there would be a huge selective disadvantage. A final question is why there are so many NCR peptides and how many are essential for development of the symbiosis. Even though the genes are relatively small, there are many genes and yet, thus far, only mutations affecting two genes have been identified (Kim M, et al. unpublished, Horva´th B, et al. unpublished). One can expect that more essential NCRs will be identified, but the low frequency of mutant identification implies either genetic redundancy or that different subsets of NCRs may act on different rhizobial strains. Perhaps thinking about how NCR peptides developed in evolution could give an insight into the diversity of the peptides. If one makes the reasonable assumption (based on symbioses with no NCR peptides) that NCR peptides are not essential for nitrogen fixation per se, why should the www.sciencedirect.com
loss of one NCR peptide block the symbiosis? Perhaps this observation is telling us that, in the absence of one NCR peptide, other NCR peptides cause lethality. If this is the case then it is possible that, in order to arrest bacteroid growth and induce differentiation without causing lethality, different NCR peptides may have to target different components on a single pathway. Identifying the functions and targets of the NCR peptides will help answer such questions.
Conclusions Only a few of the very many defensin proteins identified in diverse plants have been characterized. This has revealed antimicrobial and primarily antifungal defensins with partially elucidated modes of action. It is now evident that some defensin-like peptides modulate rhizobial growth to the benefit of some legumes. In those legumes there has been huge diversification of a subset of defensin-like genes and at least some of these control rhizobial differentiation. However we do not know the individual and collective functions of NCRs and how they induce terminal differentiation; presumably it improves the efficiency of nitrogen fixation in some legumes. Could classical defensins similarly control and sustain the survival of some endophytic micro-organisms that promote plant growth? More work on defensins and defensin-like peptides will be required to understand their functions and roles in plant–microbe interactions.
Acknowledgements Our work is supported by the ‘SYM-BIOTICS’ Advanced Grant of the European Research Council to EK (grant number 269067) and by TA´MOP4.2.2.A-11/1/KONV-2012-0035 supported by the European Union and co˝cs at the financed by the European Social Fund. We thank Attila Szu Bioinformatics Platform for his help.
Appendix A. Supplementary data Supplementary data associated with this article can be found, in the online version, at http://dx.doi.org/10.1016/j. pbi.2015.05.031.
References and recommended reading Papers of particular interest, published within the period of review, have been highlighted as: of special interest of outstanding interest 1.
Fan J, Doerner P: Genetic and molecular basis of nonhost disease resistance: complex, yes; silver bullet, no. Curr Opin Plant Biol 2012, 15:400-406.
2.
Lacerda AF, Vasconcelos EA, Pelegrini PB, Grossi de Sa MF: Antifungal defensins and their role in plant defense. Front Microbiol 2014, 5:116.
3.
Goyal RK, Mattoo AK: Multitasking antimicrobial peptides in plant development and host defense against biotic/abiotic stress. Plant Sci 2014, 228:135-149.
4.
Kereszt A, Mergaert P, Maro´ti G, Kondorosi E: Innate immunity effectors and virulence factors in symbiosis. Curr Opin Microbiol 2011, 14:76-81. Current Opinion in Plant Biology 2015, 26:57–63
62 Biotic interactions
5.
Carro L, Pujic P, Alloisio N, Fournier P, Boubakri H, Hay AE, Poly F, Franc¸ois P, Hocher V, Mergaert P et al.: Alnus peptides modify membrane porosity and induce the release of nitrogen-rich metabolites from nitrogen-fixing Frankia. ISME J 2015 http:// dx.doi.org/10.1038/ismej.2014257. This study demonstrates that Alnus glutinosa produces nodule-specific defensin-like peptides, and one of these peptides (Ag5) was shown to target the symbiotic bacterium partner Frankia. The peptide increased bacterial membrane permeability resulting in a significant release of nitrogen-containing metabolites, mainly glutamine and glutamate.
Medicago genome provides insight into the evolution of rhizobial symbioses. Nature 2011, 480:520-524. 23. Tesfaye M, Silverstein KA, Nallu S, Wang L, Botanga CJ, Gomez SK, Costa LM, Harrison MJ, Samac DA, Glazebrook J et al.: Spatio-temporal expression patterns of Arabidopsis thaliana and Medicago truncatula defensin-like genes. PLoS One 2013, 8:e58992. The authors of this article described notable differences in DEFL gene expression patterns between Arabidopsis and Medicago. The study revealed, that most of the Arabidopsis DEFLs are expressed in inflorescences, while only a few exhibited root-enhanced expression. However, Medicago DEFLs were most prominently expressed in nitrogenfixing root nodules.
6.
Gao AG, Hakimi SM, Mittanck CA, Wu Y, Woerner BM, Stark DM, Shah DM, Liang J, Rommens CM: Fungal pathogen protection in potato by expression of a plant defensin peptide. Nat Biotechnol 2000, 18:1307-1310.
7.
Park HC, Kang YH, Chun HJ, Koo JC, Cheong YH, Kim CY, Kim MC, Chung WS, Kim JC, Yoo JH et al.: Characterization of a stamen-specific cDNA encoding a novel plant defensin in Chinese cabbage. Plant Mol Biol 2002, 50:59-69.
24. Mergaert P, Nikovics K, Kelemen Z, Maunoury N, Vaubert D, Kondorosi A, Kondorosi E: A novel family in Medicago truncatula consisting of more than 300 nodule-specific genes coding for small, secreted polypeptides with conserved cysteine motifs. Plant Physiol 2003, 132:161-173.
8.
Koike M, Okamoto T, Tsuda S, Imai R: A novel plant defensin-like gene of winter wheat is specifically induced during cold acclimation. Biochem Biophys Res Commun 2002, 298:46-53.
25. Graham MA, Silverstein KAT, Cannon SB, VandenBosch KA: Computational identification and characterization of novel genes from legumes. Plant Physiol 2004, 135:1179-1197.
9.
Segura A, Moreno M, Molina A, Garcia-Olmedo F: Novel defensin subfamily from spinach (Spinacia oleracea). FEBS Lett 1998, 435:159-162.
26. Silverstein KAT, Moskal WA, Wu HC, Underwood BA, Graham MA, Town CD, VandenBosch KA: Small cysteine-rich peptides resembling antimicrobial peptides have been under-predicted in plants. Plant J 2007, 51:262-280.
10. Chen KC, Lin CY, Kuan CC, Sung HY, Chen CS: A novel defensin encoded by a mungbean cDNA exhibits insecticidal activity against bruchid. J Agric Food Chem 2002, 50:7258-7263. 11. Kanzaki H, Nirasawa S, Saitoh H, Ito M, Nishihara M, Terauchi R, Nakamura I: Overexpression of the wasabi defensin gene confers enhanced resistance to blast fungus (Magnaporthe grisea) in transgenic rice. Theor Appl Genet 2002, 105:809-814. 12. Thomma BP, Cammue BP, Thevissen K: Plant defensins. Planta 2002, 216:193-202. 13. Lay FT, Anderson MA: Defensins, components of the innate immune system in plants. Curr Protein Pept Sci 2005, 6:85-101. 14. Aerts AM, Franc¸ois IE, Cammue BP, Thevissen K: The mode of antifungal action of plant, insect and human defensins. Cell Mol Life Sci 2008, 65:2069-2079. 15. van der Weerden NL, Bleackley MR, Anderson MA: Properties and mechanisms of action of naturally occurring antifungal peptides. Cell Mol Life Sci 2013, 70:3545-3570. 16. Thevissen K, Kristensen HH, Thomma BP, Cammue BP, Franc¸ois IE: Therapeutic potential of antifungal plant and insect defensins. Drug Discov Today 2007, 12:966-971. 17. Aerts AM, Franc¸ois IE, Meert EM, Li QT, Cammue BP, Thevissen K: The antifungal activity of RsAFP2, a plant defensin from Raphanus sativus, involves the induction of reactive oxygen species in Candida albicans. J Mol Microbiol Biotechnol 2007, 13:243-247. 18. Sagaram US, Pandurangi R, Kaur J, Smith TJ, Shah DM: Structure–activity determinants in antifungal plant defensins MsDef1 and MtDef4 with different modes of action against Fusarium graminearum. PLoS One 2011, 6:e18550. 19. De Coninck B, Carron D, Tavormina P, Willem L, Craik DJ, Vos C, Thevissen K, Mathys J, Cammue BP: Mining the genome of Arabidopsis thaliana as a basis for the identification of novel bioactive peptides involved in oxidative stress tolerance. J Exp Bot 2013, 64:5297-5307. 20. Giacomelli L, Nanni V, Lenzi L, Zhuang J, Dalla Serra M, Banfield MJ, Town CD, Silverstein KA, Baraldi E, Moser C: Identification and characterization of the defensin-like gene family of grapevine. Mol Plant Microbe Interact 2012, 25: 1118-1131. 21. Silverstein KAT, Graham MA, Paape TD, VandenBosch KA: Genome organization of more than 300 defensin-like genes in Arabidopsis. Plant Physiol 2005, 138:600-610. 22. Young ND, Debelle´ F, Oldroyd GE, Geurts R, Cannon SB, Udvardi MK, Benedito VA, Mayer KF, Gouzy J, Schoof H et al.: The Current Opinion in Plant Biology 2015, 26:57–63
27. Van de Velde W, Zehirov G, Szatmari A, Debreczeny M, Ishihara H, Kevei Z, Farkas A, Mikulass K, Nagy A, Tiricz H et al.: Plant peptides govern terminal differentiation of bacteria in symbiosis. Science 2010, 327:1122-1126. 28. Hanks JN, Snyder AK, Graham MA, Shah RK, Blaylock LA, Harrison MJ, Shah DM: Defensin gene family in Medicago truncatula: structure, expression and induction by signal molecules. Plant Mol Biol 2005, 58:385-399. 29. Sagaram US, El-Mounadi K, Buchko GW, Berg HR, Kaur J, Pandurangi RS, Smith TJ, Shah DM: Structural and functional studies of a phosphatidic acid-binding antifungal plant defensin mtdef4: identification of an rgfrrr motif governing fungal cell entry. PLoS One 2013, 8:e82485. This study provides detailed analysis of the structure of MtDef4. Based on the results of mutational analyses the authors suggest that the RGFRRR loop is a translocation signal required for the internalization of the protein. Amino acid substitutions in the RGFRRR sequence abolished the ability of MtDef4 to enter fungal cells. 30. Munoz A, Chu M, Marris PI, Sagaram US, Kaur J, Shah DM, Read ND: Specific domains of plant defensins differentially disrupt colony initiation, cell fusion and calcium homeostasis in Neurospora crassa. Mol Microbiol 2014, 92:1357-1374. 31. Yount NY, Yeaman MR: Multidimensional signatures in antimicrobial peptides. Proc Natl Acad Sci USA 2004, 101:73637368. 32. Spelbrink RG, Dilmac N, Allen A, Smith TJ, Shah DM, Hockerman GH: Differential antifungal and calcium channelblocking activity among structurally related plant defensins. Plant Physiol 2004, 135:2055-2067. 33. Kaur J, Thokala M, Robert-Seilaniantz A, Zhao P, Peyret H, Berg H, Pandey S, Jones J, Shah D: Subcellular targeting of an evolutionarily conserved plant defensin MtDef4.2 determines the outcome of plant–pathogen interaction in transgenic Arabidopsis. Mol Plant Pathol 2012, 13:1032-1046. 34. Nallu S, Silverstein KA, Zhou P, Young ND, Vandenbosch KA: Patterns of divergence of a large family of nodule cysteine-rich peptides in accessions of Medicago truncatula. Plant J 2014, 78:697-705. 35. Kondorosi E, Mergaert P, Kereszt A: A paradigm for endosymbiotic life: cell differentiation of Rhizobium bacteria provoked by host plant factors. Annu Rev Microbiol 2013, 67:611-628. 36. Haag AF, Kerscher B, Dall’Angelo S, Sani M, Longhi R, Baloban M, Wilson HM, Mergaert P, Zanda M, Ferguson GP: Role of cysteine residues and disulfide bonds in the activity of a legume root nodule-specific, cysteine-rich peptide. J Biol Chem 2012, 287:10791-10798. www.sciencedirect.com
Standard defensins and defensin-like peptides Maro´ti, Downie and Kondorosi 63
In this study, the authors showed the significance of the proper formation of disulfide bridges in NCR247. They found that either cysteine replacements or S–S bond modifications influenced the activity of Medicago truncatula NCR247 against Sinorhizobium meliloti. Substitution of cysteines for serines, changing the S–S bridges from cysteines 1–2, 3– 4 to 1–3, 2–4 and oxidation of NCR247 lowered the peptide’s activity against S. meliloti.
Medicago truncatula nodulation are coupled to two transcriptome-switches. PLoS One 2010, 5:e9519. 43. Roux B, Rodde N, Jardinaud MF, Timmers T, Sauviac L, Cottret L, Carre`re S, Sallet E, Courcelle E, Moreau S et al.: An integrated analysis of plant and bacterial gene expression in symbiotic root nodules using laser-capture microdissection coupled to RNA sequencing. Plant J 2014, 77:817-837.
37. Tiricz H, Szu˝cs A, Farkas A, Pap B, Lima RM, Maro´ti G, Kondorosi E´, Kereszt A: Antimicrobial nodule-specific cysteinerich peptides induce membrane depolarization-associated changes in the transcriptome of Sinorhizobium meliloti. Appl Environ Microbiol 2013, 79:6737-6746.
44. Wang D, Griffitts J, Starker C, Fedorova E, Limpens E, Ivanov S, Bisseling T, Long S: A nodule-specific protein secretory pathway required for nitrogen-fixing symbiosis. Science 2010, 327:1126-1129.
38. O¨rdo¨gh L, Vo¨ro¨s A, Nagy I, Kondorosi E, Kereszt A: Symbiotic plant peptides eliminate Candida albicans both in vitro and in an epithelial infection model and inhibit the proliferation of immortalized human cells. Biomed Res Int 2014, 2014:320796 http://dx.doi.org/10.1155/2014/320796.
45. Du¨rgo˝ H, Klement E´, Hunyadi-Gulya´s E´, Szu˝cs A, Kereszt A, Medzihradszky KF, Kondorosi E´: Identification of nodulespecific cysteine-rich plant peptides in endosymbiotic bacteria. Proteomics 2015 http://dx.doi.org/10.1002/ pmic.201400385.
39. Sinharoy S, Torres-Jerez I, Bandyopadhyay K, Kereszt A, Pislariu CI, Nakashima J, Benedito VA, Kondorosi E, Udvardi MK: The C2H2 transcription factor regulator of symbiosome differentiation represses transcription of the secretory pathway gene VAMP721a and promotes symbiosome development in Medicago truncatula. Plant Cell 2013, 25: 3584-3601.
46. Farkas A, Maro´ti G, Du¨rgo˝ H, Gyo¨rgypa´l Z, Lima RM, Medzihradszky KF, Kereszt A, Mergaert P, Kondorosi E´: The Medicago truncatula symbiotic peptide NCR247 contributes to bacteroid differentiation through multiple mechanisms. Proc Natl Acad Sci USA 2014, 111:5183-5188. This study shows the detailed in vivo and in vitro characterization of a Medicago truncatula NCR peptide (NCR247). It was shown that NCR247 penetrates the bacteria and forms complexes with many bacterial proteins including ribosomal proteins, FtsZ and GroEL. Interaction with bacterial FtsZ required for septum formation is one of the plant host interventions for inhibiting bacterial cell division.
40. Mergaert P, Uchiumi T, Alunni B, Evanno G, Cheron A, Catrice O, Mausset AE, Barloy-Hubler F, Galibert F, Kondorosi A et al.: Eukaryotic control on bacterial cell cycle and differentiation in the Rhizobium–legume symbiosis. Proc Natl Acad Sci USA 2006, 103:5230-5235. 41. Guefrachi I, Nagymihaly M, Pislariu CI, Van de Velde W, Ratet P, Mars M, Udvardi MK, Kondorosi E, Mergaert P, Alunni B: Extreme specificity of NCR gene expression in Medicago truncatula. BMC Genomics 2014, 15:712 http://dx.doi.org/10.1186/14712164-15-712. 42. Maunoury N, Redondo-Nieto M, Bourcy M, Van de Velde W, Alunni B, Laporte P, Durand P, Agier N, Marisa L, Vaubert D et al.: Differentiation of symbiotic cells and endosymbionts in
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47. Penterman J, Abo RP, De Nisco NJ, Arnold MF, Longhi R, Zanda M, Walker GC: Host plant peptides elicit a transcriptional response to control the Sinorhizobium meliloti cell cycle during symbiosis. Proc Natl Acad Sci USA 2014, 111:3561-3566. This study demonstrates that the Medicago truncatula NCR247 peptide specifically blocks bacterial cell division and antagonizes Z-ring function. Additionally, expression of critical cell-cycle regulators, including ctrA, and cell division genes were greatly attenuated in NCR-treated cells. NCR247 treatment was also shown to affect the expression of genes and functions important in symbiosis.
Current Opinion in Plant Biology 2015, 26:57–63