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Seed defensins of barnyard grass Echinochloa crusgalli (L.) Beauv.
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Biochimie 90 (2008) 1667e1673 www.elsevier.com/locate/biochi
Research paper
Seed defensins of barnyard grass Echinochloa crusgalli (L.) Beauv. Tatyana I. Odintsova a, Eugene A. Rogozhin b, Yurij Baranov b, Alexander Kh. Musolyamov b, Nasser Yalpani c, Tsezi A. Egorov b,*, Eugene V. Grishin b a
Laboratory of Plant Genetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, ul. Gubkina 3, 117809 Moscow, Russian Federation Laboratory of Neuroreceptors and Neuroregulators, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Group of antimicrobial peptides, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russian Federation c Pioneer Hi-Bred International, Inc., 7300 NW 62nd Avenue, Johnston, IA 50131-0552, USA
b
Received 3 April 2008; accepted 16 June 2008 Available online 26 June 2008
Abstract From the annual weed barnyard grass Echinochloa crusgalli (L.) Beauv., two novel defensins Ec-AMP-D1 and Ec-AMP-D2 that differ by a single amino acid substitution were isolated by a combination of different chromatographic procedures. Both defensins were active against several phytopathogenic fungi and the oomycete Phytophthora infestans at micromolar concentrations. The Ec-AMP-D1 showed higher activity against the oomycete than Ec-AMP-D2. The comparison of the amino acid sequences of the antifungal E. crusgalli defensins with those of earlier characterized T. kiharae defensins [T.I. Odintsova, Ts.A. Egorov, A.Kh. Musolyamov, M.S. Odintsova, V.A. Pukhalsky, E.V. Grishin, Seed defensins from T. kiharae and related species: genome localization of defensin-encoding genes, Biochimie, 89 (2007) 605e612.] that were devoid of substantial antifungal activity point to the C-terminal region of the molecule as the main determinant of the antifungal activity of E. crusgalli defensins. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Weeds; Echinochloa crusgalli (L.) Beauv.; Triticum kiharae Dorof. et Migusch.; Defensins; Antifungal activity
1. Introduction Among the innate immunity mechanisms evolved by plants to protect themselves against pathogenic microorganisms and pests, the synthesis of compounds with antimicrobial properties plays a pivotal role [1]. The antimicrobial compounds include phytoalexins, and proteins and peptides that display activity against pathogens. Antimicrobial peptides (AMPs) are important components of the plant defense system [2e5]. They are cationic and amphiphilic molecules that inhibit growth of a wide range of microorganisms and pathogenic insects. In the case of microbes, their mode of action involves
Abbreviations: AMP, antimicrobial peptide; HPLC, high-performance liquid chromatography; SE, size-exclusion; RP, reversed-phase; MALDITOF MS, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry; TFA, trifluoroacetic acid. * Corresponding author. Tel.: þ7 495 3364022; fax: þ7 495 3307301. E-mail address: [email protected] (T.A. Egorov). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.06.007
the disruption of the membranes of pathogens via specific or nonspecific interactions with cell surface groups [6,7]. The deleterious effect on insects is supposed to be associated with inhibition of gut enzymes e amylases and proteases [8e10]. Most plant AMPs are cysteine-rich peptides. According to cysteine motifs (the arrangement of cysteine molecules in the amino acid sequence of the peptide) and three-dimensional scaffold, AMPs are classified into several families (thionins, defensins, lipid-transfer proteins, hevein- and knottin-like peptides, and macrocyclic peptides) [2e4]. Defensins belong to the largest and best-studied AMP family [11e14]. They were discovered in invertebrates, birds, mammals and plants [15]. More than 300 defensin-like sequences were identified in the completely sequenced Arabidopsis genome [16]. This redundancy is probably necessary to protect a plant against selections of pathogen mutants with enhanced tolerance to a particular type of a defense molecule. Plant defensins have a characteristic cysteine signature with 8 cysteine residues involved in the formation of 4 disulphide
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bridges. Despite considerable variation in amino acid sequences, the fold of the molecule is similar and involves the so-called cysteine-stabilized alpha-helix beta-sheet motif (CSab), in which two cysteine residues separated by one turn of the alpha-helix are connected with two cysteines, which are located a single amino acid apart in the third beta strand [12]. A wide range of activities has been revealed by in vitro studies of plant defensins that imply their involvement in biotic and abiotic stress responses. These include fungicidal and bactericidal activities [17e25], inhibition of proteases and amylases [8e10], the abilities to block ion channels [26, 27] and inhibition of protein synthesis [28,29]. Recently, the cytotoxic effect of some defensins on plant [30] and animal [23] cells has been shown. The protective role of defensins in vivo is supported by enhanced tolerance to infections of transgenic plants with constitutive expression of the corresponding genes. Defensins were specifically induced in cold-acclimated wheat plants during cold acclimation [31]. They were also shown to be involved in Zn tolerance in Arabidopsis [32]. Structuree function relationships in defensins remain an intriguing but still insufficiently studied area of research. The exact mechanisms of action of most defensins are poorly understood. Evidence is mounting that defensins interact with specific targets on the fungal membrane. The binding sites, fungusspecific sphingolipids, for the radish Rs-AFP2 and dahlia DmAMP1 defensins on fungal membranes were identified [33e35]. Great yield losses of crops caused by pathogenic microorganisms and pests stimulate the search for new genes conferring pathogen resistance. Defensins exhibiting broad-spectrum resistance are good candidates for genetic transformation of plants. Certain progress has already been achieved in this field. Defensins also bear great potential as models for the creation of new anti-infective drugs, both bactericidal and antimycotic, since in the last decades the incidence of microbial infections is rising, together with the number of pathogenic microorganisms resistant to conventional antibiotics [36]. Weeds, to compete with crops, have developed a highly sophisticated multicomponent adaptation system, which makes them successful as invasive species. The molecular basis of this system is formed by diverse phytotoxins, which are released into the environment and suppress growth of other plants [37], and effective pathogen resistance mechanisms providing better competitiveness and adaptation to stressful conditions. Enhanced pathogen resistance makes weeds a valuable but still poorly explored source of natural antibiotics. The aim of this work was to isolate and study seed defensins from a weed cereal species Echinochloa crusgalli (L.) Beauv. (barnyard grass) (order Poales, family: Poaceae, subfamily Pooideae, tribe Paniceae, genus Echinochloa), which is widely spread throughout the world. It reduces crop yields by removing up to 80% of the soil nitrogen. In addition, it accumulates high level of nitrates that are toxic to livestock [37]. Nevertheless, it has some important usages: hay made of this grass can be kept up for several years. In some countries, it is used as a folk remedy to cure infections [37]. Analysis of E. crusgalli AMPs contributes to the understanding of weed proteomics
and genomics necessary for the identification of genes that could improve crop yields. In this study, we isolated two novel defensins from seeds of this species, determined their amino acid sequences and demonstrated their fungicidal effect on several phytopathogenic fungi and the oomycete Phytophthora infestans. 2. Materials and methods 2.1. Biological material Seeds of E. crusgalli (L.) Beauv. (family Poaceae) were collected in the Krasnodarsky region in 2005. The fungi Fusarium graminearum, Fusarium verticillioides, Colletotrichum graminicola, Diplodia maydis were from the collection of Pioneer Hi-Bred (USA). Fusarium oxysporum, Botrytis cinerea, Alternaria consortiale, Fusarium culmorum and the oomycete P. infestans were from the collection of the Timiryazev Agricultural Academy (Moscow, Russian Federation). Helminthosporium sativum was obtained from the All-Russia Collection of Microorganisms (Pushchino, Moscow Region). 2.2. Isolation of defensins from E. crusgalii seeds Seeds of E. crusgalii (10 g) were ground into flour in a coffee mill and extracted with 10% (v/v) acetic acid in the presence of the proteinase inhibitor cocktail for plant cell extracts (Sigma, St. Louis, MO, USA) at a flour to solvent ratio of 1:10 (w/v) at room temperature for 1.5 h with constant stirring. After centrifugation at 20,000g for 25 min at 4 C, the supernatant was clarified by filtration through a paper filter. Proteins and peptides were precipitated overnight with six volumes of ice-cold acetone at 4 C, collected by centrifugation at 5000g for 15 min at 4 C, air-dried and dissolved in solvent A (0.1% TFA) for desalting. Desalting was performed by RP-HPLC on an Aquapore C8 column, 100 10 mm (Applied Biosystems, USA) equilibrated with solvent A. After elution of the unadsorbed fraction, proteins and peptides were eluted with solvent B (80% CH3CN in 0.1% TFA) at a flow rate of 1.5 ml/min and detected at 214 nm. The obtained fraction was dried on a SpeedVac concentrator (Savant, USA). For the purification of defensins from E. crusgalii seeds the protocol developed previously for the isolation of T. kiharae defensins was employed [38,39]. Desalted fraction was solubilized in 10 mM TriseHCl, pH 7.2 (buffer A) and subjected to affinity chromatography on a column (30 15 mm) of Heparin Sepharose 6 Fast Flow (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) equilibrated with buffer A. After elution of the unadsorbed fraction, proteins and peptides were eluted with a step-wise NaCl gradient in buffer A e 50, 100 and 500 mM NaCl, respectively, at a flow rate of 1 ml/min. Proteins and peptides were detected at 280 nm. The obtained fractions were desalted and dried on a SpeedVac concentrator as described above. The fraction eluted at 100 mM NaCl concentration during affinity chromatography was separated by sizeexclusion chromatography on a Superdex Peptide HR 10/30 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
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Proteins and peptides were eluted with 5% CH3CH in 0.05% TFA at a flow rate of 15 ml/h and detected at 214 nm. The peptide fraction was further separated by RP-HPLC on a ReproSil-Pur 300 ODS-3 column (250 4 mm) (Dr. A. Maisch, Ammerbuch, Germany) with a linear acetonitrile gradient (10e40% solvent B) at a flow rate of 0.7 ml/min and detection at 214 nm. 2.3. Isolation of defensins from seeds of T. kiharae The procedure for the isolation of T. kiharae defensins was described previously [38,39]. 2.4. Reduction and alkylation of defensins Defensins were reduced with dithiothreitol (DTT) and alkylated with 4-vinylpyridine as described [38]. The peptide (approx. 2 nmol) was dissolved in 20 ml of 0.5 M TriseHCl buffer, pH 7.6, containing 6 M guanidine hydrochloride and 2 mM ethylenediaminetetraacetic acid (EDTA, disodium salt). One microliter of freshly prepared aqueous DTT solution (1.4 mM DTT) was added to the mixture. The reaction was allowed to proceed under nitrogen for 4 h at 40 C. After reduction, 2 ml of 50% 4-vinylpyridine in 2-propanol (v/v) was added, mixed and allowed to react for another 20 min under nitrogen at room temperature in the dark. After the reaction, the mixture was diluted twofold with 0.1% (v/v) TFA and desalted on a ReproSil-Pur C18 column as described above. The number of cysteine residues was estimated as the mass difference between the reduced and alkylated and the nonalkylated peptide. 2.5. Digestion of defensins with Glu-C endoproteinase Reduced and alkylated defensins (approx. 1.5 nmol) were dissolved in 20 ml of 25 mM ammonium bicarbonate buffer, pH 7.8. Glu-C endoproteinase from Staphylococcus aureus V8 for protein sequencing (Sigma) dissolved in the same buffer was added to the peptide solution (1:20, w/w). The reaction proceeded for 4 h at 40 C and was stopped by the addition of 100 ml 0.1% v/v TFA. The peptides generated during the enzymatic cleavage were separated by RP-HPLC on a Reprosil column with a linear acetonitrile gradient (0e40% solvent B). Solvents A and B were the same as used for the purification of defensins. Peptides were detected at 214 nm. 2.6. Analytical methods Mass spectra were acquired on a model Ultraflex MALDITOF mass spectrometer (Bruker Daltonics, Germany). 2,5-Dihydroxy benzoic acid (Sigma, USA) was used as a matrix. The instrument was calibrated with a mixture of insulin b-chain and bovine insulin (Sigma, USA). Amino acid sequences were determined by automated Edman degradation on a model 492 Procise sequencer (Applied Biosystems) according to the manufacturer’s protocol. Amino
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acid sequence homology searches were carried out by computerized query of the GenBank/EMBL data bank. 2.7. Antifungal assays The antifungal activity of defensins was tested against the phytophathogenic fungi F. graminearum, F. oxysporum, F. verticillioides, C. graminicola, B. cinerea, H. sativum and D. maydis and carried out in microplates essentially as described [40]. Twofold dilution series of the peptides in Milli-Q water were prepared and added to the spore suspension in the growth medium. The concentration of spores varied from 4000 to 6000 spores/ml depending on the fungus. The concentration of peptides was determined from a calibration curve obtained with aprotinin dilutions. Plates were scored after 24e48 h of incubation. Inhibition of spore germination was assayed and scored from 0 to 4, with 4 representing complete inhibition of spore germination and hyphal growth and 0 denoting the absence of inhibition compared to controls. For the oomycete P. infestans, two parameters were studied: the release of zoospores after incubation at 4e6 C for 2e3 h and the development of hyphae from zoosporangia after growth at 20e22 C for 48 h. The effect of the peptide and the degree of inhibition were estimated by measuring the number of zoospores released and the zoosporangia with hyphae relative to their total number. Morphological changes in the microorganisms were also recorded. 3. Results 3.1. Isolation of defensins from E. crusgalli seeds Isolation of defensins from seeds of E. crusgalli followed the protocol earlier developed for the purification of T. kiharae defensins [38,39] and included acidic extraction of flour in the presence of the protease inhibitors with subsequent separation of the protein/peptide fraction by affinity chromatography, SE-HPLC and RP-HPLC. Since the molecular masses of plant defensins are approx. 5 kDa [12], mass spectrometry was used at each purification step to trace the defensin-containing fractions. Separation of the acid-soluble fraction from seeds of E. crusgalli by affinity chromatography is shown in Fig. 1A. Three fractions eluted at 50 mM, 100 mM, and 500 mM NaCl concentration, respectively, were collected. Molecular mass analysis showed that the fraction eluted at 100 mM NaCl concentration contained peptides in the molecular mass range of defensins, therefore this fraction was further subjected to separation by SE-HPLC (Fig. 1B). In this case, six fractions were obtained, fraction 5 contained peptides, which were further separated by RP-HPLC (Fig. 1C). As a result, two peptides Ec-AMP-D1 and Ec-AMP-D2 with molecular masses of 5098 and 5170 Da, respectively, were obtained. The number of cysteine residues in both peptides was determined by estimating the mass difference between the reduced and alkylated and the unreduced native peptides. Both peptides had 8 cysteine residues which is typical for defensins. Alkylation of unreduced peptides followed by mass measurements showed that
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Fig. 2. RP-HPLC separation of the peptides obtained by Glu-C digestion of the Ec-AMP-D1 defensin from seeds of E. crusgalli. Reconstruction of the polypeptide chain of Ec-AMP-D1 is shown on the top. Black arrows show the N-terminal sequence of the entire peptide and the C-terminal Glu-C fragment. Dashed line shows two Glu-C peptides identified by mass analysis of fraction no. 1. The position of fragments in the polypeptide chain is given in parentheses.
Fig. 1. Separation of acid-soluble proteins/peptides from seeds of E. crusgalli (L.) Beauv. (A) Affinity chromatography of the total extract on Heparin Sepharose 6 Fast Flow. (B) SE-HPLC of the 100-mM NaCl fraction on a Superdex Peptide HR 10/30 column, fraction no. 5 containing defensins is shaded. C, RP-HPLC of fraction no. 5 obtained by SE-HPLC on a Reprosil-Pur 300 ODS-3 column. For experimental details, see Section 2.2.
all cysteine residues were involved in the formation of disulphide bridges which is also a characteristic feature of plant AMPs including defensins. 3.2. Amino acid sequences of defensins The N-terminal amino acid sequence (29 amino acid residues) of the reduced and alkylated peptide Ec-AMP-D1 was determined by automated Edman degradation. To
determine complete sequence, the peptide was cleaved with Glu-C proteinase, and the reaction products were separated by RP-HPLC (Fig. 2). Two main peaks were obtained (Fig. 2). In peak 1, two molecular masses were detected: 3459.5 and 3174.3 Da. The 3459.5-Da peptide corresponded to the N-terminal part of the molecule (residues 1e27) originating from incomplete cleavage at Glu2, while the 3174.3Da peptide corresponded to the fragment 3e27 of the polypeptide chain. In peak 2, a single peptide with a molecular mass of 2503.2 Da was obtained and sequenced. It corresponded to the C-terminal part of the Ec-AMP-D1 molecule (Fig. 2). The amino acid sequence of Ec-AMP-D2 was determined similarly (data not shown). The comparison of both sequences showed that the EcAMP-D1 and Ec-AMP-D2 peptides were nearly identical, and differed by a single amino acid residue at position 45: in Ec-AMP-D2 a histidine residue was substituted for alanine (Fig. 3). For both peptides, the measured and calculated masses coincided within the accuracy of the mass spectrometric method indicating the absence of post-translational modifications except disulphide bonds. The homology search in the UniProt database revealed homology of the isolated peptides with plant defensins. In addition to amino acid sequence similarity, the cysteine motif was characteristic of defensins: 2-C10-C-5-C-3-C-8-C-6-C-1-C-3-C [14]. The alignment of amino acid sequences of Ec-AMP-D1 and Ec-AMP-D2 with some cereal defensins is given in Fig. 3. Of the defensins taken for comparison, Ec-AMP-D1 and Ec-AMP-D2 shared the highest similarity with T. kiharae D defensins characterized earlier [39]: it ranged from 45 to 65% for different molecular forms with the highest homology score for Tk-AMP-D1.1. The homology with other cereal defensins was lower: 45% with wheat g2-P and barley g1-H, 43% for wheat g1-P, sorghum SI-2 and SI-3, and maize g1-Z. It is of particular interest
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Fig. 3. Amino acid sequence alignment of E. crusgalli (Ec-AMP-D1 and Ec-AMP2), T. kiharae (Tk-AMP-D1 and Tk-AMP-D1.1) and T. turgidum (g1-P) defensins. Amino acid sequences were aligned manually. Gaps were introduced to maximize homology. Variant amino acid residues are shaded. The percent of sequence identity between Ec-AMP-D1 and other defensins is presented on the right, and in parentheses, the percentage of sequence identity between the N-terminal regions (residues 1e27) of defensins is given. The secondary structure elements (b-strands and a-helix) as determined for g1-P [14] are shown in the bottom. The variable region between Ec-AMP-D1, Ec-AMP2 and Tk-AMP-D1 discussed in the text is boxed.
that the homology between E. crusgalli defensins and most cereal defensins in the N-terminal portion of the molecule (residues 1e27, the numbering is according to Ec-AMP-D1) is even higher than for the whole molecules, reaching its maximum of 74% or 85% if we take into account synonymous substitutions for Tk-AMP-D1 (Fig. 3). With other T. kiharae D defensins it is lower (from 70 to 62%) (data not shown). These results indicate that most variation between E. crusgalli and T. kiharae D defensins resides in the C-terminal part of the molecule. 3.3. Antifungal assays Testing of the antifungal activity of Ec-AMP-D1 against phytopathogenic fungi revealed inhibitory activity against F. graminearum, F. oxysporum, F. verticillioides, and D. maydis. The peptide Ec-AMP-D1 was inactive against C. graminicola, B. cinerea, and H. sativum at concentrations below 30 mg/ml. The IC50 for inhibition of F. graminearum spore germination was 15 mg/ml, for F. verticillioides, IC50 was about 8.5 mg/ml, and for D. maydis, about 12.5 mg/ml. The Ec-AMP-D1 was only weakly active against F. oxysporum with an IC50 of 102 mg/ml. Determination of the effect of Ec-AMP-D1 on the release of P. infestans zoospores showed no effect at the concentrations tested (below 100 mg/ml). However the Ec-AMP-D1 peptide inhibited hyphal development at IC50 of 25.5 mg/ml. Increasing the peptide concentration enhanced the degree of inhibition, however complete inhibition was not achieved at all concentrations tested. In addition to the inhibition of hyphal growth, morphological changes in the oomycete including lysis of hyphae and sporangia in the presence of the peptide at concentrations above 100 mg/ml were observed. Since in this study the peptide Ec-AMP-D2 was isolated from seeds in much smaller amounts than D1, its activity was tested only against F. oxysporum and P. infestans. Antifungal assays showed that the activity of Ec-AMP-D2 against F. oxysporum was similar to that of Ec-AMP-D1 and rather weak, IC50 for both peptides was 102 mg/ml. However, the activity of Ec-AMP-D2 against the oomycete P. infestans was lower than that of Ec-AMP-D1. The IC50 for inhibition of hyphal development was 50 mg/ml. In addition, in contrast to
Ec-AMP-D1, Ec-AMP-D2 caused no morphological changes in the oomycete. Accordingly although the differences between the amino acid sequences of both E. crusgalli defensins were minimal, their biological activity in vitro was affected. Since defensins from E. crusgalli seeds showed striking similarity with T. kiharae Tk-AMP-D1 defensin especially in the N-terminal part of the molecule, we compared their antifungal activity against several phytopathogenic fungi. Testing of antifungal activity of Tk-AMP-D1 showed that it was inactive against most fungi tested at concentrations below 100 mg/ml, such as: A. consortiale, B. cinerea, H. sativum, F. culmorum, C. graminicola and D. maydis. The T. kiharae D1 defensin was only weakly active against F. graminearum and F. verticillioides at concentrations below 30 mg/ml, the inhibition of F. graminearum scored 1.5 and that of F. verticilloides, 1 at the highest tested concentrations (30 mg/ml). Thus, the activity of T. kiharae D1 defensin against the Fusarium species was much weaker than that of the E. crusgalli EcAMP-D1 defensin.
4. Discussion Plant defensins are widespread antimicrobial peptides, which share a similar tertiary structure, but differ considerably in biological functions [14]. They are regarded as novel leads for antifungal therapeutics [36] and candidates for plant transformation and production of pathogen-resistant crops. Defensins of cultivated cereals (wheat, barley, sorghum, and maize) studied so far represent a unique group in the sense that they lack substantial antifungal activity [20]. Our results confirmed the available literature data and showed that defensins of T. kiharae, a synthetic allopolyploid produced by crossing Triticum timopheevii and Aegilops squarrosa failed to inhibit growth of the filamentous fungi tested. In this work, we showed for the first time that defensins from a weed species E. crusgalli, which also belongs to the Poaceae family, in contrast to cultivated cereals studied so far, possess rather potent antifungal activity against a number of phytopathogenic fungi and the oomycete. This finding represents a spectacular example of preservation of antifungal functions in defensins of weeds to achieve fitness, competitiveness and adaptation in
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agroecosystems, which were largely lost during cultivation of cereals. The discovery of antifungal activity in E. crusgalli defensins opens new possibilities for the analysis of structureefunction relationships e.g., in particular, the identification of determinants of antifungal activity. In general, this may be achieved by several approaches: by comparing amino acid sequences of highly homologous peptides of different origin or those of a single plant species contrasting in biological (antifungal) activity, by site-directed mutagenesis, and by generating chimeric molecules combining different regions of highly homologous peptides that differ in antifungal activity (strong or weak). The pioneering works exploiting first two approaches were performed by the Belgium group [18,21] using radish defensins as a model. In this work, we isolated two nearly identical defensins from E. crusgalli. The peptides differed by a single amino acid substitution at position 45: in Ec-AMP-D2 Ala was substituted for His, thus providing the Ec-AMP-D2 molecule a higher net positive charge. This single replacement affected the biological activity of Ec-AMP-D2. Our results showed that although the activity of both peptides was similar towards the fungus F. oxysporum, it differed towards the oomycete P. infestans, Ec-AMP-D1 being more active than Ec-AMP-D2. In addition Ec-AMP-D1 caused morphological changes in the oomycete P. infestans, while Ec-AMP-D2 did not. Thus although the net positive charge of Ec-AMP-D2 was higher than that of Ec-AMP-D1 its antifungal activity against the microorganisms tested was lower. It is interesting to compare our results with those of DeSamblanx and coauthors [21]. They isolated two highly homologous radish defensins Rs-AFP1 and Rs-AFP2 [21] that differed only by 2 amino acid residues [18,21] located in beta1 and a-helix resulting in a higher net positive charge of Rs-AFP2 in comparison with Rs-AFP1. The Rs-AFP2 was much more active against the test fungus F. culmorum than Rs-AFP1 [21]. However, the replacement of amino acids at selected positions including the C-terminus in Rs-AFP2 by arginine using site-directed mutagenesis showed that in general, arginine substitutions did not show enhanced antifungal activity compared to the original peptide Rs-AFP2. This is consistent with our results demonstrating lower antifungal activity of Ec-AMP-D2. An interesting conclusion follows from the comparison of amino acid sequences of E. crusgalli defensins determined in this work and T. kiharae defensin Tk-AMP-D1 sequenced earlier [39]. As shown above, the former exhibited potent antifungal activity against the pathogens tested, while the latter conversely was only very weakly active on the same fungi. Sequence analysis showed that both peptides were highly homologous in the N-terminal half of the molecule (first 27 amino acid residues), which encompasses beta1, a-helix and the loop connecting these two secondary structure elements. The C-terminal fragment corresponding to beta3 is also very similar (two nonsynonymous substitutions are found in this region). The main variation in the amino acid sequences between Ec-AMP-D1 and Ec-AMP-D2 of E. crusgalli and Tk-AMP-D1 of T. kiharae resides in the loop connecting the
a-helix and beta2 (residues 28e30 in E. crusgalli), in beta2 (31e34 in E. crusgalli), and in the loop connecting beta2 and beta3 (35e37 the numbering is according to E. crusgalli D1 defensin). From these observations, we may speculate that just these regions are responsible for the higher antifungal activity of E. crusgalli Ec-AMP-D1 defensin in comparison with T. kiharae Tk-AMP-D1 defensin. It is of particular interest that the same region is the most variable in other wheat defensisns Tk-AMP-D1.1 and g1-P (Fig. 3). Similar conclusions were drawn from the comparison of two structurally related Medicago sativa (MsDef1) and Medicago truncatula (MtDef2) defensins [21], which shared 65% amino acid sequence homology but differed considerably in antifungal activity. Characterization of the in vitro antifungal activity of the chimeric molecules containing portions of both peptides in different combinations showed that the major determinants of the antifungal activity are located in the C-terminal part of the molecule (amino acid residues 31e45) [21]. 5. Conclusion From seeds of the weed belonging to the Poaceae family E. crusgalli L. Beauv., we have isolated and sequenced two novel highly homologous defensins Ec-AMP-D1 and Ec-AMP-D2 that differ in a single amino acid residue. We have shown that both peptides displayed antifungal activity against several phytopathogenic fungi and the oomycete P. infestans, the EcAMP-D1 peptide being more active than Ec-AMP-D2 against P. infestans. As far as we know, this is the first report on the discovery of antifungal activity in defensins of the Poaceae plants. E. crusgalli defensins exhibiting antifungal activity seem good candidates for transformation of wheat and other crops. The comparison of amino acid sequences of antifungal E. crusgalli defensins with a highly homologous T. kiharae defensin Tk-AMP-D1, which was nearly inactive against all fungi tested, showed that the antifungal determinants most likely reside in the C-terminal region of the molecule. The results obtained are also interesting from the point of view of the evolution of the Poaceae family. Very high similarity in amino acid sequences between the N-terminal regions of E. crusgalli defensins and T. kiharae defensin Tk-AMP-D1 revealed in this work indicated high conservation of this region of the molecule preserved in the evolution and remained nearly unchanged after the divergence of the tribes Paniceae (E. crusgalli) and Triticeae (T. kiharae). We may speculate that the conservation of the N-terminal portion of the molecule is vital for some universal (e.g. structural) functions of cereal defensins, while the C-terminal portion of the molecule is more variable and is responsible for functional specificity. Acknowledgments This work was supported in part by RFBR grants 06-0448874 and 08-04-00783. The authors are grateful to A. N. Smirnov for assistance with tests against P. infestans and some fungi.
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Biochimie 90 (2008) 1667e1673 www.elsevier.com/locate/biochi
Research paper
Seed defensins of barnyard grass Echinochloa crusgalli (L.) Beauv. Tatyana I. Odintsova a, Eugene A. Rogozhin b, Yurij Baranov b, Alexander Kh. Musolyamov b, Nasser Yalpani c, Tsezi A. Egorov b,*, Eugene V. Grishin b a
Laboratory of Plant Genetics, Vavilov Institute of General Genetics, Russian Academy of Sciences, ul. Gubkina 3, 117809 Moscow, Russian Federation Laboratory of Neuroreceptors and Neuroregulators, Shemyakin and Ovchinnikov Institute of Bioorganic Chemistry, Group of antimicrobial peptides, Russian Academy of Sciences, ul. Miklukho-Maklaya 16/10, 117997 Moscow, Russian Federation c Pioneer Hi-Bred International, Inc., 7300 NW 62nd Avenue, Johnston, IA 50131-0552, USA
b
Received 3 April 2008; accepted 16 June 2008 Available online 26 June 2008
Abstract From the annual weed barnyard grass Echinochloa crusgalli (L.) Beauv., two novel defensins Ec-AMP-D1 and Ec-AMP-D2 that differ by a single amino acid substitution were isolated by a combination of different chromatographic procedures. Both defensins were active against several phytopathogenic fungi and the oomycete Phytophthora infestans at micromolar concentrations. The Ec-AMP-D1 showed higher activity against the oomycete than Ec-AMP-D2. The comparison of the amino acid sequences of the antifungal E. crusgalli defensins with those of earlier characterized T. kiharae defensins [T.I. Odintsova, Ts.A. Egorov, A.Kh. Musolyamov, M.S. Odintsova, V.A. Pukhalsky, E.V. Grishin, Seed defensins from T. kiharae and related species: genome localization of defensin-encoding genes, Biochimie, 89 (2007) 605e612.] that were devoid of substantial antifungal activity point to the C-terminal region of the molecule as the main determinant of the antifungal activity of E. crusgalli defensins. Ó 2008 Elsevier Masson SAS. All rights reserved. Keywords: Weeds; Echinochloa crusgalli (L.) Beauv.; Triticum kiharae Dorof. et Migusch.; Defensins; Antifungal activity
1. Introduction Among the innate immunity mechanisms evolved by plants to protect themselves against pathogenic microorganisms and pests, the synthesis of compounds with antimicrobial properties plays a pivotal role [1]. The antimicrobial compounds include phytoalexins, and proteins and peptides that display activity against pathogens. Antimicrobial peptides (AMPs) are important components of the plant defense system [2e5]. They are cationic and amphiphilic molecules that inhibit growth of a wide range of microorganisms and pathogenic insects. In the case of microbes, their mode of action involves
Abbreviations: AMP, antimicrobial peptide; HPLC, high-performance liquid chromatography; SE, size-exclusion; RP, reversed-phase; MALDITOF MS, matrix-assisted laser-desorption/ionization time-of-flight mass spectrometry; TFA, trifluoroacetic acid. * Corresponding author. Tel.: þ7 495 3364022; fax: þ7 495 3307301. E-mail address: [email protected] (T.A. Egorov). 0300-9084/$ - see front matter Ó 2008 Elsevier Masson SAS. All rights reserved. doi:10.1016/j.biochi.2008.06.007
the disruption of the membranes of pathogens via specific or nonspecific interactions with cell surface groups [6,7]. The deleterious effect on insects is supposed to be associated with inhibition of gut enzymes e amylases and proteases [8e10]. Most plant AMPs are cysteine-rich peptides. According to cysteine motifs (the arrangement of cysteine molecules in the amino acid sequence of the peptide) and three-dimensional scaffold, AMPs are classified into several families (thionins, defensins, lipid-transfer proteins, hevein- and knottin-like peptides, and macrocyclic peptides) [2e4]. Defensins belong to the largest and best-studied AMP family [11e14]. They were discovered in invertebrates, birds, mammals and plants [15]. More than 300 defensin-like sequences were identified in the completely sequenced Arabidopsis genome [16]. This redundancy is probably necessary to protect a plant against selections of pathogen mutants with enhanced tolerance to a particular type of a defense molecule. Plant defensins have a characteristic cysteine signature with 8 cysteine residues involved in the formation of 4 disulphide
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bridges. Despite considerable variation in amino acid sequences, the fold of the molecule is similar and involves the so-called cysteine-stabilized alpha-helix beta-sheet motif (CSab), in which two cysteine residues separated by one turn of the alpha-helix are connected with two cysteines, which are located a single amino acid apart in the third beta strand [12]. A wide range of activities has been revealed by in vitro studies of plant defensins that imply their involvement in biotic and abiotic stress responses. These include fungicidal and bactericidal activities [17e25], inhibition of proteases and amylases [8e10], the abilities to block ion channels [26, 27] and inhibition of protein synthesis [28,29]. Recently, the cytotoxic effect of some defensins on plant [30] and animal [23] cells has been shown. The protective role of defensins in vivo is supported by enhanced tolerance to infections of transgenic plants with constitutive expression of the corresponding genes. Defensins were specifically induced in cold-acclimated wheat plants during cold acclimation [31]. They were also shown to be involved in Zn tolerance in Arabidopsis [32]. Structuree function relationships in defensins remain an intriguing but still insufficiently studied area of research. The exact mechanisms of action of most defensins are poorly understood. Evidence is mounting that defensins interact with specific targets on the fungal membrane. The binding sites, fungusspecific sphingolipids, for the radish Rs-AFP2 and dahlia DmAMP1 defensins on fungal membranes were identified [33e35]. Great yield losses of crops caused by pathogenic microorganisms and pests stimulate the search for new genes conferring pathogen resistance. Defensins exhibiting broad-spectrum resistance are good candidates for genetic transformation of plants. Certain progress has already been achieved in this field. Defensins also bear great potential as models for the creation of new anti-infective drugs, both bactericidal and antimycotic, since in the last decades the incidence of microbial infections is rising, together with the number of pathogenic microorganisms resistant to conventional antibiotics [36]. Weeds, to compete with crops, have developed a highly sophisticated multicomponent adaptation system, which makes them successful as invasive species. The molecular basis of this system is formed by diverse phytotoxins, which are released into the environment and suppress growth of other plants [37], and effective pathogen resistance mechanisms providing better competitiveness and adaptation to stressful conditions. Enhanced pathogen resistance makes weeds a valuable but still poorly explored source of natural antibiotics. The aim of this work was to isolate and study seed defensins from a weed cereal species Echinochloa crusgalli (L.) Beauv. (barnyard grass) (order Poales, family: Poaceae, subfamily Pooideae, tribe Paniceae, genus Echinochloa), which is widely spread throughout the world. It reduces crop yields by removing up to 80% of the soil nitrogen. In addition, it accumulates high level of nitrates that are toxic to livestock [37]. Nevertheless, it has some important usages: hay made of this grass can be kept up for several years. In some countries, it is used as a folk remedy to cure infections [37]. Analysis of E. crusgalli AMPs contributes to the understanding of weed proteomics
and genomics necessary for the identification of genes that could improve crop yields. In this study, we isolated two novel defensins from seeds of this species, determined their amino acid sequences and demonstrated their fungicidal effect on several phytopathogenic fungi and the oomycete Phytophthora infestans. 2. Materials and methods 2.1. Biological material Seeds of E. crusgalli (L.) Beauv. (family Poaceae) were collected in the Krasnodarsky region in 2005. The fungi Fusarium graminearum, Fusarium verticillioides, Colletotrichum graminicola, Diplodia maydis were from the collection of Pioneer Hi-Bred (USA). Fusarium oxysporum, Botrytis cinerea, Alternaria consortiale, Fusarium culmorum and the oomycete P. infestans were from the collection of the Timiryazev Agricultural Academy (Moscow, Russian Federation). Helminthosporium sativum was obtained from the All-Russia Collection of Microorganisms (Pushchino, Moscow Region). 2.2. Isolation of defensins from E. crusgalii seeds Seeds of E. crusgalii (10 g) were ground into flour in a coffee mill and extracted with 10% (v/v) acetic acid in the presence of the proteinase inhibitor cocktail for plant cell extracts (Sigma, St. Louis, MO, USA) at a flour to solvent ratio of 1:10 (w/v) at room temperature for 1.5 h with constant stirring. After centrifugation at 20,000g for 25 min at 4 C, the supernatant was clarified by filtration through a paper filter. Proteins and peptides were precipitated overnight with six volumes of ice-cold acetone at 4 C, collected by centrifugation at 5000g for 15 min at 4 C, air-dried and dissolved in solvent A (0.1% TFA) for desalting. Desalting was performed by RP-HPLC on an Aquapore C8 column, 100 10 mm (Applied Biosystems, USA) equilibrated with solvent A. After elution of the unadsorbed fraction, proteins and peptides were eluted with solvent B (80% CH3CN in 0.1% TFA) at a flow rate of 1.5 ml/min and detected at 214 nm. The obtained fraction was dried on a SpeedVac concentrator (Savant, USA). For the purification of defensins from E. crusgalii seeds the protocol developed previously for the isolation of T. kiharae defensins was employed [38,39]. Desalted fraction was solubilized in 10 mM TriseHCl, pH 7.2 (buffer A) and subjected to affinity chromatography on a column (30 15 mm) of Heparin Sepharose 6 Fast Flow (GE Healthcare Bio-Sciences AB, Uppsala, Sweden) equilibrated with buffer A. After elution of the unadsorbed fraction, proteins and peptides were eluted with a step-wise NaCl gradient in buffer A e 50, 100 and 500 mM NaCl, respectively, at a flow rate of 1 ml/min. Proteins and peptides were detected at 280 nm. The obtained fractions were desalted and dried on a SpeedVac concentrator as described above. The fraction eluted at 100 mM NaCl concentration during affinity chromatography was separated by sizeexclusion chromatography on a Superdex Peptide HR 10/30 column (GE Healthcare Bio-Sciences AB, Uppsala, Sweden).
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Proteins and peptides were eluted with 5% CH3CH in 0.05% TFA at a flow rate of 15 ml/h and detected at 214 nm. The peptide fraction was further separated by RP-HPLC on a ReproSil-Pur 300 ODS-3 column (250 4 mm) (Dr. A. Maisch, Ammerbuch, Germany) with a linear acetonitrile gradient (10e40% solvent B) at a flow rate of 0.7 ml/min and detection at 214 nm. 2.3. Isolation of defensins from seeds of T. kiharae The procedure for the isolation of T. kiharae defensins was described previously [38,39]. 2.4. Reduction and alkylation of defensins Defensins were reduced with dithiothreitol (DTT) and alkylated with 4-vinylpyridine as described [38]. The peptide (approx. 2 nmol) was dissolved in 20 ml of 0.5 M TriseHCl buffer, pH 7.6, containing 6 M guanidine hydrochloride and 2 mM ethylenediaminetetraacetic acid (EDTA, disodium salt). One microliter of freshly prepared aqueous DTT solution (1.4 mM DTT) was added to the mixture. The reaction was allowed to proceed under nitrogen for 4 h at 40 C. After reduction, 2 ml of 50% 4-vinylpyridine in 2-propanol (v/v) was added, mixed and allowed to react for another 20 min under nitrogen at room temperature in the dark. After the reaction, the mixture was diluted twofold with 0.1% (v/v) TFA and desalted on a ReproSil-Pur C18 column as described above. The number of cysteine residues was estimated as the mass difference between the reduced and alkylated and the nonalkylated peptide. 2.5. Digestion of defensins with Glu-C endoproteinase Reduced and alkylated defensins (approx. 1.5 nmol) were dissolved in 20 ml of 25 mM ammonium bicarbonate buffer, pH 7.8. Glu-C endoproteinase from Staphylococcus aureus V8 for protein sequencing (Sigma) dissolved in the same buffer was added to the peptide solution (1:20, w/w). The reaction proceeded for 4 h at 40 C and was stopped by the addition of 100 ml 0.1% v/v TFA. The peptides generated during the enzymatic cleavage were separated by RP-HPLC on a Reprosil column with a linear acetonitrile gradient (0e40% solvent B). Solvents A and B were the same as used for the purification of defensins. Peptides were detected at 214 nm. 2.6. Analytical methods Mass spectra were acquired on a model Ultraflex MALDITOF mass spectrometer (Bruker Daltonics, Germany). 2,5-Dihydroxy benzoic acid (Sigma, USA) was used as a matrix. The instrument was calibrated with a mixture of insulin b-chain and bovine insulin (Sigma, USA). Amino acid sequences were determined by automated Edman degradation on a model 492 Procise sequencer (Applied Biosystems) according to the manufacturer’s protocol. Amino
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acid sequence homology searches were carried out by computerized query of the GenBank/EMBL data bank. 2.7. Antifungal assays The antifungal activity of defensins was tested against the phytophathogenic fungi F. graminearum, F. oxysporum, F. verticillioides, C. graminicola, B. cinerea, H. sativum and D. maydis and carried out in microplates essentially as described [40]. Twofold dilution series of the peptides in Milli-Q water were prepared and added to the spore suspension in the growth medium. The concentration of spores varied from 4000 to 6000 spores/ml depending on the fungus. The concentration of peptides was determined from a calibration curve obtained with aprotinin dilutions. Plates were scored after 24e48 h of incubation. Inhibition of spore germination was assayed and scored from 0 to 4, with 4 representing complete inhibition of spore germination and hyphal growth and 0 denoting the absence of inhibition compared to controls. For the oomycete P. infestans, two parameters were studied: the release of zoospores after incubation at 4e6 C for 2e3 h and the development of hyphae from zoosporangia after growth at 20e22 C for 48 h. The effect of the peptide and the degree of inhibition were estimated by measuring the number of zoospores released and the zoosporangia with hyphae relative to their total number. Morphological changes in the microorganisms were also recorded. 3. Results 3.1. Isolation of defensins from E. crusgalli seeds Isolation of defensins from seeds of E. crusgalli followed the protocol earlier developed for the purification of T. kiharae defensins [38,39] and included acidic extraction of flour in the presence of the protease inhibitors with subsequent separation of the protein/peptide fraction by affinity chromatography, SE-HPLC and RP-HPLC. Since the molecular masses of plant defensins are approx. 5 kDa [12], mass spectrometry was used at each purification step to trace the defensin-containing fractions. Separation of the acid-soluble fraction from seeds of E. crusgalli by affinity chromatography is shown in Fig. 1A. Three fractions eluted at 50 mM, 100 mM, and 500 mM NaCl concentration, respectively, were collected. Molecular mass analysis showed that the fraction eluted at 100 mM NaCl concentration contained peptides in the molecular mass range of defensins, therefore this fraction was further subjected to separation by SE-HPLC (Fig. 1B). In this case, six fractions were obtained, fraction 5 contained peptides, which were further separated by RP-HPLC (Fig. 1C). As a result, two peptides Ec-AMP-D1 and Ec-AMP-D2 with molecular masses of 5098 and 5170 Da, respectively, were obtained. The number of cysteine residues in both peptides was determined by estimating the mass difference between the reduced and alkylated and the unreduced native peptides. Both peptides had 8 cysteine residues which is typical for defensins. Alkylation of unreduced peptides followed by mass measurements showed that
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Fig. 2. RP-HPLC separation of the peptides obtained by Glu-C digestion of the Ec-AMP-D1 defensin from seeds of E. crusgalli. Reconstruction of the polypeptide chain of Ec-AMP-D1 is shown on the top. Black arrows show the N-terminal sequence of the entire peptide and the C-terminal Glu-C fragment. Dashed line shows two Glu-C peptides identified by mass analysis of fraction no. 1. The position of fragments in the polypeptide chain is given in parentheses.
Fig. 1. Separation of acid-soluble proteins/peptides from seeds of E. crusgalli (L.) Beauv. (A) Affinity chromatography of the total extract on Heparin Sepharose 6 Fast Flow. (B) SE-HPLC of the 100-mM NaCl fraction on a Superdex Peptide HR 10/30 column, fraction no. 5 containing defensins is shaded. C, RP-HPLC of fraction no. 5 obtained by SE-HPLC on a Reprosil-Pur 300 ODS-3 column. For experimental details, see Section 2.2.
all cysteine residues were involved in the formation of disulphide bridges which is also a characteristic feature of plant AMPs including defensins. 3.2. Amino acid sequences of defensins The N-terminal amino acid sequence (29 amino acid residues) of the reduced and alkylated peptide Ec-AMP-D1 was determined by automated Edman degradation. To
determine complete sequence, the peptide was cleaved with Glu-C proteinase, and the reaction products were separated by RP-HPLC (Fig. 2). Two main peaks were obtained (Fig. 2). In peak 1, two molecular masses were detected: 3459.5 and 3174.3 Da. The 3459.5-Da peptide corresponded to the N-terminal part of the molecule (residues 1e27) originating from incomplete cleavage at Glu2, while the 3174.3Da peptide corresponded to the fragment 3e27 of the polypeptide chain. In peak 2, a single peptide with a molecular mass of 2503.2 Da was obtained and sequenced. It corresponded to the C-terminal part of the Ec-AMP-D1 molecule (Fig. 2). The amino acid sequence of Ec-AMP-D2 was determined similarly (data not shown). The comparison of both sequences showed that the EcAMP-D1 and Ec-AMP-D2 peptides were nearly identical, and differed by a single amino acid residue at position 45: in Ec-AMP-D2 a histidine residue was substituted for alanine (Fig. 3). For both peptides, the measured and calculated masses coincided within the accuracy of the mass spectrometric method indicating the absence of post-translational modifications except disulphide bonds. The homology search in the UniProt database revealed homology of the isolated peptides with plant defensins. In addition to amino acid sequence similarity, the cysteine motif was characteristic of defensins: 2-C10-C-5-C-3-C-8-C-6-C-1-C-3-C [14]. The alignment of amino acid sequences of Ec-AMP-D1 and Ec-AMP-D2 with some cereal defensins is given in Fig. 3. Of the defensins taken for comparison, Ec-AMP-D1 and Ec-AMP-D2 shared the highest similarity with T. kiharae D defensins characterized earlier [39]: it ranged from 45 to 65% for different molecular forms with the highest homology score for Tk-AMP-D1.1. The homology with other cereal defensins was lower: 45% with wheat g2-P and barley g1-H, 43% for wheat g1-P, sorghum SI-2 and SI-3, and maize g1-Z. It is of particular interest
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Fig. 3. Amino acid sequence alignment of E. crusgalli (Ec-AMP-D1 and Ec-AMP2), T. kiharae (Tk-AMP-D1 and Tk-AMP-D1.1) and T. turgidum (g1-P) defensins. Amino acid sequences were aligned manually. Gaps were introduced to maximize homology. Variant amino acid residues are shaded. The percent of sequence identity between Ec-AMP-D1 and other defensins is presented on the right, and in parentheses, the percentage of sequence identity between the N-terminal regions (residues 1e27) of defensins is given. The secondary structure elements (b-strands and a-helix) as determined for g1-P [14] are shown in the bottom. The variable region between Ec-AMP-D1, Ec-AMP2 and Tk-AMP-D1 discussed in the text is boxed.
that the homology between E. crusgalli defensins and most cereal defensins in the N-terminal portion of the molecule (residues 1e27, the numbering is according to Ec-AMP-D1) is even higher than for the whole molecules, reaching its maximum of 74% or 85% if we take into account synonymous substitutions for Tk-AMP-D1 (Fig. 3). With other T. kiharae D defensins it is lower (from 70 to 62%) (data not shown). These results indicate that most variation between E. crusgalli and T. kiharae D defensins resides in the C-terminal part of the molecule. 3.3. Antifungal assays Testing of the antifungal activity of Ec-AMP-D1 against phytopathogenic fungi revealed inhibitory activity against F. graminearum, F. oxysporum, F. verticillioides, and D. maydis. The peptide Ec-AMP-D1 was inactive against C. graminicola, B. cinerea, and H. sativum at concentrations below 30 mg/ml. The IC50 for inhibition of F. graminearum spore germination was 15 mg/ml, for F. verticillioides, IC50 was about 8.5 mg/ml, and for D. maydis, about 12.5 mg/ml. The Ec-AMP-D1 was only weakly active against F. oxysporum with an IC50 of 102 mg/ml. Determination of the effect of Ec-AMP-D1 on the release of P. infestans zoospores showed no effect at the concentrations tested (below 100 mg/ml). However the Ec-AMP-D1 peptide inhibited hyphal development at IC50 of 25.5 mg/ml. Increasing the peptide concentration enhanced the degree of inhibition, however complete inhibition was not achieved at all concentrations tested. In addition to the inhibition of hyphal growth, morphological changes in the oomycete including lysis of hyphae and sporangia in the presence of the peptide at concentrations above 100 mg/ml were observed. Since in this study the peptide Ec-AMP-D2 was isolated from seeds in much smaller amounts than D1, its activity was tested only against F. oxysporum and P. infestans. Antifungal assays showed that the activity of Ec-AMP-D2 against F. oxysporum was similar to that of Ec-AMP-D1 and rather weak, IC50 for both peptides was 102 mg/ml. However, the activity of Ec-AMP-D2 against the oomycete P. infestans was lower than that of Ec-AMP-D1. The IC50 for inhibition of hyphal development was 50 mg/ml. In addition, in contrast to
Ec-AMP-D1, Ec-AMP-D2 caused no morphological changes in the oomycete. Accordingly although the differences between the amino acid sequences of both E. crusgalli defensins were minimal, their biological activity in vitro was affected. Since defensins from E. crusgalli seeds showed striking similarity with T. kiharae Tk-AMP-D1 defensin especially in the N-terminal part of the molecule, we compared their antifungal activity against several phytopathogenic fungi. Testing of antifungal activity of Tk-AMP-D1 showed that it was inactive against most fungi tested at concentrations below 100 mg/ml, such as: A. consortiale, B. cinerea, H. sativum, F. culmorum, C. graminicola and D. maydis. The T. kiharae D1 defensin was only weakly active against F. graminearum and F. verticillioides at concentrations below 30 mg/ml, the inhibition of F. graminearum scored 1.5 and that of F. verticilloides, 1 at the highest tested concentrations (30 mg/ml). Thus, the activity of T. kiharae D1 defensin against the Fusarium species was much weaker than that of the E. crusgalli EcAMP-D1 defensin.
4. Discussion Plant defensins are widespread antimicrobial peptides, which share a similar tertiary structure, but differ considerably in biological functions [14]. They are regarded as novel leads for antifungal therapeutics [36] and candidates for plant transformation and production of pathogen-resistant crops. Defensins of cultivated cereals (wheat, barley, sorghum, and maize) studied so far represent a unique group in the sense that they lack substantial antifungal activity [20]. Our results confirmed the available literature data and showed that defensins of T. kiharae, a synthetic allopolyploid produced by crossing Triticum timopheevii and Aegilops squarrosa failed to inhibit growth of the filamentous fungi tested. In this work, we showed for the first time that defensins from a weed species E. crusgalli, which also belongs to the Poaceae family, in contrast to cultivated cereals studied so far, possess rather potent antifungal activity against a number of phytopathogenic fungi and the oomycete. This finding represents a spectacular example of preservation of antifungal functions in defensins of weeds to achieve fitness, competitiveness and adaptation in
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agroecosystems, which were largely lost during cultivation of cereals. The discovery of antifungal activity in E. crusgalli defensins opens new possibilities for the analysis of structureefunction relationships e.g., in particular, the identification of determinants of antifungal activity. In general, this may be achieved by several approaches: by comparing amino acid sequences of highly homologous peptides of different origin or those of a single plant species contrasting in biological (antifungal) activity, by site-directed mutagenesis, and by generating chimeric molecules combining different regions of highly homologous peptides that differ in antifungal activity (strong or weak). The pioneering works exploiting first two approaches were performed by the Belgium group [18,21] using radish defensins as a model. In this work, we isolated two nearly identical defensins from E. crusgalli. The peptides differed by a single amino acid substitution at position 45: in Ec-AMP-D2 Ala was substituted for His, thus providing the Ec-AMP-D2 molecule a higher net positive charge. This single replacement affected the biological activity of Ec-AMP-D2. Our results showed that although the activity of both peptides was similar towards the fungus F. oxysporum, it differed towards the oomycete P. infestans, Ec-AMP-D1 being more active than Ec-AMP-D2. In addition Ec-AMP-D1 caused morphological changes in the oomycete P. infestans, while Ec-AMP-D2 did not. Thus although the net positive charge of Ec-AMP-D2 was higher than that of Ec-AMP-D1 its antifungal activity against the microorganisms tested was lower. It is interesting to compare our results with those of DeSamblanx and coauthors [21]. They isolated two highly homologous radish defensins Rs-AFP1 and Rs-AFP2 [21] that differed only by 2 amino acid residues [18,21] located in beta1 and a-helix resulting in a higher net positive charge of Rs-AFP2 in comparison with Rs-AFP1. The Rs-AFP2 was much more active against the test fungus F. culmorum than Rs-AFP1 [21]. However, the replacement of amino acids at selected positions including the C-terminus in Rs-AFP2 by arginine using site-directed mutagenesis showed that in general, arginine substitutions did not show enhanced antifungal activity compared to the original peptide Rs-AFP2. This is consistent with our results demonstrating lower antifungal activity of Ec-AMP-D2. An interesting conclusion follows from the comparison of amino acid sequences of E. crusgalli defensins determined in this work and T. kiharae defensin Tk-AMP-D1 sequenced earlier [39]. As shown above, the former exhibited potent antifungal activity against the pathogens tested, while the latter conversely was only very weakly active on the same fungi. Sequence analysis showed that both peptides were highly homologous in the N-terminal half of the molecule (first 27 amino acid residues), which encompasses beta1, a-helix and the loop connecting these two secondary structure elements. The C-terminal fragment corresponding to beta3 is also very similar (two nonsynonymous substitutions are found in this region). The main variation in the amino acid sequences between Ec-AMP-D1 and Ec-AMP-D2 of E. crusgalli and Tk-AMP-D1 of T. kiharae resides in the loop connecting the
a-helix and beta2 (residues 28e30 in E. crusgalli), in beta2 (31e34 in E. crusgalli), and in the loop connecting beta2 and beta3 (35e37 the numbering is according to E. crusgalli D1 defensin). From these observations, we may speculate that just these regions are responsible for the higher antifungal activity of E. crusgalli Ec-AMP-D1 defensin in comparison with T. kiharae Tk-AMP-D1 defensin. It is of particular interest that the same region is the most variable in other wheat defensisns Tk-AMP-D1.1 and g1-P (Fig. 3). Similar conclusions were drawn from the comparison of two structurally related Medicago sativa (MsDef1) and Medicago truncatula (MtDef2) defensins [21], which shared 65% amino acid sequence homology but differed considerably in antifungal activity. Characterization of the in vitro antifungal activity of the chimeric molecules containing portions of both peptides in different combinations showed that the major determinants of the antifungal activity are located in the C-terminal part of the molecule (amino acid residues 31e45) [21]. 5. Conclusion From seeds of the weed belonging to the Poaceae family E. crusgalli L. Beauv., we have isolated and sequenced two novel highly homologous defensins Ec-AMP-D1 and Ec-AMP-D2 that differ in a single amino acid residue. We have shown that both peptides displayed antifungal activity against several phytopathogenic fungi and the oomycete P. infestans, the EcAMP-D1 peptide being more active than Ec-AMP-D2 against P. infestans. As far as we know, this is the first report on the discovery of antifungal activity in defensins of the Poaceae plants. E. crusgalli defensins exhibiting antifungal activity seem good candidates for transformation of wheat and other crops. The comparison of amino acid sequences of antifungal E. crusgalli defensins with a highly homologous T. kiharae defensin Tk-AMP-D1, which was nearly inactive against all fungi tested, showed that the antifungal determinants most likely reside in the C-terminal region of the molecule. The results obtained are also interesting from the point of view of the evolution of the Poaceae family. Very high similarity in amino acid sequences between the N-terminal regions of E. crusgalli defensins and T. kiharae defensin Tk-AMP-D1 revealed in this work indicated high conservation of this region of the molecule preserved in the evolution and remained nearly unchanged after the divergence of the tribes Paniceae (E. crusgalli) and Triticeae (T. kiharae). We may speculate that the conservation of the N-terminal portion of the molecule is vital for some universal (e.g. structural) functions of cereal defensins, while the C-terminal portion of the molecule is more variable and is responsible for functional specificity. Acknowledgments This work was supported in part by RFBR grants 06-0448874 and 08-04-00783. The authors are grateful to A. N. Smirnov for assistance with tests against P. infestans and some fungi.
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